WO1998003661A2 - Antimicrobial agents, diagnostic reagents, and vaccines based on unique apicomplexan parasite components - Google Patents

Abstract

This invention relates to uses of components of plant-like metabolic pathways not including psbA or PPi phosphofructokinase and not generally operative in animals or encoded by the plastid DNA, to develop compositions that interfere with Apicomplexan growth and survival. Components of the pathways include enzymes, transit peptides and nucleotide sequences encoding the enzymes and peptides, or promoters of these nucleotide sequences to which antibodies, antisense molecules and other inhibitors are directed. Diagnostic and therapeutic reagents and vaccines are developed based on the components and their inhibitors.

Description

ANTIMICROBIAL AGENTS, DIAGNOSTIC REAGENTS, AND VACCINES BASED ON UNIQUE APICOMPLEXAN PARASITE COMPONENTS

The U.S. government may have rights in this patent by means of partial support under: NIH NIAID TMP R01 Al 16945; NIH NIATD TMP R01 Al 27530.

This invention relates uses of components of plant-like metabolic pathways not including psbA or PPi phosphofructokinase and not generally operative in animals or encoded by the plastid DNA, to develop compositions that interfere with Apicomplexan

growth and survival. Components of the pathways include enzymes, transit peptides

and nucleotide sequences encoding the enzymes and peptides, or promoters of these nucleotide sequences to which antibodies, antisense molecules and other inhibitors are directed. Diagnostic and therapeutic reagents and vaccines are developed based on the

components and their inhibitors.

BACKGROUND Apicomplexan parasites cause the serious diseases malaria, toxoplasmosis,

cryptosporidiosis, and eimeriosis. Malaria kills more than 2 million children each year. Toxoplasmosis is the major opportunistic brain infection in AIDS patients, causes loss

of life, sight, hearing, cognitive and motor function in congenitally infected infants, and considerable morbidity and mortality in patients immunocompromised by cancer, transplantation, autoimmune disease and their attendant therapies. Cryptosporidiosis is

an untreatable cause of diarrhea in ADDS patients and a cause of epidemics of

gastrointestinal disease in immunocompetent hosts Eimeria infections of poultry lead to billions of dollars in losses to agricultural industries each year Other Apicomplexan

infections, such as babesiosis, also cause substantial morbidity and mortality Although

there are some methods for diagnosis and treatment of Apicomplexan caused diseases, these are ineffective and often toxic to the subject being treated

The tests available to diagnose Apicomplexan infections include assays which isolate the parasite, or utilize light, phase, or fluorescence microscopy, ELISAs,

agglutination of parasites or parasite components to detect antibodies to parasites, or polymerase chain reaction (PCR) to detect a parasite gene Most of the assays utilize whole organisms or extracts of whole organisms rather than recombinant proteins or

purified parasite components In many instances, the available assays have limited

ability to differentiate whether an infection was acquired remotely or recently, and are limited in their capacity to diagnose infection at the outpatient or field setting

The primary antimicrobial agents used to treat toxoplasmosis are

pyrimethamine (a DHFR inhibitor) and sulfadiazine (a PABA antagonist) The use of

pyrimethamine is limited by bone marrow toxicity which can be partially corrected by the concomitant administration of folinic acid. T. gondu cannot utilize folinic acid but

mammalian cells can. Another problem is that pyrimethamine is potentially teratogenic

in the first trimester of pregnancy The use of sulfonamides is limited by allergy,

gastrointestinal intolerance, kidney stone formation and Stevens-Johnson syndrome There are a small number of antimicrobial agents utilized less frequently to treat toxoplasmosis. These include clindamycin, spiramycin, azithromycin, clarithromycin and atovaquone. Usefulness of these medicines for treatment of toxoplasmosis is limited by toxicities including allergy and antibiotic-associated diarrhea, (especially

Clostridium difficile toxin associated colitis with clindamycin use). Lesser or uncertain efficacy of macrolides such as spiramycin, azithromycin, and clarithromycin also limits use of these antimicrobial agents. Atovaquone treatment of toxoplasmosis

may be associated with lack of efficacy and/or recrudescent disease. There are no

medicines known to eradicate the latent, bradyzoite stage of T. gondii, which is very important in the pathogenesis of toxoplasmosis in immunocompromised individuals or

those with recurrent eye disease.

Medicines used to treat malaria include quinine sulfate, pyrimethamine,

sulfadoxine, tetracycline, clindamycin, chloroquine, mefloquine, halofantrine, quinidine gluconate, quinidine dihydrochloride, quinine, primaquine and proguanil. Emergence of resistance to these medicines and treatment failures due to resistant parasites pose

major problems in the care of patients with malaria. Toxicities of mefloquine include nausea, vomiting, diarrhea, dizziness, disturbed sense of balance, toxic psychosis and

seizures. Melfoquine is teratogenic in animals. With halofantrene treatment, there is consistent, dose-related lengthening of the PR and Qt intervals in the

electrocardiogram. Halofantrene has caused first degree heart block. It cannot be used for patients with cardiac conduction defects. Quinidine gluconate or dihydrochloride also can be hazardous. Parenteral quinine may lead to severe

hypoglycemia. Primaquine can cause hemolytic anemia, especially in patients whose red blood cells are deficient in glucose 6-phosphate dehydrogenase Unfortunately,

there are no medicines known to be effective in the treatment of cryptosporidiosis

To more effectively treat Apicomplexan infections, there is an urgent need for discovery and development of new antimicrobial agents which are less toxic than those currently available, have novel modes of action to treat drug resistant parasites that

have been selected by exposure to existing medicines, and which are effective against presently untreatable parasite life cycle stages (e g , Toxoplasma gondu bradyzoites)

and presently untreatable Apicomplexan parasites (e g , Cryptosporidiu parvum) Improved diagnostic reagents and vaccines to prevent these infections are also needed

Information available on Apicomplexan parasites has not yet provided keys to solutions to health problems associated with the parasites Analogies to other organisms could provide valuable insights into the operations of the parasite There

are reports of Apicomplexan parasites having plastids, as well as the nuclear encoded

proteins, tubulin, calmodulin, PPi phosphofructokinase and enolase, which are reported to be similar in part to, or homologous with, counterparts in plant-like, lower life forms and higher plants There are reports of a plastid genome and components of a protein

synthetic system in a plastid-like organelle of Apicomplexans Plasmodium and

T. gondu plastid DNA sequences were reported to have homologies to algal plastid DNA sequences The plastid membrane of T. gondu was reported to be composed of

multiple membranes that appear morphologically similar to those of plant/algal

chloroplasts, except for the presence of two additional membranes in the T. gondu

plastid, suggesting that it may have been an ancient algal endosymbiont Some of these Apicomplexan proteins such as tubulin, calmodulin and enolase with certain plant-like features also are found in animals, and therefore may appear in the host as well as the parasite. A homologue to a gene, psbA encoding a plant protein important for

photosynthesis, also was said to be present in Apicomplexans.

Certain herbicides have been reported to inhibit the growth of Apicomplexans.

The herbicides which affect growth of Apicomplexans are known to affect plant microtubules or a plant photosynthetic protein. In addition, a compound, salicylhydroxamic acid, (SHAM), had been found to inhibit Plasmodium falciparum

(malaria) and Babesia microti.

Techniques of medicinal chemistry and rational drug design are developed sufficiently to optimize rational construction of medicines and their delivery to sites where Apicomplexan infections occur, but such strategies have not yet resulted in

medicines effective against Apicomplexans. Rational development of antimicrobial agents has been based on modified or alternative substrate competition, product competition, change in enzyme secondary structure, and direct interference with

enzyme transport, or active site. Antisense, ribozomes , catalytic antibodies, disruption

of cellular processes using targeting sequences, and conjugation of cell molecules to toxic molecules are newly discovered strategies employed to interrupt cellular functions and can be utilized to rationally develop novel antimicrobial compounds, but

they have not yet been utilized to design medicines effective against Apicomplexans.

Reagents to diagnose Apicomplexan parasite infections have been developed targeting components of Apicomplexans or immune responses to the parasites, using

ELISA, western blot, and PCR technologies, but improved diagnostic reagents, especially those that establish duration of infection or that can be used in outpatient settings are needed to diagnose Apicomplexan infections No vaccines to prevent

Apicomplexan infections are available for humans and only a live vaccine prepared for prevention of toxoplasmosis in sheep is available for livestock

To summarize, Apicomplexan parasites cause substantial morbidity and

mortality, and treatments against the parasites are suboptimal or non-existent

Improved antimicrobial compounds that attack Apicomplexan parasites are needed Because the diseases Apicomplexan parasites cause in some instances are due to recrudescence of latent parasites, an especially pressing clinical problem is that there are

no effective antimicrobial agents effective for treatment of these latent parasite life cycle

stages, especially in sequestered sites such as the brain or eye New approaches and drug targets are required Better in vitro and in vivo assays for candidate compounds are also needed Better diagnostic and therapeutic methods, reagents and vaccines to prevent these infections are needed

SUMMARY OF THE INVENTION

This invention relates uses of components of plant-like metabolic pathways (not

usually associated with animals, not encoded in the plastid genome, and not including

psbA or PPi phosphofructokinase) to develop compositions that interfere with Apicomplexan growth and survival Components of the pathways include enzymes,

transit peptides and nucleotide sequences encoding the enzymes and peptides, or

promoters of these nucleotide sequences, to which antibodies, antisense molecules and

other inhibitors are directed Diagnostic and therapeutic reagents and vaccines are

developed based on the components and their inhibitors Attenuation of live parasites through disruption of any of these components provides vaccines protective against Apicomplexans.

Transit peptides are used to identify other proteins and their organelle targeting sequences that enter and exit from unique Apicomplexan organelles. The identified components are potential for production of medicines, reagents and assays, and

vaccines. The protein which includes the transit peptide is not necessarily an enzyme in

a biochemical pathway.

The methods and compositions of the present invention arise from the

inventors' discovery that metabolic pathways, and targeting signals similar to those

found in plants and algae, especially, but not exclusively those encoded within the

nucleus, are present in Apicomplexan parasites. These plant-like pathways in Apicomplexan parasites are targetable by inhibitors, as measured by determining whether the inhibitors, either singly or in combination, are effective in inhibiting or

killing Apicomplexan parasites in vitro and/or in vivo.

The present invention includes new methods and compositions to treat,

diagnose and prevent human and veterinary disease due to Apicomplexan infections. The invention is based on applications and manipulations of components of algal and higher plant-like metabolic pathways discovered in Apicomplexan parasites. "Plant¬

like" means that products of the pathways, enzymes and nucleotide sequences encoding enzymes in the pathways, are homologous or similar to products, enzymes and

nucleotide sequences known in plants, wherein plants include algae. As used herein,

"plant-like" excludes metabolic pathways generally operative in animals and pathways involving psbA or phosphofructokinase and those encoded by the plastid genome The limits of a "pathway" are defined as they are generally known to those of skill in the art Methods to detect plant counterparts in Apicomplexan include, a) immunoassays using antibodies directed to products and enzymes known in plants, b) hybridization assays

using nucleotide probes that hybridize to specific sequences in plants; c) determining

homologies of Apicomplexan nucleotide or protein sequences with plant nucleotide or protein sequences, and/or d) substrate tests for specific enzymatic activity The "plant-like" pathways of the present invention are identified by

a) identification of metabolic pathways characteristic of plants but not generally

present in animals, b) identification and characterization of Apicomplexan enzymes, nucleic acids

and transit sequences as components similar or homologous to those in a),

c) identification and development of compounds (inhibitors) which abrogate the effect of the components of the pathways in vitro and in vivo, singly or in a plurality, against one or more types of Apicomplexan parasites and in conjoint Apicomplexan,

bacterial and fungal infections

The identified pathways are then used for a) rational design of compounds more active than the known compounds (inhibitors), with good absoφtion following oral administration, with appropriate tissue

distribution and without toxicity or carcinogenicity;

b) testing of such rationally designed compounds alone and together for safety, efficacy and appropriate absoφtion and tissue distribution in vitro and in vivo.

c) development and testing of diagnostic reagents and assays, d) development and testing of live attenuated and component based vaccines By locating new targets in Apicomplexan pathways, doors now are open for

development of more effective antimicrobial agents to treat Apicomplexan parasites in humans and agricultural animals In addition, enzymes in these plant-like pathways

provide improved diagnostic tests for diseases caused by Apicomplexans Vaccines

against infectious diseases caused by Apicomplexan parasites are derived from the

novel compositions of the invention.

A method for inhibiting an Apicomplexan parasite, includes selecting the metabolic pathway of the present invention and interfering with the operation of the

pathway in the parasite The Apicomplexan parasite is preferably selected from the

group that includes Toxoplasma, Plasmodium, Cryptosporidia, Eimeria, Babesia and Theileria The pathway may utilize a component encoded by an Apicomplexan nuclear

gene

Suitable metabolic pathways or components include a) synthesis of heme from glutamate and tRNA glu by the plant-like, heme synthesis (5 carbon) pathway (hereinafter the "heme synthesis pathway"), b) synthesis of C4 acids (succinate) by the breakdown of lipids into fatty acids

and then acetyl CoA, and their use in the glyoxylate cycle (hereinafter the "glyoxylate cycle"),

c) synthesis of chorismate from phosphoenolpyruvate and erythrose 4

phosphate by the shikimate pathway (hereinafter the "shikimate pathway"),

d) synthesis of tetrahydrofolate from chorismate by the shikimate pathway, e) synthesis of ubiquinone from chorismate by the shikimate pathway; f) electron transport through the alternative pathway with use of the alternative oxidase (hereinafter the "alternative oxidase pathway");

g) transport of proteins into or out of organelles through the use of transit

sequences; h) synthesis of aromatic amino acids (phenylalanine, tyrosine and tryptophan) from chorismate by the shikimate pathway;

i) synthesis of the menaquinone, enterobactin and vitamin Kl from chorismate by the shikimate pathway; j) synthesis of the branched chain amino acids (valine, leucine and isoleucine) from pyruvate and ketobutyrate by the plant-like branched chain amino acid

synthesis pathway; k) synthesis of the "essential" (i.e., not synthesized by animals) amino acids,

histidine, threonine, lysine and methionine by the use of plant-like amino acid synthases;

1) synthesis of linoleneic and linoleic acid; m) synthesis of amylose and amylopectin with starch synthases and Q

(branching) enzymes and their degradation; n) synthesis of auxin growth regulators from indoleacetic acid derived from

chorismate; o) synthesis of isoprenoids (diteφenes, 5 carbon units with some properties of

lipids) such as giberellins and abscidic acid by the mevalonic acid to

giberellin pathway. The interfering compositions are selected from the group consisting of enzyme inhibitors including competitors, inhibitors and competitive or toxic analogues of

substrates, transition state analogues, and products, antibodies to components of the pathways; toxin conjugated antibodies or components of the pathways; antisense molecules, and inhibitors of transit peptides in an enzyme In particular, the interfering

compositions include gabaculine, 3-NPA, SHAM, 8-OH-quinoline, NPMG

Interfering with the operation of the metabolic pathway is also accomplished by introducing a plurality of compositions to the pathway, wherein each of the compositions singly interferes with the operation of the metabolic pathway In certain

instances, the plurality of compositions inhibits the parasite to a degree greater than the

sum of the compositions used singly, that is, exhibits a synergistic effect Embodiments of a plurality of compositions include gabaculine and sulfadiazine,

NPMG and sulfadiazine; SHAM and gabaculine; NPMG and pyrimethamine, NPMG

and cycloguanil (which inhibits Apicomplexan DHFR [TS]), and other inhibitors and

competitors of interrelated cascades of plant-like enzymes Wherein the effect of inhibitors together is greater than the sum of the effects of each alone, the synergistic combination retards the selection of emergence of resistant organisms and is more

effective than the individual components alone

In various embodiments, the interfering composition acts on a latent bradyzoite

form of the parasite, or multiple infecting Apicomplexan parasites simultaneously, or on conjoint infections with other pathogenic microorganisms which also utilize the

plant-like metabolic pathway A method of determining the effectiveness of a composition in reducing the deleterious effects of an Apicomplexan in an animal, include: a) identifying a composition that inhibits growth or survival of an Apicomplexan parasite in vitro by

interfering with a plant-like metabolic pathway and b) determining a concentration of

the composition in an animal model that is non-toxic and effective in reducing the survival of the parasite in the animal host and/or the deleterious effects of the parasite

in the animal.

Developing a lead compound that inhibits an Apicomplexan parasite is

accomplished by a) identifying a plant-like metabolic pathway in an Apicomplexan parasite and b) identifying a composition that interferes with the operation of the pathway as a lead compound.

A composition which inhibits a specific life cycle stage of an Apicomplexan parasite by interfering with a plant-like metabolic pathway that utilizes a component encoded by a nuclear gene includes gabaculine; a composition including an enzyme in a

metabolic pathway in an Apicomplexan parasite that is selectively operative in a life-

cycle stage of the parasite includes the enzymes alternative oxidase, and UDP glucose starch glycosyl transferase. A composition comprising SHAM and 8-OH-quinoline

inhibits the alternative oxidase in the latent bradyzoite form of an Apicomplexan

parasite. A method to identify a plant-like gene encoding a component of a plant-like metabolic pathway in an Apicomplexan parasite is a) obtaining a strain of E. coli that is deficient for a component of the metabolic pathway, said deficiency causing the strain

to require supplemented media for growth; b) complementing the E. coli with a gene or portion of the gene encoding a component of the metabolic pathway in the

Apicomplexan parasite; and c) determining whether the complemented E. coli is able

to grow in unsupplemented media, to identify the gene.

Another method for identifying a plant-like gene product of a metabolic pathway in an Apicomplexan parasite is a) contacting the parasite with a gene probe,

and b) determining whether the probe has complexed with the parasite from which the

identity of the gene product is inferred

A method for identifying a plant-like gene product of a metabolic pathway in an Apicomplexan parasite also includes: a) cloning and sequencing the gene; and b)

determining whether the gene is homologous to a plant gene which encodes a plant

enzyme with the same function.

A method for identifying a plant-like gene product in a metabolic pathway in an

Apicomplexan parasite is a) contacting the parasite or its enzyme with a substrate for

the plant -like enzyme; b) measuring enzyme activity; c) determining whether the

enzyme is operative; and d) inhibiting activity of the enzyme in vitro with an inhibitor.

Identifying a gene or gene product in an Apicomplexan parasite which possesses an organelle transit sequence which transports a protein, wherein the protein

is not necessarily an enzyme in a metabolic pathway, but is identified because it has a characteristic organelle transit sequence is also within the scope of the invention.

The invention also relates to a diagnostic reagent for identifying the presence of

an Apicomplexan parasite in a subject, where the subject includes a domestic or

livestock animal or a human. The reagent may include all or a portion of a component of the plant-like pathway, an antibody specific for an enzyme that is a component of a plant-like metabolic pathway in the parasite, or all or part of a nucleotide sequence that hybridizes to a nucleic acid encoding a component of the pathway A diagnostic assay that identifies the presence of an Apicomplexan parasite or specific life-cycle stage of

the parasite may use the diagnostic reagents defined herein

A diagnostic reagent for identifying the presence of an Apicomplexan parasite, includes an antibody specific for an enzyme that is part of a plant-like metabolic

pathway

A diagnostic assay for the presence of an Apicomplexan parasite in a biological

sample includes a) contacting the sample with an antibody selective for a product of a plant-like metabolic pathway that operates in an Apicomplexan parasite, and b) determining whether the antibody has complexed with the sample, from which the

presence of the parasite is inferred Alternatively, the assay is directed towards a nucleotide sequence In both these cases, appropriate antibody or nucleotide sequences are selected to distinguish infections by different Apicomplexans

An aspect of the invention is a vaccine for protecting livestock animals,

domestic animals or a human against infection or adverse consequences of infection by an Apicomplexan parasite The vaccine may be produced for an Apicomplexan

parasite in which a gene encoding a component of a plant-like metabolic pathway in

the parasite is manipulated, for example, deleted or modified When the gene is

deleted or modified in the live vaccine, the component of the pathway may be replaced by the presence of the product of an enzymatic reaction in tissue culture medium The

vaccine strain can then be cultivated in vitro to make the vaccine A vaccine for protecting animals against infection by an Apicomplexan parasite

is based on an Apicomplexan parasite in which the parasite or a component of a metabolic pathway in the parasite is used

The vaccine may use a component of the pathway that is operative at a

particular life stage of the parasite A suitable component is the AroC gene from T. gondu or P. falciparum.

A method of treatment for an infection in a subject by an Apicomplexan

parasite includes the following steps a) obtaining an inhibitor of a plant-like metabolic

pathway in an Apicomplexan parasite, and b) administering an effective amount of the inhibitor to the subject

BRIEF DESCRIPTIONS OF DRAWINGS

FIG. 1A-C illustrates the heme synthesis pathway and the effect of GSAT in T. gondu

FIG. 1A diagrams the heme synthesis pathway FIGS. IB and 1C show that

uptake of tritiated uracil by tachyzoites (RH strain) is inhibited by gabaculine, an inhibitor of GSA aminotransferase P/S = pyrimethamine and sulfadiazine Note that ALA synthase is also present in T. gondu and constitutes an alternative pathway for heme synthesis

FIG. 2A-B shows unique lipid degradation in the glyoxylate cycle m T. gondu

FIG. 2 A is a schematic representation of the glyoxylate cycle FIG. 2B shows

uptake of tritiated uracil by tachyzoites (RH strain) is inhibited by 3-NPA (0 005 to 5 mg G/ML) Note this inhibitor also effects succinate dehydrogenase, so its inhibitory

effect does not unequivocally support presence of the glyoxylate pathway

FIG. 3A is a schematic representation of a pathway which demonstrates alternative oxidase as an alternative pathway for generation of energy in Apicomplexan

parasites FIG. 3B shows that uptake of tritiated uracil by tachyzoites (RH strain) is

inhibited by SHAM

FIG. 4A is a schematic representation of the pathway for conversion of shikimate to chorismate in T. gondu. The inhibitor of EPSP synthase is NMPG

FIG. 4B shows uptake of tritiated uracil by tachyzoites (RH strain) is inhibited by

NPMG Toxicity ofNPMG was assessed by its ability to prevent growth of human foreskin fϊbroblasts (HFF) after 4 days, as measured by tritiated thymidine uptake and

microscopic evaluation FIG. 4C shows product rescue of NPMG' s inhibitory effect

on EPSP synthase by PABA The effect of P ABA on sulfadiazine is similar, but the

effect on pyrimethamine, as predicted reduces the enzyme to the levels that were

present when media alone was utilized, as measured by the uracil uptake

S = sulfadiazine

PYR = pyrimethamine

PABA = para amino benzoic acid

FIG. 5 is a schematic representation of interrelationships of metabolic pathways

in Apicomplexan parasites

FIG. 6 shows inhibitory effects ofNPMG, gabaculine, SHAM 8-OH-quinoline

and on Cryptosporidia. 3NPA also inhibited Cryptosporidia FIG. 7 shows the effects of gabaculine (20 mM) on growth of tachyzoites/bradyzoites (R5) in human foreskin fibroblasts, over 8 days as determined by uracil uptake Note increased uptake of uracil by the 8^th day.

FIG. 8 shows the effect ofNPMG, pyrimethamine, and pyrimethamine plus

NPMG on survival of mice following intraperitoneal infection with 500 tachyzoites of the RH strain of T. gondii. Dosage ofNPMG was 200 mg/kg/day and pyrimethamine was 12.5 mg kg/day).

FIG. 9 shows nucleotide and deduced amino acid sequences of T. gondii

chorismate synthase cDNA. The asterisk indicates the stop codon.

FIG. 10 shows results of CLUSTAL X alignments of the deduced amino acid sequences of the putative T. gondii, chorismate synthase with the corresponding

sequences from Synechocystis, S. cerevisiae, S. lycopersicu , N. crassa and H. influenza Dashes were introduced to maximize alignment. Amino acids which are identical in all 6 organisms are underlined. The percent identity of the chorismate

synthase from each organism with the T. gondii protein was calculated to be as follows

Synechocystis (51.4%), S. cerevisiae (49.6%), S. lycopersicum (47.2%), N. crassa (45.0%) and H. influenza (44.5%). The large internal regions in the T. gondii sequence

which have no counteφarts in the chorismate synthases of other organisms, were not included in this calculation.

FIG. 11 shows the transit sequences of Zea mays and T. gondii chorismate synthases. The sequences of the transit peptide directing the transport of the wx+ protein into maize amyloplasts and chloroplasts and the portion of the T. gondii

chorismate synthase sequence which is homologous are aligned. The amino acid sequence is given in one letter code * indicates an identical amino acid in the Wx Zea

mays and T. gondu sequences • indicates homologous amino acids in the Wx Zea

mays and T. gondii sequences

The transit sequence in the Wx Zea mays protein (UDP-glucose-starch-glycosyl

transferase) begins at amino acid number 1 and ends at amino acid number 72 The

portion (amino acids 359 to 430) of P. falcφarum AroC which corresponds to the novel internal sequence of the T. gondii AroC which includes the amino acids homologous to the maize protein, is as follows

IPVΕNMSTKKESDLLYDDKGECKNMSYHSTIQ1TOEDQI NSTKGFMPPKNDKNFNNIDDYNVTFNNNEEKLL The T. gondu portion of the AroC (chorismate synthase) sequence which demonstrates 30% homology begins at amino acid number 330 and ends at amino acid number 374 The first (single) arrow indicates the processing site of Zea mays UDP glucose glycosyl transferase transit peptide and the second (double) arrow indicates the location at which

the mature protein begins FIG. 12 shows P.falciparum, chorismate synthase, cDNA and deduced amino

acid sequences

DESCRIPTION OF THE PREFERRED EMBODIMENT

This invention uses components of plant-like interrelated metabolic pathways that are essential for growth or survival of Apicomplexan parasites The pathways are

generally not operative in animals and do not include psbA or PPi phosphofructokinase

and are not encoded in the plastid Components include enzymes, products, targetting

peptides, nucleotide sequences encoding the enzymes or peptides, and promoters, as targets for specific inhibitors. Use of these pathways provide a rational and novel framework to discover, characterize and develop medicines, diagnostic reagents and vaccines for Apicomplexan parasites.

Medicines, diagnostic reagents and vaccines are based upon interrelated plant-

like enzyme cascades involved in the synthesis or metabolism or catabolism of Apicomplexan nucleic acids, amino acids, proteins, carbohydrates or lipids, energy transfer and unique plant-like properties of these enzymes which are shared with, and

provide a basis for, discovery of other parasite proteins which have unique organelle

targeting signals or unique promoter regions of the genes which encode the proteins. Synergistic combinations of inhibitors of the enzymes or proteins or nucleic acids

which encode them are particularly useful in medicines.

To select pathways for use in the invention:

a) plant textbooks and the published literature are reviewed for properties characteristic of plants, but generally not animals, databases such as Genbank or the Apicomplexan ESTs are reviewed to identify homologous Apicomplexan and plant-like

genes; and b) Western, northern and southern analyses, PCR, and ELIS.As are used to

recognize, or are based upon, for example, plant proteins and genes, to determine whether components of the pathways are present in Apicomplexans;

c) cloning, isolation and sequencing of genes and creation of gene constructs are used to identify Apicomplexan plant-like genes and their functions;

d) assays of enzyme activity are used to determined the operation of plant¬

like systems; e) functions of parasite enzymes or part of a parasite enzyme are

demonstrated by complementation of a yeast or bacteria deficient in the enzyme, or

product rescue, or other methods to demonstrate enzyme activity, f) activity of compounds, (i.e., inhibitors) known to abrogate effect of the plant-like enzyme, protein, or nucleic acid which encodes them in vitro and in vivo,

are tested singly or in a plurality, against Apicomplexan parasites alone or together, and in conjoint Apicomplexan, bacterial and fungal infections, The general compositions of this invention are A. Inhibitory compounds based on

a) targeting proteins by

(i) substrate competition and transition state analogues

(ii) product competition

(iii) alteration of active site directly or by modification of secondary structure or otherwise altering function of the active site (iv) interfering with protein function with antibody

(v) targeting an organelle or protein within an organelle using a toxic

compound linked to a targeting sequence

b) targeting nucleic acids encoding proteins (antisense, ribozymes) c) targeting a component of the protein or nucleic acid (as above)

B. Diagnostic reagents (genes, proteins, antibodies) in ELISAs, western blots,

DNA, RNA assays

C. Vaccines (live knockout, live mutated, components - genes, proteins,

peptides, parts of genes constructs, etc ) Specific examples of components of plant-like Apicomplexan pathways are in Table 1 Compounds known to inhibit these enzymes or properties in Apicomplexans

and/or other microorganisms are listed in Table 1, as are novel ways to target them in Apicomplexans.

Table 1 A Apicomplexan plant-like metabolic pathways, components and inhibitors

Function Gene Enzyme or property Known inhibitors of Basis for novel name enzymes or property inhibitor

HEME SYNTHESIS HβmL glutamate-1 -semialdehyde 3-amιno-2,3-dιhydrobenzoιc S AS R aminotransferase (GSAT) acid (Gabaculine) 4-amιno- 5-hexynoιc acid 4-amιno-5- fluoropentanoic acid 4- amιno-5-hexynoιc acid (γ acetylenic GABA), 2-amιno- 3-butanoιc acid (vinyl glycine), 2-amιno-4- methoxy-trans-3-butanoιc, -amιno-5-fluoropentanoιc

GltX glutamyl-tRNA synthase HemA glutamyl-tRNA reductase

SHIKIMATE PATHWAY

Chonsmate AroA 3-enolpyruvylshιkιmate N-(phosphonomethyl) S.AS R synthesis phosphate synthase (3- glycine (glyphosphate) phosphoshι ιmate-1 sulfosate EPSP synthase carboxyvinyitransferase) inhibitors 4 and 5 hydroxymaonate inhibitors of EPSP synthase"

AroB dehydroquinate synthase (5- dehydroquinate dyhdrolase) AroC chorismate synthase 5- enolpyruvylshikimate 3- phosphate phospholyase)

AroC-ts AroC transit sequence

AroD dehydroquinate dehydratase ArvE shikimate dehydrogenase AroF 3-deoxy-d-arabιno- heptulosonate 7 phosphate synthase

AroG chonsmate mutase (7-phospho- 2-dehydro-3-deoxy-arabιno- heptulate aldolase)

AroH 3-deoxy-d-arabιno-heptulosante 7 phosphate synthase Ami shikimate 3- phosphotransferase (shikimate kinase)

Key S, modified substrate competitor, AS, antisense, R, πbozyme, Directed at active site, D None known,

*EPSP synthase inhibitor 4 refers to 3-(phosphonooxy)-4-hydroxy-5-[N-(phosphonomethyl-2- oxoethyl)amιno-1-cyclohexene-1-carboxylιc acid (3α, 4α, 5β), compound with diethyl ethanamide EPSP synthase inhibitor 5 refers to shortened R phosphonate

**A new, aromatic analogue of the EPSP synthase enzyme reaction intermediate 1 has been identified, which contains a 3- hydroxymalonate moiety in place of the usual 3-phosphate group This simplified inhibitor was readily prepared in five steps from ethyl 3,4-dιhydroxybenzoate The resulting tetrahedrai intermediate mimic is an effective, competitive inhibitor versus S3P with an apparent K(ι) of 0 57 +/- 0 06 muM This result demonstrates that 3- hydroxymalonates exhibit potencies comparable to aromatic inhibitors containing the previously identified 3-malonate ether replacements and can thus function as suitable 3-phosphate mimics in this system These new compounds provide another example in which a simple benzene ring can be used effectively in place of the more complex shikimate ring in the design of EPSP synthase inhibitors Furthermore, the greater potency of the tetraheral intermediate mimic versus the glycolate derivative and the 5- deoxy analog, again confirms the requirement for multiple anionic charges at the dihydroxybenzoate 5-posιtιon in order to attain effective inhibition of this enzyme

The following were identified inhibition of Toxoplasma gondii (Tg), Plasmodium falαparum (Pf), and Cryptospoπdium parvum (Cp) EPSP synthase by N-phosphonomethylglycine (NPMG), Tg and Pf chorismate synthase (AroC) cDNA and deduced ammo acid sequences, a novel sequence in the Tg chorismate synthase gene (AroC-ts) a portion of which is homologous with the plastid transit sequence of Zea mays (sweet corn) The Pf chorismate synthase (AroC) also has a corresponding novel and unique internal region Cp, Eimeπa bovis (Eb) genomic DNA which hybridizes with Tg AroC (chorismate synthase) Inhibition of Tg in vitro by NPMG abrogated by para-aminobenzoate (PABA) Synergism of NPMG with pyrimethamine, with sulfadiazine and with SHAM for Tg in vitro, Synergy of NPMG with pyrimethamine against Tg in vivo, SHAM and 8-hydroxyquιnolιne inhibited Tg, Pf, Cp ιn vitro, reactivity of Tg protein of ~66Kd with 5 antibodies (monoclonal and polyclonal to VooDoo lily and 7 brucei alternative oxidases) and reduction to monomer similar to VooDoo lily and T brucei alternative oxidases on a reducing gel, Identification of Tg cDNA and genomic DNA PCR products using primers based on conserved sequences in other alternative oxidases which are probed and sequenced, Tg, Pf, Cp inhibited by high concentrations of gabaculine Reactivity of Tg protein of ~40Kd with 3 antibodies to GSAT (polyclonal α soybean, barley and synechococcus GSATs and not preimmune sera) Reactivity of Cp protein of ~40Kd with α barley GSAT Inhibition of Tg, Pf, Cp in vitro by 3NPA, Reactivity of Tg protein with polyclonal antibodies to cotton malate synthase and cotton isocitrate lyase but not preimmune sera In screening Tg cDNA library α GSAT antibody reactive clones are identified and are sequenced Tg chorismate synthase and dehydroquinase enzymatic activities are demonstrated

Table IB Components of Plant-Like Metabolic Pathways and Inhibitors

Function Gene Enzyme or Known inhibitors of enzyme or Basis for name property novel inhibitor

BRANCHED- anas acetyhydroxy Imidazolinones ιmazquιn=2-[4,5- S,AS,R

CHAIN AMINO acid synthase dιhydro-4-methyl-4-( 1 -

ACID methylethyl)-5-oxo-1 H-imidazol-

SYNTHESIS

(VALINE,

LEUCINE,

ISOLEUCINE)

nιcosulfurn=2-[[[[(4,6-dιmethoxy-

2-pyrιmιdιnyl) ammo] carbonyl] amιno]sulfonyl]-N,N-dιmethyl-3- pyndinecarboxamide, prιmιsulfuron=2-[[[[(4,6- bιs(dιfluoromethoxy)-2- pyπmidinyl) ammo] carbonyl] amιno]sulfonyl]benzoιc acid, thιfensulfuron=3-[[[[(4-methoxy-6- methyl-1 ,3,5-tnazιn-2-yl) am o] carbonyljammo] sulfonyl]-2- thiophene-carboxylic acid, trιbenuron=2-[[[[(4-methoxy-6- methyl-1 ,3,5-trιazιn-2- yl)methylamιno]carbonyl]amιno]s ulfonyl]benzoιc acid, sulfometuron=2-[[f[(4,6-dιmethyl-

2-pyrιmιdιnyl) am o] carbonyl] amιno]sulfonyl]benzoιc acid, metsulfuron=2-[[[[(4-methoxy-6- methyl-1 ,3,5-tπazιn-2- yl)amιno]carbonyl]amιno]sulfonyl] benzoic acid, halosulfuron=,

Sulfonaπilides flumetsulam=N-

(2,6-dιfluorophenyl)-5- methyl[1,2,4]tπazolo[1 ,5- a]pyrιmιdιne-2-sulfonamιde

Kar Keto-acid HOE 704 reducto isomerase ipd isopropylmalate 0-oιsobutenyl oxalhydroxamate dehydrogenase

Key S, modified substrate competitor, AS, antisense, R, πbozyme, D, direct inhibitor, alteration of target These are suitable because they are unique to Apicomplexans Unique to Apicomplexans means that either they do not exist in animals (e g , acetohydroxyacid synthase, linoleic acid synthase, starch-amylose or amylopectin synthase, Q or branching enzyme, UDP glucose, starch glycosyl transferase or have unique antigenic or biochemical properties distinct from those of animals (e g acetylco A carboxylase)

*Also present in animals

♦Other enzymes in these pathways unique to Apicomplexans

Additional herbicides which disrupt cell membranes include Diphenyl ethers [nitro phenyl ethers=] (acιfluorfen=5-[2-chloro-4-(trιfluoromethyl)phenoxy]-2-nιtrobenzoιc acid, fomeasafen=5-[2- chloro-4-(tπfluoromethyl)phenoxy]-N-(methylsulfonyl)-2-nιtrobenzamιde, lactofen=()-2-ethoxy-1- methyl-2-oxoethyl 5-[2-chloro-4-(trιfluoromethyl)phenoxy]-2-nιtrobenzoate, oxyflurfen=2-chloro-1-(3- ethoxy-4-nιtrophenoxy)-4-(tπfluoromethyl)benzene), Other bentazon=3-(1-methylethyl)-(1H)-2, 1 3- benzothιadιazιn-4(3H)-one 2,2-dιoxιde above Additional herbicides which disrupt pigment production include clomazone=2-[(2-chlorophenyl)methyl]-4,4-dιmethyl-3-ιsoxazolιdιnone, amιtrole=1H-1 2,4- trιazol-3-amιne, norflurazoπ=4-chloro-5-(methyl amιno)-2-(3-(trιfluoromethyl) phenyl)-3(2H)- pyπdazinone, flurιdone=1-methyl-3-phenyl-5-[3-(tπfluoromethyl)phenyl]-4(1H)-pyrιdιnone

Enzymes in the heme synthesis [with a default ALA synthase pathway],

shikimate pathway, alternative generation of energy and glyoxylate cycle are

exemplified (Table IA) and the others (Table IB) are suitable for the practice of the invention

As outlined succinctly above, the present invention includes new methods and

compositions to treat, diagnose and prevent human and veterinary disease due to Apicomplexan parasites Apicomplexan infections include those due to Toxoplasma gondii (toxoplasmosis), Plasmodia (malaria), Cryptosporidia (cryptosporidiosis), Eimeria (eimeriosis), Babesia (babesiosis), Theileria (theileriosis), Neospora canmum,

and others An Apicomplexan parasite, Toxoplasma gondu, is a representative of other Apicomplexan parasites because Apicomplexan parasites appear to be phylogenetically related and have organelles and enzymes which are critical for their growth and

survival The presence of plant-like pathways/enzymes is confirmed in Apicomplexans

by a) the effect of known inhibitors of the pathways in plants using in vitro and in vivo assays, b) Western, Northern and Southern hybridization analyses, c) isolation and

comparison of relevant genes, d) demonstration of enzymatic activity, e) demonstration of immunologically reactive proteins which cross-react with proteins in plants,

f) complementation of organisms which lack a gene or part of the gene encoding an enzyme with a parasite gene which encodes the enzyme, and/or g) recognition of plant¬

like transit sequences In vitro assays include product rescue (i.e., complete or partial abrogation of the effect of an inhibitor by providing the product of the reaction and thus

bypassing the need for the enzyme which catalyzes the reaction The assays are based

on inhibition of the parasite i.e. restriction of growth, multiplication or survival of the parasite Another measure of infection is "parasite burden" which refers to the amount (number) of parasites present as measured in vivo in tissues of an infected host

Another measure of infection is destruction of host tissues by the parasites Inhibitors

reduce parasite burden and destruction of host tissues caused by the parasites Preferably the inhibitors must not be toxic or carcinogenic to the parasites' host and for in vitro assays not be toxic to cells in culture

Enzymes of the newly detected plant-like pathways provide novel, unique and useful targets for antimicrobial therapy These unique pathways and enzymes are within the plastid, glyoxosomes, cytoplasm or mitochondria In addition, not suggested

before for these parasites, some enzymes used in these pathways are encoded by genes

within the nucleus Plant-like pathways detected in Apicomplexan parasites include a) the 5-carbon heme biosynthesis pathway that utilizes glutamate as a carbon skeleton for synthesis

and requires the unique enzyme glutamate- 1-semialdehyde aminotransferase, b) the

mobilization of lipids in the glyoxylate cycle which is a unique pathway that includes the enzymes isocitrate lyase and malate synthase, c) the generation of energy by an

alternative pathway which includes a unique alternative oxidase and/or other unique

pathways and enzymes for generating energy in the mitochondria or plastid, and, d) the

conversion of shikimate to chorismate utilized in the synthesis of ubiquinone, aromatic

amino acids and folate by plants, but not humans The shikimate pathway includes the enzyme 3-phospho-5-enolpyruvylshikimate (EPSP) synthase, chorismate synthase, and chorismate lyase, as well as a number of enzymes unique to plants, fungi, bacteria, and mycobacteria, but not to animals. Inhibitors of some of these enzymes also provide information about the functioning and targeting of the enzymes.

The heme synthesis pathway involves enzymes encoded in the nucleus and imported to the plastid. This pathway is present in Apicomplexans including 7. gondii, P. falciparum, and Cryptosporidia parvum . Inhibitors of the enzyme GSAT in the

pathway include gabaculine (3-amino-2,3-dihydro benzoic acid), 4-amino-5-hexanoic

acid, and 4-amino-5-fluropentanoic acid. The glyoxylate cycle, reported to be present in plants, fungi, and algae, is also present in T. gondii. The cycle uses lipids and converts them to C4 acids through a

series of biochemical reactions. One of the last steps in this series of reactions is

dependent on the isocitrate lyase enzyme and another on the malate synthase enzymes. Inhibitors of these enzymes include 3-nitropropionic acid and itaconic acid. The alternative respiratory pathway, present in a range of organisms including

some bacteria, plants, algae and certain protozoans (trypanosomes), is present in T. gondii, Cryptosporidia parvum, and Plasmodium falciparum (in the latter parasite, two clones designated W2 and D6 were inhibited). The pathway is inhibited by a range of compounds including salicylhydroxamic acid, 8-hydroxyquinoline, Benzyhydroxamic

acid (BHAM), m-Chlorohydroxamic acid (m-CLAM), Propylgallate, Disulfliram and

others.

Enzymes involved in the synthesis of chorismate, including those which convert

shikimate to chorismate, and enzymes which generate folate, aromatic amino acids and ubiquinone from chorismate in plants, are present in T. gondu, Plasmodium falciparum,

Cryptosporidium parvum, and Eimeria Inhibitors include N-(phosphonomethyl)

glycine (glyphosate, sulfosate and others) A full-length T. gondu cDNA sequence encoding a chorismate synthase from this pathway and the deduced amino acid

sequence provide information useful in developing novel antimicrobial agents The

T. gondu chorismate synthase has features in common with other chorismate synthases

and entirely unique features as well The unique features are novel sequences not shared with chorismate synthases from other organisms but with homology to an amyloplast/chloroplast transit sequence of Zea mays (sweet corn) A P. falciparum

cDNA sequence encoding chorismate synthase and its deduced amino acid sequence also provide information useful for developing novel antimicrobial agents

The genomic sequences provide information about regulation of the gene (e g , unique promoter regions) and such unique regions enable targeting their regulatory elements with antisense

A part of the novel internal sequence (i e , SCSFSESAASTIKHERDGSAATLS E

RASDGRTTSRHEEEVERG) in the T. gondii AroC (chorismate synthase) gene has homology

with the chloroplast/amyloplast targeting sequence of Zea mays (sweet corn) wx (UDP

glucose-starch-glycosyl transferase) protein (i e , MAAIATSQLVATRAGLGVPDASTFRRG

AAQGLRGARASAAADTLSMRTSARAAPRHQQQARRGGRFPSLWC) This transit sequence provides a

novel way to target T. gondu enzymes that move from the cytoplasm into the plastid

and is generally applicable to targeting any subcellular organelle The P. falciparum

AroC (chorismate synthase) has a corresponding novel internal sequence Additional pathways found in Apicomplexan parasites include the synthesis of

branched chain amino acids (valine, leucine and isoleucine) and acetohydroxy acid synthase is the first enzyme in the branched chain amino acid synthesis pathway, inhibited by sulfonylureas and imidazolinones, as well as the synthesis of other

"essential" amino acids, such as histidine, methionine, lysine and threonine. Starch synthesis, including starch synthases, the UDP-glucose-starch glycosyl transferase, and debranching enzymes and enzymes of lipid, terpene, giberellin and auxin synthesis, are

part of other pathways in Apicomplexan parasites. Down modulation of the UDP-

glucose starch glycosyl transferase pathway leads to a switch from amylose to amylopectin synthesis and thus the bradyzoite phenotype.

Demonstration of presence of one enzyme or the gene that encodes it in a

known pathway implies presence of the full pathway. Thus, enzymes in parasite metabolic pathways that can be inhibited include: glutamyl-tRNA synthetase; glutamyl-

tRNA reductase; prephenate dehydrogenase; aromatic acid aminotransferase (aromatic transaminase); cyclohexadienyl dehydrogenase; tryptophan synthase alpha subunit;

tryptophan synthase beta subunit; indole-3-glycerol phosphate synthase (anthranilate isomerase), (indoleglycerol phosphate synthase); anthranilate phosphoribosyltransferase; anthranilate synthase component I; phosphoribosyl anthranilate isomerase; anthranilate synthase component II; prephenate dehydratase

(phenol 2-monooxygenase); catechol 1,2-deoxygenase (phenol hydroxylase);

cyclohexadienyl dehydratase; 4-hydroxybenzoate octaprenyltransferase; 3-oxtaprenyl-4-

hydroxybenzoate carboxylyase; dehydroquinate synthase (5-dehydroquinate hydrolase);

chorismate synthase (5-enolpyruvylshikimate 3-phosphate phosph-lyase); dehydroquinate dehydratase, shikimate dehydrogenase, 3-deoxy-d-arabino-

heptuloonate 7 phosphate synthase, chorismate mutase (7-phospho-2-dehydro-3-

deoxy-arabino-heptulate aldolase), 3-deoxy-d-arabino-heptuloonate 7 phosphate

synthase, shikimate 3-phosphotransferase (shikimate kinase), UDP glucose starch glycosyl transferase, Q enzymes, acetohydroxy acid synthase, glutamate- 1-

semialdehyde 2, 1 -aminotransferase, chorismate lyase, malate synthase, isocitrate lyase, and 3-enolpyruvylshikimate phosphate synthase (3-phosphoshikimate-l carboxyvinyltransferase)

Recombinant protein produced by constructs with genes encoding these

enzymes in E coli or in other expression systems is useful for producing antibodies and obtaining a crystal structure Native enzyme is isolated The expressed and native proteins are used to design and test new inhibitors in enzyme assays Expressed and

native (from varied life-cycle stages) proteins are used and the expressed protein is a

source of the enzyme, and the enzyme assay is carried out in the presence and absence

of the inhibitors, either alone or in combination and controls include the buffer for the enzyme alone The crystal structure is useful for characterizations of enzyme active

site(s), secondary structure, transit sequence, substrate and product interactions The

design of additional inhibitors is carried out using published methods such as modifying substrates as had been done with inhibitors of EPSP synthase

Certain pathways are shown to be affected by inhibitors which are synergistic in

vitro Examples of synergistic inhibitors in vitro are gabaculine (heme synthesis) and

SHAM (alternative energy generation), NPMG and SHAM; NPMG and sulfadiazine; and NPMG and pyrimethamine. Gabaculine and sulfadiazine are an additive combination in vitro.

An aspect of the invention is identifying potential targets for therapeutic intervention by considering nuclear as well as organellar genes as part of the production of enzymes for unique plant-like pathways. For example, the protein synthesis of plant¬

like proteins that is also demonstrated in Apicomplexan parasites suggests not only

conservation of plastid genes but also conservation of nuclear genes which encode enzymes that act inside or outside the plastid, from an ancestor that is common to Apicomplexan parasites and algae. Many vital metabolic pathways of algae (often

shared with their evolutionary relatives, higher plants) also are conserved in the

Apicomplexan parasites, whether or not the pathways involve the plastid. Consequently, Apicomplexan parasites are sensitive to inhibitors that block several of these unique pathways. Combined attack on multiple targets retards the emergence/selection of resistant organisms. Considering nuclear and organellar genes

has the dual advantage of rapidly identifying conservation of specific pathways and simultaneously identifying both target sites and lead compounds for therapeutic drug development.

An aspect of the invention is a plurality of inhibitors, singly or in combination,

directed against enzymes and/or genes encoding a different metabolic pathway. Examples of inhibitors suitable for practice of the present invention include GSAT,

3NPA, SHAM, 8-OH-quinoline, and NPMG, sulfonylureas, imidazolinones, other

inhibitors of EPSP synthase or chorismate synthase which include competitive substrate analogues, transitional state inhibitors and direct active site inhibitors as well as other known compounds (Table I). Some pluralities of inhibitors produce synergistic effects.

Improved treatments against Apicomplexan parasites result from a variety of

options:

1. some compositions may inhibit the operation of more than one pathway, thereby producing a strong effect and lessening the probability of resistance to the drug emerging because more than one mutation may be required;

2. some compositions may inhibit more than one step in a pathway;

3. some pluralities of compositions may have synergistic effects, producing more effective drugs; and

4. some compositions may target pathways operative exclusively during a life cycle of the parasite, making them more selective e.g. against the latent phase.

5. some compositions may inhibit other microorganisms (including other Apicomplexans.) An additional detail of the invention is that representative Apicomplexan

parasites, notably T. gondii, are used for assaying candidate inhibitors. The invention is directed at effects of inhibitors of the unique plant-like pathways in Apicomplexan,

alone and in combination. Organisms used for the assays include T. gondii tachyzoites,

bradyzoites and a mutant that expresses 50% tachyzoite and 50% bradyzoite antigens.

Unique plant enzymes and pathways that were found to be inhibited by compounds shown to inhibit plant pathways in Apicomplexans include: (1) glutamate- 1

semialdehyde amino transferase, an enzyme important in heme synthesis, (2) isocitrate

lyase, an enzyme important in utilization of lipids, (3) alternative oxidase enzyme complex, enzymes important in energy production and (4) 3-phospho-5-

enolpyruvylshikimate synthase (EPSP synthase), an enzyme important in conversion of

shikimate to chorismate which is a precursor for synthesis of folate, ubiquinone, and certain amino acids essential for survival

The invention provides a rational, conceptual basis for development of novel

classes of antimicrobial agents that inhibit Apicomplexan parasites, unique diagnostic

reagents, and attenuated vaccines The inhibitors provide lead compounds for the development of antimicrobial agents Conserved enzyme active sites or parts of the molecules or genes that encode the protein which are targeted by the inhibitors provide

the basis for development of new but related ways to target the enzymes, such as related protein inhibitors, intracellular antibodies, antisense DNA, and ribozymes

Inhibitors are effective against more than one parasite (e.g. T. gondu, P. falciparum and C. parvum) and enzymes in these pathways also are present in other

bacterial and fungal pathogens such as Pneumocystis carinii, Mycobacterium tuberculosism Staphylococcus aureus, and Hemophilus influenza, but not animals

Thus, inhibitors of these pathways affect susceptible microorganisms which concurrently infect a host Because enzymes are utilized differentially in different

parasite life-cycle stages, stage-specific inhibitors are within the scope of the invention Genes encoding the enzymes in Apicomplexans are identifiable The genes encoding

the enzymes are effectively knocked out in these parasites by conventional techniques "Knockout" mutants and reconstitution of the missing genes of the parasite demon¬

strate the importance of gene products to the varying life-cycle stages of the parasite which are identified using antibodies to proteins and ability to form cysts in vivo which define the life cycle stages The parasites in which a gene is knocked out are a useful basis for an attenuated vaccine The genes encoding the enzymes or parts of them (e g , a novel targeting sequence) or the proteins themselves alone or with adjuvants

compnse a useful basis for a vaccine The pathways and enzymes of the invention are useful to design related antimicrobial agents The sequences and definition of the active sites of these enzymes, and pathways, and organelle (e g , plastid) targeting sequences

provide even more specific novel and unique targets for rational design of antimicrobial

agents effective against Apicomplexan parasites For example, proteins which interact with the enzyme and interfere with the function of the enzyme's active site, or are competitive substrates or products or intracellular antibodies (i.e., with a gene encoding the Fab portion of an antibody that targets the protein the antibody recognizes), or

antisense nucleic acid or targeted ribozymes that function as inhibitors are useful, novel antimicrobial agents Enzymes of the invention are a novel basis for unique diagnostic

tests Because some of these pathways are important in dormant parasites, or in selecting the dormant or active life cycle stages, they are especially important as

antimicrobial agent targets for life cycle stages of the parasite for which no effective antimicrobial agents are known or as diagnostic reagents which ascertain the duration

of infection

Identification of the pathways in Apicomplexan parasites provides additional

enzyme targets present in these pathways which are not present in or are differentially expressed in animal cells Identification of the interrelatedness of these pathways with

each other provides the basis for the development and demonstration of combinations

of inhibitors which together have an effect which is greater than the expected additive effect (i.e., synergistic) The meaning of synergism is that compound A has effect A',

compound B has effect B', compounds A+ B have an effect greater than A'+ B' Synergism is characteristic of inhibitors of these pathways because an initial pathway affected by an inhibitor often provides a product used as a substrate for another pathway so the inhibition of the first enzyme is amplified These pathways or their

products are interrelated Therefore, the enzymes or DNA which encodes them are targeted by using two or more inhibitors leading to an additive or synergistic effect

Examples include the additive effect of gabaculine and sulfadiazine and the synergistic

effects ofNPMG and sulfadiazine and NPMG and pyrimethamine One or more of the

inhibitors preferentially affect one of the life cycle stages of Apicomplexan parasites Some enzymes are preferentially used by specific stages of the parasites Detection of an enzyme of this type or a nucleic acid encoding it offers a novel

diagnostic test not only for presence of a parasite, but also for identification of the stage

of the parasite Genes encoding enzymes in pathways of the present invention are "knocked out" using techniques known in the art A parasite with a gene knocked out is said to

be attenuated either because the gene expression of the enzyme is stage specific so the parasite cannot become latent, or because the knocked out enzyme is essential for

parasite survival The importance of an enzyme's functions in various life-cycle stages is determined using a mutant-knockout-complementation system In the former case,

the attenuated parasite is useful as a vaccine because the "knocked out" gene is critical for the parasite to establish latency Its administration to livestock animals results in

immunity without persistence of latent organisms Mutants with the gene "knocked out" also can be selected because when the parasites are grown in vitro they are grown

in the presence of product of the enzymatic reaction to allow their survival. However, such attenuated parasites do not persist in vivo in the absence of the product and,

consequently they are useful as vaccines, for example, in livestock animals. The genes that encode the protein also are used in DNA constructs to produce proteins themselves or the proteins or peptides are used in immunized animals. These constructs

are used to elicit an immune response and are used for vaccines alone or with

adjuvants. Specific examples are incorporation of the gene for alternative oxidase or chorismate synthase in a construct which has a CMV promoter and expresses the protein following intramuscular injection (i.e., a DNA vaccine). This type of construct,

but with genes not identified or described as plant-like, has been used as in a vaccines that protect against bacterial and protozoal infections

Plant-like pathways in Apicomplexans were inhibited in vitro. An Apicomplexan GSAT enzyme that is part of a heme synthesis pathway was targeted

with inhibitors. A gene with homology to ALA synthase was identified by analysis of the T. gondii ESTs (Washington University T. gondii gene Sequencing project), indicating that T. gondii has alternative methods for synthesis of ALA. An

Apicomplexan glyoxylate cycle was analyzed to determine the sensitivity of tachyzoites

and bradyzoites to glyoxylate cycle inhibitors. Specifically, Apicomplexans have

isocitrate lyase and malate synthase which present a unique pathway for lipid

metabolism that is targeted by inhibitors. Apicomplexan alternative oxidase is targeted,

as evidenced by effects of inhibitors of alternative oxidase on this pathway and its

expression and immunolocalization in tachyzoites and bradyzoites; Apicomplexan parasites have a metabolically active EPSP synthase enzyme involved in conversion of

shikimate to chorismate These four metabolic pathways, i e., heme synthesis, shikimate pathway, alternative generation of energy, and the glyoxylate cycle are all

exemplified in T. gondii To show that inhibition was specific for key enzymes in these

pathways that are generally absent or used only rarely in mammalian cells, product

inhibition studies were used in vitro For example, growth of T. gondii is sensitive to NPMG that inhibits the synthesis of folic acid via the shikimate pathway Because mammalian hosts lack the entire shikimate pathway, it is unlikely that the parasites can

obtain either PABA or its precursor chorismate from the host cells so provision of PABA circumvents the need for the substrate pathway for folate synthesis and rescues the EPSP synthase inhibition by NPMG

Further proof of the presence of the plant-like pathways arises from biochemical

assays for an enzyme in analogous plant pathways and isolation of encoding genes

Genes are identified by search of available expressed sequence tags (ESTs, i e , short, single pass cDN A sequences generated from randomly selected library clones) by PCR

amplification using primer sequences derived from published conserved sequences of

plant genes with parasite genomic DNA or parasite DNA libraries (Chaudhun et al , 1996), by the screening of Apicomplexan DNA expression libraries with antibodies to previously isolated homologous proteins or the DNA which encodes them and by

complementation of E. coli or yeast mutants deficient in an enzyme Genes isolated by

these techniques are sequenced which permits identification of homologies between plant and Apicomplexan genes using sequence databases such as Genbank These

assays confirm that an enzyme and the gene encoding it are present in Apicomplexan parasites. E. coli mutants and yeast deficient in the enzyme are complemented with plasmid DNA from T. gondii cDNA expression libraries or the isolated gene or a

modification (e.g., removing a transit sequence) of the isolated gene which allows the

production of a functional protein in the E. coli or yeast, demonstrating that the gene encoding the enzyme is functional. Homologous genes in T. gondii, P. malaria,

Cryptosporidia, Neospora, and Eimeria are identified when relevant plant or T. gondii

genes are used as probes to DNA obtained from these organisms and the genes are identified either by cloning and sequencing the DNA recognized by the probe or by using the probe to screen the relevant parasite libraries Genomic DNA is sequenced

and identifies unique promoters which are targeted Unique parts of the genes were identified in the sequences and provide additional antimicrobial agent targets, diagnostic reagents and vaccine components or bases for vaccines Clade and bootstrap analyses (Kohler et al., 1997) establish the phylogenetic origin of novel, sequenced, parasite

genes and this indicates other related antimicrobial agent targets based on components, molecules, and pathways of phylogenetically related organisms Gene products are

expressed and utilized for enzyme assays and for screening novel inhibitors, for making antibodies for isolation of native protein, for x-ray crystallography which resolves

enzyme structures and thus establishes structure-function relationships and enzyme

active sites which are useful for the design of novel inhibitors lmmunoelectronmicroscopy using antibodies to enzymes such as chorismate

synthase, alternative oxidase, malate synthase or isocitrate lyase immunolocalizes the

enzymes within the parasite and determines their location, in particular whether they are in plant-like organelles. Apicomplexan transit peptides are identified by their homology to known transit peptides in other species Attachment of reporter proteins to the wild type transit peptide, or deletion or mutations of the transit peptide or portion of the peptide or gene encoding it, and then characterization of targeting of these constructs

alone or in association with reporter constructs establishes that the amino acid

sequences of the transit peptide determine intracellular localization and site of function of proteins with this sequence Stage specificity of these enzymes is determined in vitro by using antibodies to stage-specific antigens in inhibitor-treated cultures, by Western

or Northern analyses (detection), by enzyme assays using selected parasite life cycle

stages, by using RT PCR (Kirisits, et al, 1996) and a DNA competitor as an internal standard to quantitate the amount of mRNA in parasite samples, by ELISA

(quantitation) and by determining whether a parasite with the gene knocked out can

develop a bradyzoite phenotype in vitro in the appropriate bradyzoite inducing culture conditions Stage specificity in vivo is determined by observing effects of the inhibitors on different life cycle stages in acutely vs chronically infected mice and by determining

whether a parasite with the gene knocked out can form cysts in vivo Useful techniques

to develop diagnostic reagents for detection of these proteins or nucleic acids include ELISAs, Western blots, and specific nucleotides used as probes

EXAMPLES

Example 1: Novel In Vitro Assay Systems to Assess Antimicrobial Effects on T. gondii

New in vitro and in vivo assay systems were developed to determine whether

plant metabolic pathways are present in Apicomplexans. New elements include use of

longer culture times (e.g., extending the duration of the assay to > 6 days is also a unique and useful aspect of this invention, because it allows demonstration of antimicrobial effect for compounds which have to accumulate prior to exerting their effect), use of Me49 PTg and R5 strains in vitro, employing synergistic combinations of

NPMG and low dosage pyrimethamine in vivo, and assays of parasitemia m vivo using competitive PCR

Improvements were developed in the assays reported by Mack et al (1984) and Holfels et al. (1994) to measure T. gondii replication in tissue culture The

improvements are based on microscopic visual inspection of infected and inhibitor treated cultures, and on quantitation of nucleic acid synthesis of the parasite by measuring uptake of ³H uracil into the parasite's nucleic acid Uracil is not utilized by

mammalian cells Parasites present as tachyzoites (RH, Ptg, a clone derived from the

Me49 strain), bradyzoites (Me49), and R5 mutants (mixed tachyzoite/bradyzoites of the Me49 strain that can be stage switched by culture conditions) (Bohne et al , 1993,

Soete et al , 1994, Tomovo and Boothroyd, 1995 , Weiss et al. , 1992) are suitable for

assay systems used to study effects of inhibitors Only the RH strain tachyzoites,

cultured for up to 72 hours, had been used in previously reported assays The use of Me49, Ptg, and R5 mutant are unique aspects of the methods used in these assays in this invention

Results using the assay systems are shown in FIGS. 4, 6-8 In these assays

toxicity of a candidate inhibitor was assessed by its ability to prevent growth of human

foreskin fibroblasts (HFF) after 4 days and after 8 days as measured by tritiated thymidine uptake and microscopic evaluation Confluent monolayers of HFF were infected with tachyzoites or bradyzoites Inhibitor was added one hour later Non-

toxic doses were used in parasite growth inhibition assays Parasite growth was measured by ability to incorporate tritiated uracil during the last 18 hours of culture Example 2: Detection of Plant-like Pathways in Apicomplexans

Using assays disclosed herein, some of which were novel, Apicomplexan

parasites were found to contain at least four metabolic pathways previously thought to be unique to plants, algae, bacteria, dinoflagellates, and fungi. Specifically, the presence of a unique heme synthesis pathway, an alternative oxidase pathway, a glyoxylate cycle and a pathway necessary for the biosynthesis of chorismate and its

metabolites were explored Growth of the parasite, T. gondii, depends upon these pathways To examine T. gondii for the presence of plant-like and algal metabolic

pathways, certain inhibitors of metabolic pathways are suitable to apply because of their ability to prevent growth of the parasite in tissue culture

Pathways which are present in Apicomplexans were analyzed as follows First,

T. gondii tachyzoites were tested to see if they were sensitive in vitro to inhibition by

specific inhibitors of target pathways. Then bradyzoites are tested Positive results for

each pathway provided presumptive evidence that the inhibitor targets were present and that their activities are important for parasite survival and growth The inhibitors

effective in vitro were screened for activity in vivo in mice An example of an effective combination in vivo is NPMG and low dosage pyrimethamine

The presence of an enzyme was further confirmed by product rescue in vitro, in

which the product abrogates the need for its synthesis by the enzyme An example was

rescue by PABA for the reaction catalyzed by EPSP synthase Other methods to

demonstrate the presence of an enzyme and thus the pathway include functional enzyme assays, complementation of mutant E. coli strains, PCR, screening of a T. gondu expression library with antibodies or DNA probes, and immunostaining of Western

blots For some enzymes, identification of a partial sequence of a gene in an EST library in the gene database led to cloning and sequencing the full length gene Demonstration of the enzymes also is diagnostic for presence of the parasites

Examples are demonstration of T. gondii and C. parvum GSAT and T. gondii

alternative oxidase and T. gondii isocitrate lyase and malate synthase by Western analysis and cloning and sequencing of the 7. gondii and P. falciparum chorismate

synthase gene Reagents (gene probes and antibodies) obtained during characterization of genes from T. gondii are used to detect homologous enzymes and pathways in other

Apicomplexan parasites Examples were using the T. gondii chorismate synthase gene to probe P. falciparum, Eimeria bovis and Cryptosporidium parvum genomic DNA

Other examples are using heterologous plant DNA to detect Apicomplexan GSAT,

isocitrate lyase, malate synthase, and alternative oxidase genes Such genes are used as

DNA probes to screen libraries to clone and sequence the genes to identify PCR

products Example 3: Effects of Inhibitors In Vitro on T. gondii

Using the assays described in Example 1, five compounds that restrict the growth of T. gondii in vitro were identified

(i) Gabaculine

(ϋ) NPA

(Hi) SHAM (Salicylhydroxamic Acid),

(iv) 8-hydroxyquinoline

(v) NPMG Specifically these inhibitors act as follows i. The Effect of Gabaculine, An Inhibitor Of The 5-Carbon Heme

Synthesis Pathway, On the Growth of T. gondii

FIG. 1A compares heme biosynthesis in plants, algae and bacteria with heme biosynthesis in mammals In higher plants and algae, ALA is produced in the plastid by

the action of GSA aminotransferase on glutamate 1-semialdehyde In mammals, ALA

is formed through the condensation of glycine and succinyl CoA ALA is subsequently

converted to heme In one dinoflagellate and 7. gondii both pathways are present

Inhibitors of plant heme synthesis pathway restrict the growth of Toxoplasma

gondii in vitro As shown in FIG. 1A, the synthesis of δ-aminolevulinic acid (ALA),

the common precursor for heme biosynthesis, occurs in the plastid of plants, algae and Apicomplexan parasites by the 5-carbon pathway and ALA synthesis occurs by a

different pathway in animals The pathway in animals involves the condensation of

glycine and succinyl CoA The data in FIG. 1B-C and a Western blot utilizing an

antibody to the homologous soybean, and barley, and synechococcus GSATs, demonstrate that Toxoplasma gondii utilizes the 5-carbon pathway for ALA synthesis

and therefore heme biosynthesis. 3-amino 2,3-dihydroxybenzoic acid (gabaculine) inhibits GSA in the heme synthesis pathway

First the toxicity of gabaculine was assessed by its ability to prevent growth of

human foreskin fibroblasts (HFF) as measured by ³H-thymidine uptake and microscopic

evaluation. Non-toxic doses were used in parasite growth inhibition assays In vitro parasite growth inhibition assays included confluent monolayers of HFF infected with

tachyzoites (RH) or mutant Me49 (R5). Gabaculine was added 1 hour later Parasite

growth was measured by the ability to incorporate ³H-uracil during the last 18 hours of

culture. In addition, parasite growth was evaluated microscopically in Giemsa stained slides.

Toxoplasma organisms were grown in human foreskin fibroblasts alone and in

the presence of different concentrations of gabaculine (3-amino-2,3-dihydrobenzoic acid) Growth was measured by the ability of T. gondii to incorporate tritiated uracil This compound was effective at inhibiting the growth of T. gondii at the 20mM

concentration FIG. IB demonstrates the ability of gabaculine (a specific inhibitor of

GSA aminotransferase) to restrict the growth of T. gondii in an in vitro assay over a 4 day period. T. gondii growth is measured by ability of the parasites to incorporate

tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis The X-axis

describes how the T. gondii cultures were treated. Cultures that were grown in

medium (medium) produced a CPM of around 45,000 If no T. gondii were added to

the cultures (no RH), a CPM of around 2,000 was observed Pyrimethamine (0 1

μm/ml) and sulphadiazine (12 5 μg/ml) added to cultures resulted in a marked reduction in CPM compared with untreated cultures At a dose of 5 mM gabaculine

restricted around 50% of CPM and at a dose of 20 mM it almost completely inhibited parasite growth, with counts of about 5,000 CPM

FIG. IC demonstrates the ability of gabaculine to inhibit the growth of T. gondii over 8 days in culture T. gondii growth is measured by ability of the

parasites to incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis The X-axis represents days post infection Parasite growth was evident in the cultures where no drug was added (medium) over the entire time course Parasite

growth was restricted in cultures with 20 mM gabaculine (gabaculine) over the 8 day

time course Similarly, parasite growth was restricted in cultures with pyrimethamine and sulphadiazine (P/S) over the 8 day time course Similar concentrations showed no toxicity to the foreskin fibroblasts indicating the specificity of this compound for

T. gondii Parallel cultures, fixed and stained with Giemsa and examined by

microscopy, clearly demonstrated that T. gondii growth was substantially inhibited in the presence of 3-amino-2,3-dihydrobenzoic acid The results in FIGS. IB and IC indicate that T. gondii utilizes the 5-carbon ALA synthesis pathway

FIG. 7 demonstrates the ability of gabaculine to inhibit the growth of the

mutant R5 strain of T. gondii over 8 days in culture This mutant strain is atovaquone resistant and possesses certain characteristics of the tachyzoite stage and certain

characteristics of the bradyzoite stage T. gondii growth is measured by their ability to

incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis

The X-axis represents days post infection Parasite growth was evident in the cultures where no drug was added (medium) over the entire time course Parasite growth was restricted in cultures with 20mM gabaculine (gabaculine) over the first 6 days of culture, after which a marked increase in parasite growth was detected. Furthermore

groups of proliferating organisms which resembled tissue cysts were observed in

similarly treated cultures. Parasite growth was restricted in cultures with pyrimethamine and sulphadiazine (P/S) over the entire 8 day time course. Residual R5 organisms in treated cultures at 8 days begin to incorporate uracil again and some of

them appeared cyst-like. Therefore, T. gondii cyst-like structures are selected by

gabaculine treatment of cultures. Specific immunostaining of such cultures treated with gabaculine for tachyzoite and bradyzoite specific antigens demonstrates that gabaculine selects bradyzoites. Table 2 is a schematic representation of experiments designed to test the hypothesis that tachyzoites utilize both conventional oxidase and alternative oxidases, but bradyzoites only use alternative oxidases, therefore interfering with generation of iron sulfated proteins by gabaculine treatment will select bradyzoites. The design and predicted results of stage specific immunostaining (Kasper et al, 1983)

if the hypothesis were correct are shown in Table 2 and confirm the hypothesis These

results suggest that T gondii has stage-specific utilization of alternative oxidases which

are utilized when cell cultures are treated with gabaculine because it depletes heme and

thus depletes iron sulfated proteins used in conventional respiration.

In summary, 3-amino-2,3-dihydrobenzoic acid (gabaculine) is an inhibitor of the 5 carbon heme synthesis pathway present in Apicomplexan parasites. Heme synthesis occurs by a different pathway in mammalian cells and is therefore unaffected by 3-

amino-2,3-dihydrobenzoic acid. Table 2. Gabaculine treatment of cultures selects bradyzoites.

Antibody Treatment Tachy- BradyIFA result on culture day used for of zoite zoite IFA culture Control Control 0 2 6

α BSAG Media (expressed on bradyzoites one day after stage Gabaculine switch)

α BAG5 Media (expressed on bradyzoites by five day Gabaculine after stage switch in culture)

IFA is immunofluorescent assay. SAG1 is surface antigen 1. BSAG is bradyzoite surface antigen 1. BAGS is bradyzoite antigen 5. A. Hypothesis. B. Design and predicted results of stage specific immunostaining if hypothesis were to be correct. < indicates no specific fluorescence of the organism; ^ indicates specific surface fluorescence of the organism due to presence of the antigen recognized by the antibody (e.g., αSAGI or αBSAG); ^^ indicates specific internal fluorescence in the organism due to presence of the antigen within the parasite recognized by the antibody (e.g., αBAGδ).

ii. An inhibitor of the glyoxylate cycle restricts the growth of

T. gondii in vitro.

3-Nitropropionic acid is an inhibitor of isocitrate lyase in the degradation of

lipid to C4 and inhibits replication of T. gondii in vitro FIG. 2A illustrates how the

glyoxylate cycle manufactures C4 acids Acetyl CoA, a byproduct of lipid breakdown combines with oxaloacetate to form citrate By the sequential action of a series of enzymes including isocitrate lyase, succinate is formed Glyoxalate, the byproduct of

this reaction is combined with a further molecule of acetyl CoA by the action of malate synthase Malate is then converted to oxaloacetate, thus completing the cycle 3-NPA

and itaconic acid are inhibitors of this pathway FIG. 2B demonstrates the ability of

3-NPA (an inhibitor of isocitrate lyase) to restrict the growth of T. gondii in an in vitro

assay over a 4 day period This result indicates it is likely that T . gondu degrades lipids using isocitrate lyase 7^". gondii growth is measured by their ability to incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis The X-axis

described how the T gondu cultures were treated Cultures that were grown in medium (medium) produced a CPM of about 30,000 If no T. gondu were added to

the cultures (no RH), a CPM of about 2,000 was observed Pyrimethamine (0 1 μg/ml)

and sulphadiazine (12 5 μg/ml) added to cultures resulted in a marked reduction in

CPM compared with untreated cultures A dose of 0 006 mg/ml 3-NPA (3-NPA)

restricted around 60% of CPM 3-NPA inhibits the glyoxylate cycle (isocitrate lyase) and/or succinate dehydrogenase in Apicomplexan parasites iii. and iv. Effect of SHAM and 8-hydroxyquinoline on alternative

oxidase in T. gondii

There is a metabolic pathway found in most plants and algae and in

Apicomplexans, but absent in most multicellular animals. FIG. 3A describes the electron transport respiratory chain that normally occurs on the inner membrane of

mitochondria In animals, NADH and succinate produced by the action of the citric acid cycle diffuse to the electron transport chain. By a series of oxidation reactions mediated in part through the cytochromes, free energy is released This free energy

yields the potential for the phosphorylation of ADP to ATP. In plants, in addition to

the conventional electron transport chain complexes, there is an alternative pathway of respiration Alternative pathway respiration branches from the conventional pathway at ubiquinone and donates released electrons directly to water in a single four electron

step An important feature of this pathway is that it does not contribute to

transmembrane potential and thus free energy available for the phosphorylation of ADP to ATP The pathway provides a source of energy and is preferred for conditions with relatively low ATP demands A key enzyme in this pathway is an alternative oxidase

that is cyanide insensitive and does not require heme Toxoplasma gondii utilizes the alternative oxidase for respiration

FIG. 3B demonstrates the ability of SHAM (a specific inhibitor of alternative oxidase) to restrict the growth of T. gondii in an in vitro assay over a 4 day period

The ability of these compounds to inhibit the growth of T. gondii was examined by the assay described in Example 1. T. gondii growth is measured by their ability to

incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis The X-axis describes how the T. gondii cultures were treated Cultures that were grown in medium (medium) produced a CPM of around 54,000 If no 7. gondii were

added to the cultures (no RH), a CPM of around 1,000 was observed Pyrimethamine

(0 1 μg/ml) and sulphadiazine (12 5 μg/ml) added to cultures resulted in a marked

reduction in CPM compared with untreated cultures A dose of 0 19 μg/ml SHAM

(0 19) restricted around 50% of CPM and at a dose of 0 78 μg/ml it essentially

inhibited parasite growth, with counts of about 8,000 CPM

Salicylhydroxamic acid (SHAM) and 8-hydroxyquinoline are inhibitors of the alternative oxidase and are also effective against T. gondu, presumably by inhibiting the alternative pathway of respiration Salicylhydroxamic acid and 8-hydroxyquinoline inhibit the alternative oxidase of T. gondii tachyzoites Since alternative oxidative respiration does not occur in mammals, this makes antimicrobial compounds targeting this pathway therapeutic candidates

v Effect of NPMG The shikimate pathway is common to plants, fungi and certain other

microorganisms and Apicomplexan parasites, but it is not present in mammalian cells

FIG. 4A details the events that result in the production of tetrahydrofolate, aromatic

amino acids and ubiquinone in plants, algae, bacteria and fungi In this pathway,

chorismate is formed through the sequential action of a number of enzymes including EPSP-synthase and chorismate synthase EPSP-synthase is inhibited by NPMG

Chorismate is further processed to yield tetrahydrofolate or ubiquinone by a further

series of enzymatic reactions This pathway has not been described in mammals which

are dependent on diet for folate and therefore for tetrahydrofolate production This pathway is required for the synthesis of certain aromatic amino acids and aromatic

precursors of folic acid and ubiquinone. It is likely that Toxoplasma gondii utilizes the shikimate pathway for synthesis of folic acid, ubiquinone and aromatic amino acids. N-(phosphonomethyl) glycine, an inhibitor of 3-phospho-5-

enolpyruvylshikimate (EPSP) synthase and thus an inhibitor of shikimate to chorismate

conversion, affects the pathway (Table 1). The ability of this compound to inhibit the growth of T. gondii was examined by the assay described in Example 1. FIG. 4B demonstrates the ability ofNPMG (a specific inhibitor of EPSP-synthase) to restrict the growth of T. gondii in an in vitro assay over a 4 day period. T. gondii growth is

measured by their ability to incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis. The X-axis describes how the T. gondii cultures

were treated. Cultures that were grown in medium (medium) produced a CPM of

around 72,000. If no T. gondii were added to the cultures (no RH), a CPM of around

2,000 was observed. Pyrimethamine (0.1 μg/ml) and sulphadiazine (12.5 μg/ml) added

to cultures resulted in a marked reduction in CPM compared with untreated cultures. At a dose of 3.12 mM NMPG (3.12) restricted around 60% of CPM and at a dose of 4.5 mM it inhibited parasite growth by around 80%, with counts of about 12,000 CPM.

In FIG. 4C the ordinate shows uptake of tritiated uracil into T. gondii nucleic

acids; inhibitory effects ofNPMG on nucleic acid synthesis is shown; where PABA at

increasing concentrations is added to such cultures, PABA abrogates the inhibitory

effects ofNPMG on EPSP synthase restoring nucleic acid synthesis. vi. Branched Chain Amino Acid Synthesis

Imidazolinones and sulfonylureas inhibit acetohydroxy acid synthase in

Apicomplexan parasites.

vii. Starch (a ylopectin) Synthesis and Degradation UDP glucose starch glycosyl transferase is inhibited by substrate competition in

Apicomplexan parasites

viii. Transit Sequences

Antisense, ribozymes, catalytic antibodies, (Pace et al. , 1992; Cate et al., 1996, Charbonnier 1997; Askari et al., 1996) conjugation with toxic compounds allow targeting of parasite molecules using transit sequences

Identification of transit sequences in Apicomplexans provides many means for disruption of metabolic pathways. Antisense or ribozymes prevent the production of

the transit peptide and associated protein. Alternatively production of transit peptide

sequences, and the conjugation to toxic molecules, allow disruption of organellar

function. Catalytic antibodies also are designed to destroy the transit sequence These antisense compounds or ribozymes or toxic molecules targeted to transit sequences with intracellular antibodies are used as medicines to inhibit the parasite.

Example 4: Plant-like Pathways and Enzymes in Apicomplexan Parasites

Plasmodium falciparum and Cryptosporidia parvum Based on the effects of inhibitors of plant-like pathways, abrogation of inhibitor

effects, and detection of specific enzymes and/or genes, Apicomplexans, in general, have plant-like pathways. Results shown in this example broaden the observations of the presence of plant-like pathways in Apicomplexans beyond the representative parasite T. gondii.

i. Heme Synthesis

Gabaculine inhibited the heme synthesis pathway (GSAT) in Apicomplexan parasites (FIGS. IB and IC, T. gondii, FIG. 6, Cryptosporidia) but with modest or no affect on P. falciparum (Table 3, Malaria).

FIG. 6 demonstrates the effect ofNPMG, gabaculine, SHAM and 8-hydroxyquinoline and 3-NPA on Cryptosporidia in vitro. C. parvum oocysts at

50,000/well were incubated at 37° C (8% CO₂) on confluent MDBKF5D2 cell

monolayers in 96 well microtiter plates with the following concentrations of each drug.

The concentrations used were: SHAM (0.2% ETOH was added) 100, 10, 1, 0.1 μg/ml; 8-hydroxyquinoline 100, 10, 1, 0.1 μg/ml; NPMG 4.5, 0.45, 0.045 μg/ml;

gabaculine 20, 2, 0.2 μg/ml. The level of infection of each well was determined and

analyzed by an immunofluorescence assay at 48 hours using an antibody to C. parvum

sporozoites made in rabbits at a concentration of 0.1%. Fluorescein-conjugated goat anti-rabbit antibody was used at a concentration of 1%. 95% Cl count was the mean parasite count per field when 16 fields counted at lOx magnification ± s.d. of the mean. The approximate 95% Cl counts were as follows: media and ethanol ~ 1200;

paromomycin (PRM) and ethanol ~ 100; SHAM 100 μg/ml ~ 400; SHAM

10 μg/ml ~ 1100; SHAM 1 μg/ml ~ 1100; SHAM 0.1 μg/ml ~ 1200; media alone ~ 800 μg/ml; PRM -200; 8-OH-quinoline 100 μg/ml; -300; 8-OH-quinoline 10 μg/ml; - 900; 8-OH-quinoline 1 μg/ml ~1100; 8-OH-quinoline 0.1 μg/ml - 1300; NPMG 4.5 μg/ml - 900; NPMG 0.45 μg/ml - 1200; NPMG 0.045 - 1200; gabaculine

20 μg/ml - 200; gabaculine 2 μg/ml -600; and gabaculine 0.2 μg/ml - 1300. Thus

each of these compounds are promising lead compounds as antimicrobial agents effective against Cryptosporidia.

ii. Glyoxylate cycle

3-NPA inhibited the glyoxylate cycle (isocitrate lyase) and/or succinate dehydrogenase in Apicomplexan parasites (FIG. 2B, T. gondii) and also inhibited P. falciparum and C. parvum.

To determine whether there is an Apicomplexan glyoxylate cycle, to analyze the sensitivity of T. gondii tachyzoites and bradyzoites to glyoxylate cycle inhibitors and to determine whether Apicomplexan parasites have isocitrate lyase which presents a

unique pathway for lipid metabolism that can be targeted with inhibitors, the following methods are suitable.

The inhibitor of isocitrate lyase is 3-nitropropionic acid (concentration ranging from 0.005 to 5mg/ml in vitro, and 5 to 50 mg/kg/day in vivo). Mutants [Yale Stock

Center] used for complementation are as follows: E. coli strains; DV 21A01 (aceA

which lacks isocitrate lyase) and DV21 A05 (aceB which lacks malate synthase). Plant gene sequences suitable for comparison are those described by Kahn et al. (1977),

Maloy et al. (1980); and Maloy et al. (1982). A biochemical assay for isocitrate lyase

activity is the method of Kahn et al. (1977). The polyclonal antibodies to cotton malate

synthase and cotton isocitrate lyase which hybridize to T. gondii proteins of approximately 60 kd are used to identify these enzymes in other Apicomplexan

parasites.

in. Alternative Oxidase

SHAM and 8-hydroxyquinoline inhibited the alternative pathway of respiration, i.e., the alternative oxidase in Apicomplexan parasites [FIG. 3, T gondii; FIG. 6,

Cryptosporidia parvum, Table 3, Plasmodium falciparum (clones W2, D6),

pyrimethamine resistant or sensitive clones Because Cryptosporidia appear to lack

mitochondria, the plastid is a likely site for the alternative pathway of respiration

Effect of SHAM on wild type malaria in vitro had been described earlier (Fry and Beesley, 1991) However, this observation was presented without knowledge that SHAM affected alternative oxidase function

IV. Shikimate/Chorismate

NPMG inhibited the shikimate pathway in Apicomplexan parasites (FIG. 4B,

T. gondii, Table 4, Mαlαriα; FIG. 6, Cryptosporidia) Presence of a product of the enzymatic reaction in the pathways of the present invention abrogates the effect of the inhibitor on a specific enzyme because the product

no longer has to be made by enzyme catalysis of a substrate. Thus, addition of the

product proves the specificity of the effect of the inhibitor on the enzyme. The addition of PABA abrogates the exogenous effect ofNPMG which is an inhibitor of EPSP

synthase (FIG. 4B, T. gondii). Because PABA ablates the effect of the inhibitor

NPMG on EPSP synthase, the presence of the shikimate pathway in Apicomplexan parasites is demonstrated.

Other specific methods to determine whether Apicomplexan parasites have a metabolically active EPSP synthase enzyme involved in conversion of shikimate to

chorismate and further characterize this metabolic pathway in T. gondii are as follows: Use of the inhibitor N-(phosphonomethyl) glycine (concentrations of 3.125mM

in vitro and 100 mg/kg/day in vivo). The product rescue assays are performed with PABA. The mutants for complementation are as follows: E. coli, AroA; E. coli, AroC,

and yeast, AR. [Yale Stock Center] Plant gene sequences for comparison are outlined by Klee et al. (1987). A biochemical assay for EPSP synthase activity in cellular lysates is as described by Mousdale and Coggins (1985). Other enzymes in this pathway also

are characterized (Nichols and Green, 1992). The full length nucleotide sequence of

chorismate synthase was obtained following restriction digestion and primer-based sequencing of the Tg EST zyllc05.rl clone obtained from the "Toxoplasma EST Project at Washington University" and of P. falciparum EST czap PFD d2.1 clone

obtained from the "malaria EST project," D Chakrabarti, Florida. The Toxoplasma

gondii sequence has substantial homology with tomato and several other chorismate synthases and a region of the 71 gondii protein has 30% identity and 45% homology with the transit sequence of Zea mays (sweet com). Other inhibitors of EPSP synthase

are Inhibitors 4 and 5, sulfosate (Marzabadi et al., 1996). Other inhibitors of enzymes in this pathway also have been developed by others and provide a paradigm for the

rational synthesis of competitive substrate inhibitors of Apicomplexan parasites. v. Branched Chain Amino Acid and Other Essential

Amino Acid Synthesis

Acetohydroxy acid synthase is an enzyme present in plants but not animals and

is inhibited by imindazolinones and sulfonylureas in Apicomplexan parasites. Inhibitors of histidine synthesis restrict growth of Apicomplexan parasites. vi. Starch (Amylose/Amylopectin) Synthesis and Degradation

UDP glucose starch glycosyl transferase, starch synthetase and Q (branching)

enzymes are inhibited by substrate competitors in Apicomplexan parasites. vii. Lipid Synthesis

The plant-like acetyl coA decarboxylase is inhibited by a number of inhibitors

shown in Table IB. Linoleic acid and linoleneic acid synthases are inhibited by newly

designed competitive substrates. viii. Auxins and Giberellins The known auxin mimics and Giberellin synthesis and Giberellin inhibitors

inhibit Apicomplexan parasites' growth. ix. Glutamine/Glutamate Synthesis

Glufosinate inhibits Apicomplexan glutamine/glutamate synthesis because the critical enzyme is plant-like. x. Transit Sequence

The transit sequence is conjugated with toxic molecules such as ricins and used

to disrupt plastid function in Apicomplexans. Other strategies, such as antisense, ribozymes or the use of catalytic antibodies prevent translation of DNA to protein or catalyze the destruction of mature protein. This interferes with functioning of the

molecule and thus the parasite's growth and survival.

Example 5: The Combined Effects of lnhibitors of Apicomplexan Parasites The effect of enzymes in pathways "in parallel" are additive and in "series" are more

than the additive effect of either inhibitor used alone (i.e., synergistic). FIG. 5

demonstrates the inter-relationship of the shikimate pathway and heme synthesis with the electron transport chain. The shikimate pathway produces 3,4-dihydroxybenzoate which is converted to ubiquinone, an essential component of the electron transport

chain. Thus, NPMG, an inhibitor of EPSP-synthase, indirectly affects ubiquinone production and, thus, the electron transport chain. Similarly, heme is required for the production of cytochromes in the electron transport chain. Thus, inhibition of heme

production by gabaculine also indirectly affects the conventional electron transport chain. This scheme allows synergistic combinations of drugs. Thus, NPMG and

sulphadiazine (a competitive PABA analogue) which act at different points of the folate synthesis pathway are predicted to be synergistic, whereas the effects of gabaculine and sulphadizine (a competitive PABA analogue) which act on different pathways, are predicted to be additive Similarly, gabaculine and SHAM are a predicted synergistic combination of inhibitors Table 4 demonstrates the additive inhibitory effect of

sulphadiazine and gabaculine on the growth of T. gondii over 4 days in culture T. gondii growth is measured by their ability to incorporate tritiated uracil and is expressed as counts/minute (CPM) Cultures that were grown in medium (medium)

produced a CPM of about 36,000 If no T gondii were added to the cultures (no RH),

a CPM of about 2,000 was observed Pyrimethamine (0 1 μg/ml) and sulphadiazine

(12 5 μg/ml) added to cultures resulted in a marked reduction in CPM compared with

untreated cultures The growth of T. gondii was inhibited by about 60% in cultures

treated with 5 mM gabaculine (gabaculine) The growth of T. gondii in cultures treated

with sulphadiazine (1 56 μg/ml) was reduced by approximately 60% When this dose

of sulphadiazine was used in combination with 5 mM gabaculine, as expected, the

combined effect of gabaculine plus sulfadiazine is additive because the pathways are in

parallel In contrast, NPMG and sulfadiazine combine in a synergistic manner

Because heme is needed for conventional mitochondria! respiration, it is expected that if both the heme synthesis and alternative oxidase pathways are present, then 3-amino- 2,3-dihydrobenzoic acid and SHAM will demonstrate synergy Similarly, ubiquinone or

end products of the shikimate pathway are needed for mitochondrial respiration and

NPMG plus SHAM therefore demonstrate synergy Table 4 also shows that, the

effects of gabaculine and SHAM are not synergistic as would be predicted by this

simple model The likely reason for this is that ALA synthase is present in T. gondii

and provides a default pathway for the synthesis of δ-aminolevulinic acid Thus, the

effects of gabaculine plus SHAM are not synergistic Cycloguanil which affects the plant-like DHFR-TS of T. gondii (McAuley et al, 1994) also is synergistic with NPMG and other inhibitors of enzymes in the shikimate pathway which provides an improved, novel method to treat this infection. Use of synergistic combinations provide an

improved strategy for the development of new medicines for the treatment of disease

and eradication of the parasite.

Example 6: Effects of Inhibitors In Vivo

Candidate inhibitors are administered to animals by daily intraperitoneal

injection or by addition to the drinking water To inhibit EPSP synthase, in vivo,

NPMG is administered at a dose of 1 OOmg/kg/day

a) Survival Five hundred tachyzoites of the RH strain are administered intraperitoneally to BALB/c mice Cumulative mortality is followed in groups of mice given inhibitor compared to untreated controls

b) Formation of Cysts. C3H/HeJ mice that have been infected perorally with the Me49 strain of T. gondii for 30 days are treated with the inhibitor for 30 days

Cyst burden and pathology in the brains of inhibitor-treated and control mice are compared using methods described previously (Roberts, Cruickshank and Alexander,

1995, Brown et al., 1995, McLeod, Cohen, Estes, 1984, McLeod et al, 1988) Cyst numbers present in a suspension of brain are enumerated, or cyst numbers in formalin fixed paraffin embedded sections are quantitated

c) Persistence of Cvsts C3H/HeJ mice are infected orally with 100 cysts

of T. gondii (Me49 strain) Inhibitors are administered to groups of mice from day 30 post infection to day 50 post infection Cyst burden, mortality and pathology are compared in treated and control mice on days 30 and 50 post infection and in mice that

receive antibody to gamma interferon which leads to recrudescence of disease

d) Synergy If marked synergistic effect is demonstrated in vitro by showing that the subinhibitory concentrations used together exert an effect greater than the additive effects of each used separately, for any combinations, their effect alone and

together in vivo is compared e) New Assays Which Determine the Effects of Antimicrobial Agents on T. gondii In Vivo

Previously reported assay systems measure protection against death following

intraperitoneal infection if an animal is infected with the virulent RH strain of T. gondii Novel aspects of the assay systems in the present invention are using the Me49 (AIDS

repository) strain of T. gondii to determine the effect on brain cyst number following acute peroral infection by an Apicomplexan parasite, the effect on the established number of brain cysts during subacute/chronic infection, and use of the Me49 and RH strains to demonstrate synergy of inhibitors of plant-like pathways of the present invention which are "in series," and a novel system to demonstrate reduction of parasitemia which is quantitated using a competitive PCR technique In this

competitive PCR method the T. gondii B 1 gene is amplified by PCR in the presence of a construct which produces a product slightly smaller than the wild type B 1 gene The

amount of construct can be quantitated to semiquantitate the amount of the competing

wild type gene For example, presence of a greater amount of the wild type gene will

result in lesser use of the competitor f) Effect of Antimicrobial Agents on Apicomplexan Parasites In Vivo

A demonstration of the effect of inhibitors of plant-like metabolic pathways

m vivo is the synergistic effect ofNPMG and low dosage pyrimethamine NPMG is an inhibitor of infection and promotes survival of mice infected with the virulent RH strain of T. gondii when utilized in conjunction with a low dose of pyrimethamine, whereas neither low dosage pyrimethamine nor NPMG alone are protective Sulfadiazine reduced manifestations of infection in vivo SHAM affects parasitemia and number of

brain cysts

FIG. 8 demonstrates the ability ofNPMG and pyrimethamine administered in combination to protect mice from an otherwise lethal challenge with the virulent RH strain of T. gondii Mice were infected intraperitoneally with 500 tachyzoites and left

untreated (control) or treated by the addition of pyrimethamine (PYR), NPMG

(NPMG) or both pyrimethamine and NPMG (PYR NPMG) to their drinking water Percent survival is marked on the Y-axis and days post infection on the X-axis

Untreated mice and those treated with either pyrimethamine or NPMG died between

day 7 and 9 post infection In contrast 66 percent of mice treated with pyrimethamine and NPMG survived until day 9 post infection and 33 percent survived until the conclusion of the treatment (day 30 post infection) After the withdrawal of treatment, all of these mice survived until the conclusion of the experiment (day 60 post infection)

Example 7: Presence of an Enzyme in a Specific Life Cycle Stage Predicts

Efficacy of lnhibitors of the Enzyme on this Stage of the Parasite The effect of candidate inhibitors on different life cycle stages and their effect

on stage conversion is of considerable interest and clinical importance The bradyzoite

form of T. gondu was studied by electron microscopy and was found to have a plastid

Intraparasite immunolocalization of the enzymes is also performed Gabaculine treated

cultures are stained with antibodies to tachyzoites and bradyzoites Tachyzoites of the RH strain are grown in the peritoneum of ND4 mice for 3 days Tachyzoites are

harvested in saline (0 9%) from the peritoneal cavity of euthanized mice and purified by filtration through a 3 μm filter. Bradyzoites are isolated as described herein in the Material and Methods The tachyzoites are pelleted by centrifugation and the pellet is fixed in 2 5% glutaraldehyde Cysts and bradyzoites are purified from the brains of

C57BL10/ScSn mice as described herein in the Materials and Methods and then fixed in 2.5% glutaraldehyde

lmmunoelectronmicroscopy is as described by Sibley and Krahenbuhl (1988) using gold particles of different sizes with antibodies to the enzymes to identify the

enzyme localization in different organelles which are identified morphologically lmmunoelectronmicroscopy localization is accomplished with Amersham Immunogold

kit and cryosectioning using standard techniques in the electronmicroscopy facility at

the University of Chicago or at Oxford University, Oxford, England Extracellular

organisms are studied as well as tachyzoites and bradyzoites at intervals after invasion Morphology of the parasites, their ultrastructure and the localization of the intracellular

gold particles conjugated to the antibodies is characterized Invasion is synchronized

by placing tachyzoites and bradyzoites with P815 cells at 4°C, then placing cultures at

37°C Intervals to be studied are before 1, 5, and 10 minutes and 4 hours after

invasion

Immunostaining and immunoelectronmicroscopy using an antibody to soybean,

or synechococcus, or barley GSAT indicate whether the enzyme is present or absent in both the tachyzoite and bradyzoite life cycle stages and localizes the enzyme in the

parasite. a) Immunostaining for tachyzoites and bradyzoites

Immunostaining of tachyzoites and bradyzoites is evaluated with fluorescent

microscopy. This is performed on cultures of fibroblasts in Labtech slides infected with

tachyzoites (RH strain) or bradyzoites and permeabilized using triton, or saponin or methanol, as described by Weiss et al., 1992; Dubremete and Soete, 1996; Bohne et al. (1996). Slides are stained 1, 2, 4, 6, and 8 days post infection with anti-BAG (Weiss

et al., 1992) and anti-SAGl (Mineo et al., 1993; McLeod et al., 1991; Roberts and

McLeod, 1996). b) Antibodies

Antibodies to the bradyzoite antigens (Weiss et al., 1992; and Bohne et al.,

1993) and monoclonal and polyclonal antibodies to SAG1 (Kasper et al. 1983) as a

marker for tachyzoite stage specific antigens are used for immunostaining of parasites to establish stage of the parasite. Transgenic parasites with bradyzoite genes with reporter genes are also useful for such studies.

c) Inhibitors and Stage Switching

The effect of inhibitors of conventional (KCN, Rotenone, Antimycin A or

Myxothiazol) respiration and alternative respiration on inhibition of growth of

tachyzoites and bradyzoites are compared using standard inhibition experiments in

conjunction with immunostaining techniques. Tachyzoites use conventional and alternative pathways of respiration whereas the bradyzoite stage relies on alternative respiration Inhibitors of conventional respiration favor tachyzoite to bradyzoite

switching whereas inhibitors of alternative respiration inhibit tachyzoite and bradyzoite

stages d) Synergy studies, gabaculine treatment Synergy studies with gabaculine are of particular interest because heme is used in the conventional oxidase pathway If there is synergy, iron influences stage switch¬

ing For alternative oxidase, immunostaining for bradyzoites and tachyzoite antigens is performed using gabaculine treated and control cultures This is especially informative concerning whether bradyzoites utilize alternative oxidases exclusively, because gabaculine treatment of cultures would limit use of conventional oxidases and thereby

select bradyzoites. e) Western Blot Analysis, and ELISAs to determine stage specific expression of enzymes

Bradyzoites and tachyzoites also are compared directly for the relative amounts

of alternative oxidase, using northern blot analyses, enzyme assays of parasites, isolation of mRNA and RT-PCR, using a competitor construct as an internal standard,

and by Western blotting and ELISAs using antibodies to the enzymes (e.g., alternative

oxidase) UDP-glucose-starch glycosyl transferase, chorismate synthase, isocitrate lyase, GSAT also are studied in a similar manner

Example 8: Probing Apicomplexan DNA with Homologous Plant-like Genes or

Potentially Homologous Genes From Other Parasites The presence of the gsa genes, alternative oxidase genes, EPSP synthase genes,

chorismate synthase genes, isocitrate lyase genes, and malate synthase genes are identified by probing, and then sequenced. For example, the cDNA clone of soybean gsa is labeled for chemiluminescent detection (ECL) or ³²P detection to identify

homologous gsa sequences in T. gondii. Probes are used on a membrane containing the genomic DNA of T. gondii and soybean (positive control). When T. gondii genes are isolated, they are used to probe other Apicomplexan DNA. Thus, the gsa genes of Cryptosporidia, Eimeria, and Malaria are detected in the same manner as the T. gondii

gsa. In addition, DNA probes complementary to Trypanosome alternative oxidase

DNA are used to probe the Apicomplexan DNA. The gene for T. gondii alternative oxidase is identified by screening T. gondii cDNA expression libraries using the 7D3

monoclonal antibody or the tobacco alternative oxidase gene used as a probe and thus detecting the gene expressing the relevant protein. This gene is used to detect the

alternative oxidase genes of other Apicomplexan parasites by Southern analysis and screening other Apicomplexan cDNA libraries.

A nucleotide sequence generated from random sequencing of a T. gondii

tachyzoite cDNA library and placed in the Genbank database was found to encode a protein with homology to tomato chorismate synthase. The EST was obtained, cloned

and the full length sequence of the T. gondii chorismate synthase gene and deduced

amino acid sequences were obtained (FIGS. 9 and 10). This provides evidence for

these plant-like pathways and information useful in preparing a probe to isolate and sequence this full gene from other Apicomplexan parasites as well. This gene was used as a probe and identified a chorismate synthase in Eimeria bovis DNA and

Cryptosporidium parvum DNA. A P. falciparum EST has also been cloned and sequenced. Probes for gsa (soybean) alternative oxidase (soybean and tobacco),

isocitrate lyase (cotton), UDP glucose starch glycosyl transferase (sweet com), and acetohydroxy acid synthase (sweet co ) also are used to screen for clone, and

sequence Apicomplexan genes. Large numbers of T. gondii genes from tachyzoite and bradyzoite cDNA libraries are being sequenced and deposited in Genbank. Putative

homologous genes encoding plant enzymes are used to compare with these sequences to determine whether they are identified in the libraries and if so to determine whether

the enzymes are encoded in the nucleus or plastid.

Example 9: Identification of Genes Encoding Enzymes of the Plant-Like Biochemical Pathways in Apicomplexan Genes are isolated from a cDNA library by hybridization using specific probes

to genes known to encode enzymes in metabolic pathways of plants, (see Example 9) Genes are cloned by complementation from a T. gondii cDNA expression library using

a series of E. coli mutants that lack these enzymes and thus depend on the addition of exogenous additives for their optimal growth. Transformed bacteria are used to isolate

and sequence plasmid DNA and from those sequences, probes are generated to determine whether other Apicomplexans have genes homologous to those in T. gondii.

1 ) cDNA libraries A cDNA library was constructed by Stratagene from

mR A isolated from T. gondii tachyzoites of the Me49 strain of T. gondii using the Uni-ZAP XR cDNA library system. The titer of the amplified library is 1-2 X lθ'7ml. Other cDNA libraries also are utilized.

The phagemids were excised with R408 or VCS-M13 helper phage and

transduced into XLl-Blue Cells. The plasmid DNA was purified using the Qiagen maxiprep system. Other libraries, e.g. , early Me49 bradyzoite, in vivo Me49

bradyzoite, and Me49 tachyzoite libraries also are suitable, as are other tachyzoite and bradyzoite libraries prepared by Stratagene.

2) Screening of library for genes. This is done in a standard manner using monoclonal or polyclonal antibodies or a radiolabeled gene probe. 3) cDNA expression libraries are probed with DNA from the genomes of: a) Toxoplasma gondii;

b) Plasmodium malar iae; c) Cryptosporidium parvum; d) Eimeria.

The existence of plant-like pathways is confirmed in members of the

Apicomplexa by demonstrating the existence of genes encoding the enzymes required for the pathways. Genomic DNA is examined by Southern blot analysis for the

presence of the sequences encoding enzymes required for specific algal or plant metabolic pathways. Genomic DNA is extracted from Apicomplexan parasites by

proteinase K digestion and phenol extraction. DNA(5-10μg) is digested with restriction enzymes, electrophoresed through 1% Agarose and transferred to a nylon

membrane. The ECL (Amersham) random prime system is used for labeling of DNA probes, hybridization and chemiluminescence detection Alternatively, the Boehnnger

Mannheim Random Prime DNA labeling kit is used to label the DNA with ³²P with unincorporated nucleotides removed using G-50 Sephadex Spin columns Hybridiza¬

tion with the ³²P-labeled probe is carried out in [1M NaCl, 20 mM NaH₂ PO4 pH 7 0, 1% SDS, 40% formamide, 10% dextran sulfate, 5 mg/ml dry milk, 100 μg/ml salmon

sperm DNA] at 37°C Washes are optimized for maximum signal and minimum background Probes are prepared from T. gondii cDNA clones obtained and characterized as described in Example 9 If lack of overall sequence conservation limits

ability to detect homology, highly conserved regions are useful For example, two highly conserved regions of the gsa gene are useful to generate oligonucleotide probes (Matters et al., 1995)

4) PCR An alternative approach for identifying genes encoding enzymes of the present invention is by using PCR with primers selected on the basis of homologies already demonstrated between plant protein sequences for the relevant gene For example, for the gsa gene, polymerase chain reaction technology is used to amplify homologous sequences from a T. gondii cDNA library or T. gondii genomic

DNA using primers generated from two highly conserved regions of GSAT The

Neurospora crassa alternative oxidase gene has been isolated using degenerate primers designed from conserved regions in alternative oxidase sequences from plant species

(Li et al., 1996) These primers are used to detect and clone the alternative oxidase

gene from T. gondii Candidate PCR products are cloned using the Invitrogen TA

cloning kit. 5) Sequencing DNA from candidate cDNA clones is extracted using the Promega Wizard Miniprep System Clones of interest are purified in large scale using

the Maxiprep Protocol (Qiagen) and are sequenced by modified Sanger method with an

automated sequencer (ABI Automated Sequencer) by the University of Chicago Cancer Research Center DNA Sequencing Facility

6) Homology Search to determine whether there is homology of isolated

genes with other genes, e.g. gsas, sequences are compared against those in Genbank

using the BLASTN (DNA → DNA) and BLASTX (DNA → Protein) programs

T. gondu sequence data is available in Genbank Sequences for plasmodia also are available as are some isolated sequences for the other Apicomplexan parasites

T. gondii sequences are searched for homologies to the known plant genes gsa,

glutamyl-tRNA reductase, isocitrate lyase, malate synthase, alternative oxidase, EPSP

synthase, and chorismate lyase using the BLASTN (DNA→ DNA) and TBLASTN

(Protein — > Conceptual Translation of DNA Sequence) programs The conserved plant

gene sequences for the shikimate pathway are those described by Kahn et al. ( 1977) and Maloy et al. (^"1 80, 1982) Conserved plant gene sequences for comparison of

homologies are outlined by Klee et al. (1987) Similar libraries and sequence data for

Plasmodia also are compared for homologies in the same manner

7) Complementation To isolate T. gondii genes or to demonstrate that a gene encodes a functional enzyme product, plasmids from the cDNA library detailed above, or modified constructs, are used to complement E. coli mutant strains GE1376

or GE1377 (hemL) and RP523 (hemB) from the Yale E. coli genetic stock center and SASX41B (hemA) from D. Soil. This strategy has been successful for cloning gsa genes from plants and algae (Avissar and Beale, 1990; Elliott et al, 1990; Grimm, 1990; Sangwan and O'Brian, 1993). The hemA gene encodes glutamate-tRNA

reductase, an enzyme important in the C5-pathway for heme synthesis. The hemB gene

encodes ALA dehydratase, an enzyme common to both heme biosynthesis pathways

that should be common to all organisms and is included as a positive control. Mutant bacteria are made competent to take up DNA with CaCl₂ treatment and are transformed with plasmids from the cDNA library. Briefly, chilled bacteria

(O.D. 550nm ~0.4-0.5) are centrifuged to a pellet and resuspended in ice-cold 0. IM CaCl₂ and incubated for 30 minutes on ice. Following further centrifugation, the

cells are resuspended in 0.1 M CaCl₂, 15% glycerol and frozen at -80°C in

transformation-ready aliquots. 0.2ml ice-thawed competent bacteria are incubated on

ice for 30 minutes with approximately 50ng plasmid DNA. Cells are placed at 43°C for 2.5 minutes and cooled on ice for 2 minutes. Following the addition of 0.8ml Luria Broth, cells are incubated at 37°C for 1 hour and 0.1ml is plated onto M9 minimal

media plates. The M9 (Ausubel et al, 1987) medium contains 0.2% glycerol as the

carbon source, 1 mM MgSO₄, O. lmM CaCl , 1 mM IPTG, 0.2 mg/ml Ampicillin, and

40 μg/ml threonine, leucine, and thiamine. Nonselective medium contains 25 μg/ml δ-

aminolevulinic acid (hemL and hemA) or 4 μg/ml hemin (hemB). Alternatively, bacteria can take up DNA by electroporation. Chilled bacteria are prepared by a repetition of

centrifugation and resuspension. The cells are washed in an equal volume of cold

water, a VΪ volume of cold water, a 1/50 volume of cold 10% glycerol, and finally in a 1/500 volume of cold 10% glycerol and frozen in 0.04 ml aliquots at -80°C. Cells are

thawed at room temperature and chilled on ice. Cells are mixed with the DNA for 0.5-1 minutes and then pulsed at 25μF and 2.5 KV. The cells are rapidly mixed with SOC medium and grown at 37°C for 1 hour. Cells are plated in the same way as for

CaCl transformation.

Successful complementation of the E. coli mutants with a T. gondii gene is

determined by plating the transformed bacteria onto minimal medium which lacks the supplement required for optimal growth of theE. coli mutant. Growth on the selective

medium is compared to growth on nonselective medium, which contains 25 :g/ml δ-

aminolevulinic acid (hemL or hemA) or 4 μg/ml hemin (hemB). Clones that complement each E. coli mutant are tested for their ability to complement each of the

other mutants. Clones of putative T. gondii gsa and glutamate-tR A reductase should

complement only hemL and hemA mutants, respectively. Clones that suppress more than one hem mutation are candidates for alternative oxidase gene clones. A cDN A clone containing the entire soybean gsa gene was able to transform the

E. coli hemL mutant from auxotrophic to prototrophic for δ-aminolevulinic acid

(ALA). Thus the system for obtaining T. gondii genes that complement E. coli mutants is available.

For the glyoxylate cycle the mutants used for complementation are as follows:

DV21 AOl (aceA which lacks isocitrate lyase) and DV21 A05 (aceB which lacks malate synthase). For the shikimate pathway the mutants for complementation are available and used as follows. E. cob, AroA and yeast AR

The same procedures are used for Plasmodium falciparum and Plasmodium knowlesii, Cryptosporidium and Eimeria complementation When transit sequences lead to production of a protein which does not fold in such a manner that the protein

can be expressed in E. cob or yeast constructs that lack these sequences are prepared to use for complementation that lack these sequences

Example 10: Analysis of Alternative Oxidases in T. gondii

T. gondii bradyzoites use unique alternative oxidases Alternative oxidases are necessary and sufficient for bradyzoite survival Methods to characterize plant alternative oxidases are as described (Hill, 1976, Kumar and Soil, 1992; Lambers, 1994; Li et al, 1996, Mclntosh, 1994)

For in vitro studies, cell lines that lack functional mitochondria are used These

cell lines are used to allow the study of inhibitors effective against the conventional or alternative respiratory pathways within the parasite, but independent from their effects on the host cell mitochondria SHAM, an inhibitor of the alternative respiratory

pathway is used at concentrations between 0 25 and 2 μg/ml in vitro, and 200

mg/kg/day orally or parenterally in vivo alone or in conjunction with other inhibitory compounds Other approaches include complementation of alternative oxidase-

deficient E. cob mutants to isolate and sequence the alternative oxidase gene,

immunostaining using antibodies for potentially homologous enzymes, enzymatic assay and the creation of mutant-knockouts for the alternative oxidase gene and studying stage specific antigens in such knockouts

1) Cell lines Two cell lines, a human fibroblast cell line (143B/206)

lacking mitochondrial DNA, and the parental strain (143B) which possess functional

mitochondria are used These cell lines have been demonstrated to support the growth of T. gondii (To avo and Boothroyd, 1995)

2) Inhibitor studies Inhibitor studies are carried out as described herein

SHAM concentrations are 0 25 to 2 mg/ml in vitro and 200 mg/kg/day in vivo

3) Immunostaining for tachyzoite and bradyzoites Immunostaining is performed on cultures of fibroblasts in Labtech slides infected with tachyzoites (RH strain) as described herein Slides are stained 1, 2, 4, 6 and 8 days post infection with

anti-BAG and antiSAGl

4) RT-PCR is as performed using the protocol of Hill (Chaudhuri et al ,

1996) with degenerate primers based on consensus sequences The product is cloned,

sequenced and homology with known alternative oxidases documents its presence

5) Complementation and alternative oxidase gene cloning Complementation is used to demonstrate function and is an alternative approach to isolate the gene Proper function of the complementation system is demonstrated by

using complementation with a plant alternative oxidase gene Mutants suitable for use

are hemL, hemA, hemB The alternative oxidase gene, AOX, is cloned from a T gondii cDNA expression library by complementation of the E. cob hemL mutant HemL

mutants of E. cob cannot synthesize heme and are therefore deficient in respiration This cloning strategy has been successful in isolating AOX genes from Arabidopsis

(Kumar and Soil, 1992) The procedure employed for recovering transformants is

identical to that used for cloning the T. gondii gsa gene The distinction between the gsa and AOX genes is that the AOX gene should restore function not only to hemL

mutants but also to other hem mutants of E. cob In addition, respiratory growth of E.

cob on the alternative oxidase should be antimycin-insensitive and SHAM-sensitive Clones recovered are tested for complementation of hemL, hemB and hemA mutants Growth is tested for inhibitor sensitivity Sequences of cDNA clones that provide

functional alternative oxidase activity by these tests are compared with known AOX gene sequences (Mclntosh, 1994)

The Escherichia cob strain XL 1 -Blue was prepared for infection with the 7. gondii phage library according to Stratagene manufacturer's protocol The RH

tachyzoite library, in the λ-ZAP vector system was titred, and 10⁶ pfu are added to the

XL 1 -Blue preparation Approximately 6 X 10⁵ plaques are plated on agar onto

150 mm² petri dishes containing NZY medium, and grown at 42°C for 3 5 or 8 hours,

depending upon which screening method is employed If antibodies are used for screening, IPTG-soaked nitrocellulose filters are placed on the plates after the short incubation period, and the growth of the plaques is allowed to proceed for an equivalent period of time Filters are blocked in BLOTTO overnight Screening is

carried out under the same conditions which had been optimized during Western

blotting with that primary antibody, and the appropriate secondary antibody If DNA probes are used for screening, the plaques are grown for 8 hours post-infection, and placed at 45°C for 2 hours to overnight. Nitrocellulose filters are placed on the plates,

and all subsequent steps for lysis and fixing of the DNA are as specified in the

Stratagene protocol. Filters are placed into a pre-hybridization solution containing

Denhardts, SSC, SDS, and denatured salmon sperm DNA, as directed in Ausubel et al.

(1987). Blots are hybridized to ³²P-labeled probe overnight. Low stringency washes, containing 5X SSC and 0.1% SDS are performed twice at room temperature, and high stringency washes with 0.2X SSC and 0.1% SDS are performed at a temperature

dependent upon the degree of homology between the probe and the T gondii DNA.

6) Assays for the presence of genes: Evidence for the presence of the genes which encode the novel enzymes is obtained by demonstrating enzyme activity and/or Western blot analysis of Apicomplexan whole cell lysates and/or polymerase

chain reaction and/or probing the genomic DNA of the parasite with the homologous DNA. Identification of the genes is accomplished by screening an Apicomplexan cDNA library with the antibody to homologous enzymes from plants or other

microorganisms or probes which recognize the genes which encode them and/or complementation of mutant bacteria lacking the enzyme with Apicomplexan DNA.

7) Mutant-Knockouts : The alternative mitochondrial oxidase pathway is the preferred oxidative pathway for bradyzoites and is likely to be important for their

survival. The genetic system used to examine the function of the gene via targeted

gene knock-outs and allelic replacements essentially as described (Donald & Roos,

1993, 1994, 1995). The alternative oxidase is not absolutely required for growth when

cytochrome oxidase can be active and mutants are recoverable. The AOX-null strains may be hypersensitive to GSAT inhibitors, both in vitro and in vivo. The ability of the

AOX-null strains to switch stages, both in vitro and in vivo is determined. The AOX- null strains are examined for stage specific antigens. Virulence and ability to form cysts are assessed in vivo in C3H HeJ mice as described herein.

Knockouts with a bradyzoite antigen reporter gene are produced and these

constructs and organisms with the genes knocked out are cultured under conditions that would ordinarily yield a bradyzoite phenotype. These are used to determine whether expression of the "knocked out" gene is critical for bradyzoite antigen expression and the bradyzoite phenotype.

8) Similar "knockouts" of EPSP synthase or chorismate synthase are produced.

9) Similar procedures are used for other Apicomplexan parasites. For example, a similar genetic system is available for P. falciparum.

Example 11: Production, Testing, and Use of Vaccines against Apicomplexa "Knock out" organisms (e.g., lacking GSAT, or alternative oxidase or EPSP-

synthase or chorismate synthase or UDP-glucose starch glycosyl transferase) are

produced as described herein. The knock-out vaccine strain in some cases is cultivated in tissue culture because components which are deficient are provided by a single product or a plurality of products. DNA constructs and proteins are produced and

tested as described herein (see Materials and Methods) using unique genes and

sequences and assay systems and methods which are known to those of skill in the art

and disclosed herein. Briefly, they are used to immunize C3H mice, and tissues of immunized and control mice are subsequently examined for persistence of parasites.

These immunized mice and controls are challenged perorally with 100 cysts of Me49

strain or intraperitoneally with 500 RH strain tachyzoites. Effect of immunizations on

survival, and tissue parasite burden are determined (McLeod et al, 1988). Parasite burden refers to quantitation of numbers of parasites using PCR for the Bl T. gondii

gene, quantitating numbers of cysts in brain tissue, quantitating numbers of parasites by inoculating serial dilutions of tissues into uninfected mice when the RH strain of T. gondii is utilized and assessing survival of recipient mice as 1 parasite of the RH strain of T. gondii is lethal. Ability to prevent congenital transmission and to treat

congenital infections is also a measure of vaccine efficacy. Vaccines are useful to prevent infections of livestock animals and humans. Standard methods of vaccine development are used when substantial prevention of infection is achieved in murine

models.

Example 12: Nucleotide and Deduced Amino Acid Sequence of T. eondii Chorismate Synthase cDNA

Animals and most protista (e.g. Leishmania) rely exclusively on exogenous folates. Previous studies which demonstrate the efficacy of anti-folates for the

treatment of toxoplasmosis have implied that T. gondii has the enzymes necessary to synthesize folates. For this purpose, T. gondii uses PABA. The biochemical events

that lead to PABA production in T. gondii or any other Apicomplexan have not been

previously characterized. In algae, plants, certain bacteria and fungi, the shikimate

pathway facilitates the conversion of shikimate to chorismate, a three step reaction catalyzed by three enzymes, shikimate kinase, 3-phospho-5-enolpyruvyl shikimate synthase (EPSP synthase) and chorismate synthase Chorismate is then used as a

substrate for the synthesis of PABA In plants, EPSP-synthase and chorismate synthase

are encoded in the nucleus In plants, algae and bacteria, chorismate is not only an essential substrate for the synthesis of folate, but it is required for the synthesis of ubiquinone and certain aromatic amino acids The shikimate pathway may occur both inside and outside of the plastid For example, EPSP synthase exists in two forms in

Euglena, one associated with the plastid of those grown in the light and the other found in the cytosol of those grown in the dark Apicomplexan parasites utilize the shikimate pathway for folate synthesis An

inhibitor of the EPSP synthase, an essential enzyme in this pathway, restricts the growth

of T. gondu, P. falciparum and C. parvum in vitro This inhibitor, NPMG, synergizes with pyrimethamine and sulfadiazine to prevent T gondii multiplication NPMG also synergizes with pyrimethamine to protect mice against challenge with the virulent RH

strain of 7^'. gondii The sequence of a T. gondii gene that encodes a putative

chorismate synthase, that has considerable homology with chorismate synthases from other organisms, provides information useful in developing novel antimicrobial agents

A partial cDNA sequence of approximately 250 bases was identified from the

"Toxoplasma EST Project at Washington University " This sequence, when translated, had approximately 30% homology with chorismate synthase from a number of

organisms Both strands of the corresponding clone were sequenced and found to be

2312 bases in length (FIG 9) Analysis revealed a large open reading frame of 1608 base pairs which would encode a 536 amino acid protein Homology was determined

by the use of CLUSTAL X, a computer program that provides a new window base user interface to the CLUSTAL W multiple alignment program (Thompson, 1994) The deduced amino acid sequence has considerable identity (44.5 to 51 4%) with

chorismate synthases of diverse species (FIG. 10) The putative T. gondu protein

differs from other known chorismate synthases in length Chorismate synthases from other organisms range in length from 357-432 amino acids. The larger size of the T. gondii protein is due to an internal region that has no counterpart in other known

chorismate synthases and is novel. The function of this region remains to be

determined The T. gondii chorismate synthase sequence was used in a search with the BLAST program An EST from a Plasmodium falciparum cDNA library was located

that has considerable homology with the T. gondii sequence Chorismate synthase is

also present in Mycobacterium tuberculosis

The nucleotide sequence of the cDNA which encodes a putative T. gondu chorismate synthase and the amino acid sequence deduced from it is shown in FIG. 9. The deduced amino acid sequence of putative T. gondii chorismate synthase has

substantial homologies with chorismate synthases from diverse organisms including Solanum lycospersicum (tomato), Synechocystis species, Hemophilus influenza,

Saccharomyces cerevisiae, and Neurospora crassa. (FIG. 10)

The Apicomplexan data base in Genbank was searched for homologies to the

T. gondu chorismate synthase gene. A homologous P. falciparum EST (FIG. 11) was identified. It was sequenced This provided additional evidence that at least a component of the shikimate pathway also was present in P. falciparum

Sequencing Method

Characterization of Insert and Design of Sequencing Strategy.

Clone TgESTzyl lc05 rl was obtained from the Toxoplasma project at

Washington University and supplied in the Bluescript SK vector as a phage stock

Phagemid DNA was excised by simultaneously infecting XLl-Blue cells with the phage stock and VCS-M13 helper phage Purified phagemids were used to infect XL 1 -blue cells Infected XLl-Blue cells were grown in LB media and plasmid DNA purified using Qiagen maxi-prep kits The cDNA insert was excised using EcoR I and Xho I restriction enzymes and found to be approximately 2 4KB Initial sequencing of the 5 prime end of the insert's plus strand and its translation, revealed 30% homology with

previously described chorismate synthases from other organisms However, sequencing

of the 5 prime end of the minus strand yielded a sequence that when translated had little

apparent homology with any known protein A series of restriction digestion experiments were performed to establish a restriction map of the insert Restriction

fragments were electrophoresed through a 1% agarose gel and fragments visualized by

ethidium bromide staining and ultra-violet illumination Due to the lack of available

restriction enzyme sites within the insert, sequencing with the conventional technique of using sub-cloned overlapping restriction fragments as templates would prove to be

laborious and time consuming To circumvent this potential problem and facilitate

rapid sequencing, a strategy was designed that used both conventional sub-cloned overlapping restriction fragments with standard vector annealing primers and the full

length clone with custom designed primers Thus, sequencing was first carried out by using sub-cloned restriction fragments and the information obtained used to custom design unique sequencing primers These primers allowed efficient sequencing of the

internal regions and the external 3 prime end of each strand The customized primers

were

CUSTOMIZED PRIMERS:

CS1 5' TGT CCA AGA TGT TCA GCC T 3'

CS2 5' AGG CTG ATC ATC TTG GAC A 3' CS2 5' TCG GGT CTG GTT GAT TTT 3'

CS4 5' GAG AGA GCG TCG TGT TCA T 3'

CS5 5' ATG AAC ACG ACG CTC TCT C 3'

CS6 5' CAT GTC GAG AAG TTG TTC 3'

CS7 5' GAA CAA CTT CTC GAC ATG 3' CS8 5' ACT TGT GCA TAC GGG GTA C 3'

CS9 5' GTA CCC CGT ATG CAC AAGT 3'

CS10 5'TGAATGCAACTGAACTGC3'

CS11 5' GCA GTT CAG TTG CAT TCA 3'

CS12 5' AGCCGTTGGGTGTATAATC3' CS13 5'CTACGGCACCAGCTTCAC3'

CS14 5' CGT CCT TCC TCA ACA CAG TG3' CS 15 5 ' GTG AAG CTG GTG CCG TAG 3 ' CS16 5' CGC CTC TGA TTT GGA AGT G 3'

CS17 5' TCT GCC GCA TTC CAC TAG 3'

CS18 5' GAA GCC AAG CAG TTC AGT T 3'

Sub-cloning

Sub-clones were made from restriction fragments isolated by agarose gel

electrophoresis and purified using the Qiaex gel extraction kit Qiagen, Chatsworth CA Double digestions of the plasmid with Hinc II and Pst I resulted in 4 fragments of 500, 800, 300 and 4000 base pairs The 800 bp fragment, corresponding to the base pairs 800-1600 was ligated into the bluescript KS vector The 1600-2400 base pair portion of the insert was obtained in a similar manner using Pst 1 and Xho I restriction enzymes and ligated into the bluescript KS vector Ligations were performed for 12 hours at 18

degrees centigrade on a PTC 100, programmable thermal cycler, MJ Research Inc

Watertown, Massachusetts. Plasmids containing the restriction fragments were used to transform DH5α competent cells. Plasmid DNA was purified using Qiagen maxi-prep

kits.

Primer Sequence Design

Primers were designed based on the sequencing information obtained from

restriction enzyme fragments To facilitate sequencing of a region on the same strand

and 5 prime to an already sequenced portion of insert, primers were designed from an

area approximately 200-300 nucleotides 5 prime into the last obtained sequence For sequencing of the complementary strand, primers were designed to be the complement

and reverse of the same region Primers were designed to be 18-25 nucleotides in length and have a Tm of 55-60 degrees centigrade G plus C content was 45-55

percent Primers were designed to have minimal self annealing and to have a low

propensity for primer to primer annealing Primers with the ability to form stable

secondary structures were not designed These criteria for the design of primers were based on theoretical considerations and results of other experiments which found that primers which had Tms of much less than 55 degrees centigrade failed to work or

performed poorly, producing ambiguous sequences of low quality

Sequencing and Assembly of Sequence Information

All sequencing was performed using a Perkin Elmer automated sequencer The three purified plasmids containing the entire cDNA or a restriction fragment were used

as templates for sequencing reactions with the standard Ml 3 and reverse primers The sequences obtained were used to design primers which allowed sequencing of the

internal regions of the inserts This process was repeated until both strands of the entire clone were sequenced. Chromatograms were critically edited and controlled for

quality using Sequencher software. Edited chromatograms of excellent quality were assembled with the same software package and a consensus sequence obtained The consensus sequence was analyzed for open reading frames using Macvector software package Kodak International Biotechnology, Inc , New Haven, CT Example 13: Transit Sequence of T. gondii Chorismate Synthase

Homology with other peptides was sought using the Genbank database and the

unique sequence in the T. gondu chorismate synthase (amino acids 284 to 435,

Figure 11) There was thirty percent identity and forty-five percent homology, with a number of conserved motifs, between this unique sequence of T gondii chorismate synthase and the amyloplast/chloroplast transit (translocation) sequence of the Waxy

protein (UDP-glucose starch glycosyl transferase) of Zea mays (sweet corn) The same methods whereby the Zea mays transit sequence was analyzed (Klosgen and Well, 1991), i.e , construction of the transit sequence with a reporter protein, immunolocalization of the protein, creation of the construct with deletions or mutations of the transit sequence and subcellular immunolocalization using immunoelectronmicroscopy are useful for proving that this is a transit sequence in the

T gondii chorismate synthase A useful reporter protein for a chimeric construct is β

glucoronidase of E. cob, expressed under the control of the 355 promoter of

cauliflower mosaic vims The β glucoronidase alone is expressed, in parallel The

transit peptide chimeric construct is found in the plastid The control β glucoronidase

is found in the cytoplasm Antibodies to the chorismate synthase protein are also used

to detect the presence of the product of the gene (with the transit sequence) in the

plastid and the product of a construct in which the transit sequence is not present in the cytoplasm only Further mutations and deletions are made which identify the minimal

transit sequence using the same techniques as described above for the entire peptide

Antisense, ribozyme or intracellular antibodies directed against the transit sequence nucleic acid or translated protein are useful as medicines. The amino acid or nucleic acid which encodes the transit sequence are the bases for diagnostic reagents and

vaccine development. This transit sequence is useful for the construction of ribozyme,

antisense nucleic acids, intracellular antibodies which target a key parasite protein, and creation of constructs with accompanying molecules which are lethal to the parasites (Roush, 1997; Mahal et al, 1997). This transit sequence also is useful because it

provides a general extension of the concept of transit and targeting sequences in

Apicomplexan parasites that enable targeting of other parasite organelles in addition to plastids. The transit sequence of Zea mays and T. gondii are shown in Figure 11

Example 14. Nucleotide and Deduced Amino Acid Sequences of P. falciparum

Chorismate Synthase EST. Sequencing of P. falciparum chorismate synthase EST followed the same

pattern as described above for sequencing the T. gondii chorismate synthase gene with the following exceptions: There was difficulty in obtaining sequence from the 3 ' region

of the cDNA due to an unstable polyA tail. This made it necessary to do all sequencing approaching from the 5' end using gene walking techniques and subcloning of

restriction fragments. The AT richness of P. falciparum genes increased the

complexity of design of the customized primers. The customized primers utilized were:

PFCS 1 AGC TAT TGG GTG GATC PFCS2 TCC ATGTCC TGGTCT AGG

PFCS3 ATA AAA ACA CAT TGA CTA TTC CTT C PFCS4 GGG GAT TTT TAT TTT CCA ATT CTT TG

PFCS5 TTG AAT CGT TGA ATGATA AGA C

PFCS6 TTT TAG ATC AGC AAT CAA ACC

PFCS7 AAC TTT TTA TCT CCATAC TTT G PFCS8 GAA GGA ATA GTC AAT GTG TTT TTA T

PCFS9 GTA TTT TAC CAA GAT TAC CAC CC

PFCS10 CCC CCA ACA CTA TGT CG

PFCS11 CAGTGGGCAAAA TAA AGA

PFCS12 CCAGTGGGC AAA ATAA PFCS13 GGA AGA GAA ACA GCC AC

PFCS14 TGC TGC TGG GGC GTG

The gene and deduced amino acid sequences are in Figure 12 Example 15: Southern Blotting Demonstrates Presence of Chorismate Synthase (and bv Inference all of the Shikimate Pathway)

in Apicomplexan Parasites

Southern blotting using the T. gondii chorismate synthase gene as a ³ P labeled probe demonstrated homology at moderate stringency (e g 0 2 x SSC, 0 1% SDS at

42°C) [more stringent conditions define greatest relatedness of genes] with Eimeria

bovis and Cryptosporidium parvum DNA This T. gondii cDNA also comprises a probe for screening cDNA libraries of all

other Apicomplexa to identify their chorismate synthase genes. The same principles are

applicable to all the other enzymes in Table 1.

Example 16: Gene Expression. Recombinant Protein. Production of Antibody and Solving the T. gondii and P. falciparum Crystal Structures of chorismate synthase to establish their active site and secondary

structure.

These are done using standard techniques. The gene construct is placed within a competent E. coli. Recombinant enzyme is identified by homologous antibody reactivity and purified using affinity chromatography. Protein is injected into rabbits

and antibody specific to the protein is obtained and utilized to purify larger amounts of

native protein for a crystal structure. The crystal structure provides information about enzyme active site and facilitates rational drug design (Craig and Eakin, 1997).

Example 17: Other Uses (e.g. in diagnostic reagents and vaccines) of the

Chorismate Synthase Gene as a Representative Example of Uses of

Each of the Genes and Enzymes in These Pathways That are not Present or Rarely Present in Animals. These uses include T gondii genes and proteins used as diagnostic reagents and

as a vaccine to protect against congenital infection. Recombinant protein (all or part of the enzyme) is produced and is used to elicit monoclonal antibodies in mice and

polyclonal antibodies in rabbits. These antibodies and recombinant protein (e.g., to T-gondii chorismate synthase) are used in ELISA (e.g. antibody to human IgG or IgM, or IgA or IgE attached to ELISA plate + serum to be tested + antibody conjugated to

enzyme + enzyme substrate) The recombinant proteins, pooled human sera from known uninfected individuals (5 individual sera pooled) and infected individuals (5

individuals with acute infection sera pooled, 5 individuals with chronic infection sera

pooled) are the controls This test is useful with serum or serum on filter paper

Another example of a diagnostic reagent are primers to amplify the target transit sequence or another portion of the chorismate synthase sequence unique to T. gondu

PCR with these primers is used with whole blood to detect presence of the parasite

Such assays have proven to be useful using the T. gondii ^' Blgene (Kirisits, Mui, Mack, McLeod, 1996)

Another example of a diagnostic reagent is useful in outpatient settings such as an obstetrician's office or in underdeveloped areas of the world where malaria is

prevalent FABs of monoclonal antibodies (which agglutinate human red cells when ligated) (Kemp, 1988) are conjugated to antibodies to the target sequence or selected

enzyme Antigen conjugated anti-red cell Fab also is used to detect antibody to the component. A positive test occurs when the enzyme or antibody is circulating in the

patient blood and is defined by agglutination of red cells (in peripheral blood from the patient) mixed with the conjugated antibodies. Controls are the same as those specified

for the ELISA Examples of vaccines are protein, peptides, DNA encoding peptides or proteins

These are administered alone or in conjunction with adjuvants, such as ISCOMS These vaccine preparations are tested first in mice then primates then in clinical trials Endpoints are induction of protective immune responses, protection measured as enhanced survival, reduced parasite burden, and absent or substantial reduction in

incidence of congenital infection (McLeod et al., 1988).

Example 18: T. gondii Chorismate Synthase Genomic Sequence Genomic clones are isolated from commercially available genomic libraries

(AIDS repository) using the identified cDNA clones as probes in the screening process.

The genomic library, as λ phage, is isolated onto NZY agar plates using XLl-Blue E.

coli as the host, resulting in plaques following a 37°C incubation. The cDNA sequence is radiolabeled with ³²P and hybridized to nylon membranes to which DNA from the

plaques has been covalently bound. Plasmids from candidates are excised and their

restriction enzyme-digested inserts sequenced. Experimental details are as described in

Ausubel e/ α/. (1987).

Example 19: P. falciparum Chorismate Synthase Genomic Sequence.

This will be done with a gene specific subgenomic library as described in

Example 18.

Other examples of enzymes and the genes which encode them and which are characterized as outlined above include: glutamyl-tRNA synthetase; glutamyl-tRNA

reductase; prephenate dehydrogenase aromatic acid aminotransferase (aromatic

transaminase); cyclohexadienyl dehydrogenase tryptophan synthase alpha subunit; tryptophan synthase beta subunit; indole-3 -glycerol phosphate synthase

(anthranilateisomerase), (indoleglycerol phosphate synthase); anthranilate

posphoribosyltranferase; anthranilate synthase component I; phosphoribosyl anthranilate isomerase anthranilate synthase component II, prephenate dehydratase

(phenol 2-monooxygenase) catechol 1,2-deoxygenase (phenol hydroxylase), cyclohexadienyl dehydratase, 4-hydroxybenzoate octaprenyltransferase, 3-oxtaprenyl-

4-hydroxybenzoate carboxylyase dehydroquinate synthase (5-dehydroquinate

hydrolase), chorismate synthase (5-enolpyruvylshikimate 3-phosphate phosph-lyase), dehydroquinate dehydratase, shikimate dehydrogenase, 3-deoxy-d-arabino- heptuloonate 7 phosphate synthase, chorismate mutase (7-phospho-2-dehydro-3- deoxy-arabino-heptulate aldolase), 3-deoxy-d-arabino-heptuloonate 7 phosphate

synthase, shikimate 3-phosphotransferase (shikimate kinase), UDP glucose starch glycosyl transferase, Q enzymes, acetohydroxy acid synthase, chorismate synthase malate synthase, isocitrate lyase, 3 -enolpyruvylshikimate phosphate synthase (3- phospho shikimate- 1 carboxyvinyltransferase)

Example 20: T. gondii Chorismate Synthase Activity is Demonstrated

An assay for chorismate synthase in T. gondu is performed and demonstrated

such activity

Example 21: T. gondii Dehydroquinase Activity is Demonstrated

An assay for dehydroquinase in 71 gondu was performed and demonstrated such activity

Example 22: GSAT activity is demonstrated in T. gondii tachyzoite lysates An enzymatic assay (Sangwan and O'Brian, 1993) demonstrates GSAT

activity in T. gondii lysates The buffer contains 0.1 M MOPS (3-[N-

mo holino]propanesulfonic acid), pH 6 8, 0 3M glycerol, 15 mM MgCl₂, 1 mM dithiothreitol, 20 μM pyridoxal phosphate, 1 mM PMSF (phenylmethylsulfonyl

fluoride) The MOPS, glycerol and MgCl₂ are combined and then pH'd This is

important because the glycerol alters the pH, so it must be added first This is filter

sterilized and has a long shelf life When the buffer is needed, DTT, pyridoxal

phosphate and PMSF are added immediately prior to use The protein extract stock should be -10 mg/ml if possible The principle of the assay is conversion of substrate

which produces a change in color due to the reactant

Example 23: Isocitrate lvase activity is demonstrated in T. gondii tachyzoite lysates An enzymatic assay demonstrates isocitrate lyase activity in T. gondu isolates

prepared by disruption of the parasite membranes using french press or a lysis buffer Demonstration that the lysis buffer does not alter enzyme activity is carried out by performing the assay with known substrate and enzyme in the lysis buffer and

documenting presence of enzyme activity

Example 24: Alternative oxidase activity is demonstrated in T. gondii preparations. T. gondu tachyzoites and bradyzoites are assayed for alternative oxidase

activity and such activity is found to be present in greater amounts in bradyzoites

Example 25: Novel Substrate Competitors and Transition State Analogues of

Enzymes Inhibit Apicomplexan Enzymes

Some inhibitors are competitive substrates or transition state analogues and

they are utilized in the enzyme assay, in vitro with tachyzoite and bradyzoite preparations and with native enzyme, tissues culture assays and in in vivo models as described above These provide a model paradigm for designing inhibitors of any of

the enzymes specified above Briefly, inhibitors are produced as follows Competitive substrates are produced by designing and synthesizing compounds similar to known

compounds but modified very slightly For example, inhibitors related to glyphosate are known The structures of glyphosate, sulfosate and the precursor for EPSP have similarities (please see below) Inhibitors are designed by modifying substrates in such

a manner that the modification interferes with the enzyme active site This can be

performed using molecular modeling software Similarly, halogenated substrates for other enzymes have functioned effectively as nontoxic inhibitors The principles are applicable to the design of inhibitors for any of the unique enzymes with well characterized substrates and active sites

The approaches to rational design of inhibitors include those standard in the art (Craig and Eakin, 1997, Ott et al, 1996) These methods use information about

substrate preference and three-dimensional structure of the target enzyme (e g , chorismate synthase or EPSP synthase)

In one approach, the structure of the target is modeled using the three- dimensional coordinates for amino acids in a related enzyme An example of this is

that the crystal structure of GSAT from a plant has been solved and its active site is

known

In another part of this approach, expression of high levels of recombinant

enzyme is produced using cDNA (e.g , the chorismate synthase of 71 gondii or P. falciparum) and quantities of protein adequate for structural analysis, via either NMR

or X-ray crystallography are obtained.

Drug resistant mutants are produced in vitro following mutation with nitrosoguanidine and culture with the inhibitor. The surviving organisms have acquired resistance to the inhibitor. This process is carried out either with^* the Apicomplexan

parasite or with bacteria or yeast complemented with the gene encoding the enzyme or

part of the gene (e.g., without the transit sequence). PCR amplifies the relevant cDNA and this cDNA encoding the resistant enzyme is cloned and sequenced. The sequence is compared with that of the enzyme that is not resistant. With the information about

the inhibitor target and three-dimensional structure, the point mutations which oause resistance are analyzed with computer graphic display. This information provides the mechanism for altered binding of the drug, and the inhibitory compound is then modified to produce second generation medicines designed to treat resistant pathogens

prior to their development in nature. An example of the use of toxic analogues to kill parasites used by others provides a means whereby there is production of analogues toxic to parasites. Specifically, the purine analogue prodrugs, 6 sulfanylpurinol, 6 thioguanine, 6

thioxanthine and allopurinol interact with hypoxanthine phosphoribosyltransferase which is responsible for salvage of purines used to produce AMP and GMP. Such

toxic analogues are effective against the plant-like enzymes in the pathways (see Table 1) in Apicomplexans. Transit state analogues bind with extraordinarily high efficiency to the enzyme active site and are predicted from the three-dimensional structure and kinetic information Analogues that mimic the structural properties and electrostatic surface

potentials for the transition state are designed and synthesized Empirical testing using recombinant enzyme demonstrates that these transition state analogues are good leads with high affinity for the active site of the target enzyme

Multisubstrate analogues are useful because they markedly enhance the

binding affinity to the enzyme Similarly, if enzymes in a cascade are linked in such a manner that the substrate for one reaction provides the substrate for the next reaction, multisubstrate analogues are more useful

Selective inhibitor design and lead refinement: Co-crystallization of

inhibitors with target enzymes of host and pathogen enable three-dimensional analysis of molecular constructs and atomic interactions between inhibitors and enzymes and

redesign of inhibitors (leads) to enhance their affinity for the pathogen enzyme

Iterative crystallography, lead redesign and inhibitor testing in vitro and in vivo enable

design and development of potent selective inhibitors of the target of the pathogen enzyme Recombinant methods for screening large numbers of analogues for those

that bind selectively to the enzymes of specific parasites provide justification for

inclusion of the analogues which bind best in the design of transition-state or

multisubstrate analogues

Additional examples (included to illustrate principles employed) but already patented

by others include Inhibitor of EPSP synthase have been designed based on the similarities of the inhibitor to the substrate. Based on molecular modeling algorithms

additional inhibitors are designed. Phosphoenolpyruvate = CH O l ii C-O-P O°

Co₂ ³ O^e

O O

II glyphosate = HO-C-CH₂ N-CH₂-P-OH

I \

H OH

Inhibitors that effect components of these pathways are halogenated substrates or analogues which are effective competitors.

Inhibitors of Ubiquinone: Modifications (substitutions) of benzhydroxamic acids

produce CoQ (ubiquinone) analogues such as esters of 2, 3 and 3,4 dihydroxybenzoic

acid and structurally related compounds.

Inhibitor of Isoleucine/valine biosynthetic pathway: These are noncompetitive

inhibitors as is shown by the lack of relatedness of the inhibitors (e.g., imidazolinones, sulfonylureas) to the target enzymes.

Inhibitors of GSAT

The following acids (5 amino- 1,3 cyclohendienyl carboxylic acid, 4 amino 5

hexynoic acid ( acetylenic, GABA), 4 amino 5 hexonoic acid ( vinyl GABA) 2 amino 3

butanoic acid (vinyl glycine), 2 amino 4 methoxy-trans-3 butenoic acid, 4 amino 5 fluoropentanoic acid alter catalysis dependent formation of a stable covalent adduct Example 26: Modifications of Inhibitory Compounds to Improve Oral

Absorption Tissue Distribution (especially to brain and eve).

Tissue distribution is characterized using radiolabeled inhibitor administered to mice with its disposition to tissues measured by quantitation of radiolabel in tissues Compounds are modified to improve oral absoφtion and tissue distribution by standard

methods

Example 27. Efficacy of Antimicrobial Compounds Alone. Together and In Conjoint Infections in Murine Models

Inhibitors of plant-like pathways are effective against the Apicomplexan

infection alone, together with the bacterial and/or fungal infections and also treat the

bacterial and fungal infections alone

Presence of inhibitory activity of new antimicrobial compounds is tested using Apicomplexans, bacteria and fungi in enzymatic assays, in vitro, and in vivo assays as described above and known to those of skill in the art Infections are established in murine models and the influence of an inhibitor or

combination of inhibitors on outcomes are determined as follows Infections Infections with Toxoplasma gondu, Pneumocystis carinii, Mycobacterium

tuberculosis, Mycobacterium avium intracellular and Cryptosporidium parvum are established alone and together using an immunosuppressed rodent model Endpoints

in these infections are

Survival Ability of an inhibitor to protect the infected animal is measured as prolonged survival relative to the survival of untreated animals.

Parasitemia: Is a measure using isolation of R A and RT-PCR. A competitive inhibitor is used for quantitation.

Tissue Parasite Burden: Is determined by quantitating brain and eye cyst numbers.

Inflammatory Response: This is noted in histopathologic preparations.

Representative combinations of inhibitors are NPMG and sulfadiazine, SHAM

and atovaquone, NPMG and pyrimethamine, NPMG and SHAM.

Example 28. Establishing Efficacy, Safety, Pharmakokinetics. and Therapeutic/Toxic Index: The testing in murine models includes standard Thompson tests. Testing of

antimicrobial agents for efficacy and safety in primate models for malaria is performed. Dosages are selected based on safety information available from data bases of

information concerning herbicides and the literature. Measurements of serum and

tissue levels of antimicrobial compounds are performed using assays which detect

inhibitor concentrations and concentrations of their metabolites. Representative assays are high performance liquid chromatography, and assaying tissues for percentage of

radiolabeled compounds administered, using liquid scintillation, and other assays also

are used. Example 29. Determining whether there is Carcinogenicitv and Teratogenicity:

Standard assays to evaluate carcinogenicity and teratogenicity include

administration of medicines as described above to rodents and observation of offspring for teratogenic effects and carcinogenicity (i e development of malignancies) Observation includes general physical examination, autopsy and histopathologic studies

which detect any teratogenic or carcinogenic effects of medicines

Example 30. Constructs to Measure Parasitemia:

Portions of genes are deleted and the shorter gene is used as an internal

standard in RT PCR assays to measure amount of parasites present (Kirisits, Mui, McLeod, 1996)

Example 31. Vaccine Constructs and Proteins and their Administration:

These are prepared, as described They include DNA constructs (Ulmer, Donnelly and Liu, 1996) with the appropriate gene or portions of the gene alone or

together, with adjuvants. Representative adjuvants include ISCOMS,

nonionicsurfactant, vesicles, cytokine genes in the constructs and other commonly used adjuvants Native and recombinant proteins also are used in studies of vaccines

Protection is measured using immunologic in vitro assays, and assessing enhanced

survival, reduction of parasitemia tissue and parasite burden and prevention of congenital infection [McLeod et al , 1988]

Example 32: Stage-Specific Expression of Proteins

This is evaluated by enzyme assays, northern or western analysis, ELISA,

semi-quantitation of R A using RT-PCR with a competitor as internal standard in gene-knockout organisms using culture conditions (e.g. alkaline pH, increased

temperature, nitric oxide exposure) which ordinarily elicit a bradyzoite phenotype, or engineering a reporter construct and characterizing presence of the reporter in stage specific expression of antigens. Ability to change between life cycle stages or to

persist in a particular life cycle stage is affected by presence or absence of particular plant-like genes and by treatment of inhibitors with plant-like processes. Suitable examples of plant-like enzymes which make parasites less able to switch from or persist in a specific life cycle stage include: alternative oxidase, enzymes critical for

amylopectin synthesis such as starch synthases, UDP glucose-glucosyl starch transferase and branching (Q) enzymes.

Example 33. Preparation of Diagnostic Test Reagents and Diagnostic Tests: These assays are as described (Boyer and McLeod, 1996). Sensitivity and

specificity are established as is standard in the field. Tests and reagents include ELISAs in which antibodies to the proteins or peptides and recombinant proteins of this invention such as chorismate synthase (Aroc) are used and PCR methodology in

which primers to amplify DNA which encodes the enzymes, or parts of this DNA, are

used. A test useful in an outpatient setting is based on conjugation of a monoclonal antibody to human red blood cells with antibody to plant-like peptides or proteins based on an assay described by Kemp et al. (Kemp et al, 1988). The red cells are

cross linked via the monoclonal antibody moiety, resulting in agglutination of the red blood cells in the blood sample if the antigen or antibody to the parasite component is present in the blood sample. ELISA and PCR can be utilized with samples collected on filter paper as is standard in Newborn Screening Programs and also facilitates

outpatient and field use

Example 34. Development and use of Antisense Oligonucleotides in Design and Use of Medicines to Protect Against Apicomplexans: Antisense oligonucleotides directed against the nucleic acids which encode the

enzymes of the essential parasite metabolic process described herein are effective

medicines to treat these infections Antisense oligonucleotides also are directed against transit sequences in the genes Antisense oligonucleotides are short synthetic stretches of DNA and RNA designed to block the action of the specific genes described above, for example, chorismate synthase of 71 gondii or P. falciparum, by

binding to their RNA transcript They turn off the genes by binding to stretches of their messenger RNA so that there is breakdown of the mRNA and no translation into protein When possible, antisense do not contain cytosine nucleotides Antisense reagents have been found to be active against neoplasms, inflammatory disease of the bowel (Crohn's Disease) and HIV in early trials Antisense will not contain cytosine nucleotides followed by guanines as this generates extreme immune responses (Roush,

1997) Antisense oligonucleotides with sequence for thymidine kinase also is used for

regulatable gene therapy

Example 35. Ribozymes and Other Toxic Compounds as Antimicrobial Agents: Ribozymes are RNA enzymes (Mack, McLeod, 1996) and they and toxic

compounds such as ricins (Mahal et al. 1997) are conjugated to antisense oligonucleotides, or intracellular antibodies, and these constructs destroy the enzyme

or other molecules.

Example 36. Intracellular Antibodies to Target Essential Enzymes Proteins and Organelles:

Intracellular antibodies are the Fab portions of monoclonal antibodies directed

against the enzymes of this invention or portions of them (e.g., anti-transit sequence antibodies) which can be delivered either as proteins or as DNA constructs, as described under vaccines.

Example 37. Development of New Antimicrobial Compounds Based on Lead Compounds:

The herbicide inhibitors comprise lead compounds and are modified as is standard. Examples are where side chain modifications or substitutions of groups are

made to make more active inhibitors (Table 1). Their mode of action and stmcture as well as the enzyme and substrate stmctures are useful in designing related compounds which better abrogate the function of the enzymes. Examples of such substrate or active site targeting are listed in Table 1.

Native or recombinant protein used in enzymatic assays and in vitro assays

described above are used to test activity of the designed newly synthesized compounds. Subsequently, they are tested in animals. Example 38. Trials to Demonstrate Efficacy of Novel Antimicrobial Agents for Human Disease:

Trials to demonstrate efficacy for human disease are performed when in vitro

and murine and pπmate studies indicate highly likely efficacy and safety They are standard Phase 1 (Safety), Phase II (small efficacy) and Phase III (larger efficacy with outcomes data) trials For medicines effective against T. gondii tachyzoites, resolution

of intracerebral Toxoplasma brain lesions in individuals with HIV infection with no other therapeutic options available due to major intolerance to available medicines is the initial strategy for Phase II trials Endpoints for trials of medications effective against 71 gondii bradyzoites include absence of development of toxoplasmic encephalitis in individuals with HIV HIV infected patients who also are seropositive for 71 gondii infection are evaluated Evaluation is following a one-month treatment

with the novel anti 71 gondii medicines Observation is during a subsequent 2 year

period when the patients peripheral blood CD4 counts are low Effective medicines

demonstrate efficacy measured as absence of 71 gondii encephalitis in all patients Otherwise, 50% of such individuals develop toxoplasmic encephalitis When

medications efficacious against bradyzoites and recmdescent toxoplasmic encephalitis

in patients with AIDS are discovered and found to be safe, similar trials of efficacy and

safety for individuals with recurrent toxoplasmic chorioretinitis are performed All such trials are performed with informed consent, consistent with Institutional NIH, and

Helsinki guidelines applicable to treatment trials involving humans - I l l -

Example 39. Vaccine Trials for Humans

After vaccine efficacy in rodent models to prevent congenital and latent

Toxoplasma infection are established, for component vaccines only, trials to establish safety and efficacy in prevention of congenital and latent infection are performed. They follow standard procedures for phase I, II and III trials as outlined above and as

is standard for vaccine development.

Endpoints for vaccine effect and efficacy are development of antibody and cell- mediated immunity to 71 gondii (effect) and most importantly, prevention of 71 gondii

congenital infections. After establishing in phase I trials that the vaccine is entirely

safe, nonpregnant women of childbearing age will be vaccinated with recombinant

vaccine. Assay for efficacy is via a serologic screening program to detect newborn congenital toxoplasmosis (described in Boyer and McLeod, 1996) with usual testing to document whether seropositive infants are infected (described in Boyer and McLeod,

1996). Example 40. Vaccine Efficacy and Safety for Livestock Animals

The efficacy of candidate vaccines is tested in sheep as previously described (Buxton et al, 1993). Vaccines are live attenuated, genetic constmcts or recombinant

protein. The most efficious routes and frequency of inoculation is assessed in a serious of experiments as described below. Intra-muscular, sub-cutaneous and oral are the

preferred routes, although intravenous, intraperitoneal and intradermal routes may also

be used. Scottish blackface or/and swaledale ewes, four to six years old are tested for

IgG antibodies to Toxoplasma gondii using an ELISA assay. Only sero-negative animals are used for the study Three groups of 10- 15 ewes are used for each experiment Groups 1 are vaccinated, while group 2 and 3 are not Three months later

all ewes are synchronized for estrous and mated At 90 days gestation the ewes in

groups 1 and 2 are given 2000 sporulated oocyst of 71 gondii

The outcome of pregnancy is monitored in all groups Aborted lambs or those dying soon after birth are examined histologically and by PCR for the B 1 gene or sub- inoculation into mice or tissue culture, for the presence of 71 gondii All placentas are

examined histologically and as above for parasites. Lambs are weighed at birth Pre- colostral semm is taken from each lamb Congenital transmission is assessed by performing ELISA assays on the semm for IgG or IgM Protection is measured as a decrease in congenital transmission, a decrease in the incidence or severity of

congenital disease, or a decrease in abortion

MATERIALS AND METHODS

A. Methods to Assay Candidate Inhibitors

1 Inhibitors of Toxoplasma gondii

a) Cell lines Fibroblasts Human foreskin fibroblasts (HFF) are

grown in tissue culture flasks in Iscoves' Modified Dulbecoes Medium (IMDM),

containing 10% fetal bovine semm, L-glutamine and penicillin/streptomycin at 37°C in

100%) humidity and a 5% CO₂ environment. Confluent cells are removed by trypsinization and washed in IMDM They are used in a growth phase for toxicity assays or when 100% confluent for parasite inhibition assays

b) Tachyzoites Tachyzoites of the RH and pTg strains of 71 gondu

are passaged and used for in vitro studies (McLeod et al, 1992) The R5 mixed tachyzoite/bradyzoite mutant was derived from mutagenesis with nitrosoguanidine in

the present of 5 hydroxynapthoquinone These organisms are used for in vitro

experiments at a concentration of 2 x 10³, 2 x 10⁴, or 2 x 10⁵ organisms per ml, dependent upon the planned duration of the experiment (i.e., larger inoculations for shorter duration experiments)

c) Bradyzoites: Bradyzoites are obtained as described by Denton et al (1996b) Specifically, C57BL10/ScSn mice are infected intraperitoneally

with 20 cysts of the Me49 strain of T. gondii Their brains are removed 30 days later and homogenized in PBS by repeated passage through a 21 gauge needle Aliquots

containing the equivalents of 3-4 brains are diluted in PBS and 6.5 mis of 90% percoll

added to the mixture which is allowed to settle for 30 mins. 2mls of 90% Percoll is then added as a bottom layer and the mixture centrifuged for 30 mins at 2500xg. The cysts are recovered from the bottom layer and a small portion of the layer above. After

the removal of Percoll by centrifugation, the contaminating red blood cells are removed

by lysis with water followed by the addition of 1 ml of lOxPBS per 9 ml brain

suspension in water. Bradyzoites are released from the purified cysts by digestion in a 1% pepsin solution for 5 minutes at 37°C. This method routinely permits recovery of greater than 90% of the cysts present which yields approximately 100 bradyzoites per

cyst. Bradyzoites are used at concentrations of 2x10³, 2xl0⁴ and 2xl0⁵ per ml in parasite growth inhibition assays. pH shock is also used to retain organisms in bradyzoite stage when such pH does not interfere with inhibitor activity. d) Inhibitors: Inhibitor compounds are tested over a range of

concentrations for toxicity against mammalian cells by assessing their ability to prevent cell growth as measured by tritiated thymidine uptake and inspection of the monolayer

using microscopic evaluation. A range of concentrations that are non-toxic in this

assay are tested for their ability to prevent the growth of 71 gondii and also other

Apicomplexans within these cells. i.) Heme Synthesis: The inhibitor of the heme synthesis pathway, gabaculine (Grimm, 1990; Elliot et al, 1990; Howe et al,

1995; Mets and Thiel, 1989; Sangwan and O'Brian 1993; Matters and

Beale, 1995) is used at a concentration of 20 mM [which has been demonstrated to be effective against tachyzoites of the RH and R5

strains]. Other inhibitors of this pathway include 4 amino- 5 -hexynoic acid and 4-aminofluoropentanoic acid which provide additional corroborative evidence that this pathway is present

ii) Glyoxylate Cycle The inhibitor of isocitrate lyase is 3 nitropropionic acid (ranging from 0 005 to 5 mg/ml in vitro)

iii) Alternative Oxidase 71 gondii bradyzoites use unique

alternative oxidases Alternative oxidase is necessary and sufficient for

bradyzoite survival Methods to characterize plant alternative oxidases are described (Hill, 1976, Kumar and Soil, 1992, Lambers, 1994, Li et al, 1996, Mclntosh, 1994).

For the in vitro studies, cell lines that lack functional mitochondria are

used These cell lines are used to allow the study of inhibitors effective against the conventional or alternative respiratory pathways within the parasite, but independent of

their effects on the host cell mitochondria Two cell lines, a human fibroblast cell line

(143B/206) lacking mitochondrial DNA, and the parental strain (143B) which poses functional mitochondria are used These cell lines have been demonstrated to support the growth of 71 gondii (Tomavo S and Boothroyd JC, 1995) SHAM, an inhibitor of

the alternative respiratory pathway is used at concentrations between 0.25 and 2 μg/ml

in vitro iv) Shikimate Pathway For EPSP synthase, the inhibitor is

N-(phosphonomethyl) glycine (concentrations of 3.125mM in folate deficient media) e) Culture assay systems for assessing inhibitor effect i) Toxicity assays: Aliquots of cells (HFF) are grown in 96-well tissue culture plates until 10% confluent. Cells are incubated with various

concentrations of drug for 1, 2, 4 and 8 days. Cultures are pulsed with tritiated

thymidine (2.5 μCi/well) for the last 18 hours of the culture after which the cells are

harvested using an automated cell harvester and thymidine uptake measured by liquid scintillation.

ii) In vitro parasite growth inhibition assays: Confluent monolayers

of HFF cells, grown in 96-well plates are infected with T. gondii tachyzoites of the RH strain and serial dilutions of anti-microbial compound are applied 1 hour later 71 gondii growth is assessed in these cultures by their ability to incoφorate tritiated uracil (2.5 μCi/well) added during the last 18 hours of culture After harvesting cells

with an automatic cell harvester, uracil incorporation is measured by liquid scintillation Alternatively, confluent HFF cells are grown in the chambers of Labtech slides and parasite growth is assessed microscopically following fixation in aminoacridine and

staining in 10% Giemsa (McLeod et al , 1992).

f) Product rescue assays to evaluate specificity of the inhibitor: To attempt to demonstrate specificity of the site of action of the inhibitor, growth

inhibition assays are performed in the presence of varying concentrations of product,

e.g., in the case where gabaculine is the inhibitor, ALA is added simultaneously to determine whether product rescue occurs. This type of study is only inteφretable when rescue is demonstrated because it is possible that exogenous "product" is not transported into the 71 gondii within host cells. For EPSP synthase, product rescue

assay is performed with PABA. g) Assays for synergy in vitro. This is an assay in which < 50% inhibitory

concentrations of two antimicrobial agents are added alone and together to determine whether there is an additive, synergistic or inhibitory interaction. All other aspects of

this assay are as described herein.

2. Inhibitors of Cryptosporidia parvum

C. parvum oocysts at 50,000/welI were incubated with each dmg (PRM=paromomycin which is the positive control, NPMG, gabaculine, SHAM, 8-

hydroxyquinoline) at 37°C (8% carbon dioxide) on confluent MDBKF5D2 cell monolayers in 96 well microtiter plates. The level of infection of each well was

determined and analyzed by an immunofluorescence assay at 48 hours using as an antibody C. parvum sporozoite rabbit anti-semm (0.1%), and using fluorescein-

conjugated goat anti-rabbit antibody (1%). Data are expressed as mean parasite count/field when 16 fields counted at lOx magnification "s.d. of the mean. (FIG. 6)

3. Inhibitors of Plasmodium falciparum

This assay is performed in folate deficient RPMI 1640 over a 66 hour

incubation in plasma as described by Milhous et al. (1985). Both the W2 clone DHFR resistant phenotype and the D6 clone are used (Odula et al, 1988) (Table 3). 4. Inhibitors of Eimeria tenella

Susceptibility of Eimeria tenella in vitro is analyzed by a method similar to that described by McLeod et al, 1992 or for Cryptosporidium as disclosed hereia 5. In vivo studies, measurement of parasitemia of Toxoplasma gondii

A method to measure the amount of parasitemia in mouse peripheral blood has been developed. Briefly, the target for PCR amplification is the 35 fold repetitive B 1

gene of T. gondii and the amplification was performed using primers previously reported. In order to semiquantitate the PCR product and to avoid false negative results, a competitive internal standard is generated using a linker primer and the

original B 1 primers. Competitive PCR was performed by spiking individual reactions

(containing equal amounts of genomic DNA) with a dilution of the internal standard Since this internal control contains the same primer template sequences, it competes with the B 1 gene of 71 gondii for primer binding and amplification The sensitivity of the PCR reaction in each sample can be monitored Following competitive PCR, the

PCR products are distinguished by size and the amount of products generated by the target and internal standard can be compared on a gel The amount of competitor DNA

yielding equal amounts of products gives the initial amount of target gene.

6. Interpretation of Data/Statistical Analysis of Data:

In vitro studies are performed with triplicate samples for each treatment group

and a mean ± sd determined as shown in the FIGs. All in vivo studies utilize at least 6

mice per group. Statistical analysis performed by Students' t-test or the Mann- Whitney

U-test. A p value of < 0.05, is considered statistically significant.

B. Western Blots Demonstrate Plant-Like Enzymes

Western analysis for GSAT, isocitrate lyase, malate synthase, alternative

oxidase and EPSP is used to demonstrate the presence of plant-like enzymes in many Apicomplexan parasites, e.g., Plasmodia, Toxoplasma, Cryptosporidia, Malaria and

Eimeria

Tachyzoites and bradyzoites (McLeod et al 1984, 1988, Denton et al, 1996a,

b), or their mitochondria and plastids are isolated as previously described Equivalent

numbers of tachyzoites and bradyzoites are separately solubilized in 2x sample buffer

and boiled for 5 minutes Samples are electrophoresed through a 10 percent SDS- polyacrylimide gel Proteins are transferred to a nitrocellulose membrane at 4°C, 32V with 25mM Tris and 192mM glycine, 20% v/v methanol, pH 8 3 Blots are blocked in

PBS (pH 7.2) containing 5% powered milk and 0 1% Tween 20 for 2 hours at 20°C

After washing in PBS (pH 7 2), 0 1% Tween 20, blots are stained with polyclonal or monoclonal antibodies specific for alternative oxidases in PBS (pH 7 2) containing 0 1% Tween 20 for 1 hour at 20°C Following washing in PBS (pH 7.2) containing

0 1% Tween 20, blots are incubated with an appropriate secondary antibody conjugated

to HRP at a dilution to be determined by methods known in the art After further washes, binding is visualized by chemoilluminescence (Amersham)

Antibodies to various enzymes, e.g., soybean GSAT, barley GSAT,

synechococcus GSAT, plant and/or trypanosome alternative oxidase, cotton isocitrate lyase, cotton malate synthase, soybean malate synthase, petunia EPSP synthase were

used to determine whether homologous enzymes are present in 71 gondii tachyzoites,

bradyzoites, mitochondrial and plastid enriched preparations Antibodies used include

monoclonal antibodies to Trypanosoma bruceu and Voo Doo Lily (Chaudhuπ et al. 1996) alternative oxidase and polyclonal antibody to Trypanosoma bruceu alternative oxidase The hybridizations with antibodies to plant and related protozoan alternative

oxidases demonstrated the relatedness of 71 gondu metabolic pathways to those of

plants and other non-Apicomplexan protozoans The products GSAT and alternative

oxidase were demonstrated by Western analysis Both polyclonal and monoclonal antibodies were reacted with alternative oxidase to confirm this observation

C. Probing Other Parasite Genes. The genes isolated from 71 gondii as

described herein are used to probe genomic DNA of other Apicomplexan parasites including Plasmodia, Crypiospor odium, and Eimeria.

D. Genomic Sequence. Genomic clones are identified and sequenced in the same manner as described above for cDNA except a genomic library is used Analysis of unique promoter regions also provide novel targets

E. Enzymatic Activity Demonstrates Presence of Plant-Like Enzymes in Metabolic pathways

The presence of the enzymes putatively identified by inhibitor studies is

confirmed by standard biochemical assays Enzyme activities of GSAT, isocitrate lyase, malate synthase, alternative oxidase, and EPSP synthase, chorismate synthase, chorismate lyase, UDP-glucose starch glycosyl transferase and other enzymes listed

herein are identified using published methods Representative methods are those of

Jahn er α/., 1991; Weinstein and Beale, 1995, Kahn et al, 1977, Bass e/ al, 1990, Mousdale and Coggins (1985) In addition, enzyme activity is used to determine in

which of the tachyzoite and bradyzoite life cycle stages each pathway is operative

Tachyzoites and bradyzoites are purified as described herein The parasites are lysed in 50mM HEPES (pH7.4) containing 20% glycerol, 0.25% Triton X-100 and proteinase inhibitors (5mM PMSF, 5FM E64, 1FM pepstatin, 0.2mM 1,10-phenanthroline). This

method has proven successful for measurement of phosphofructokinase, pyruvate

kinase, lactate dehydrogenase, NAD- and NADH-linked isocitrate dehydrogenases and succinic dehydrogenase activity in tachyzoites and bradyzoites of 71 gondii (Denton

et al, 1996a,b).

1 ) GSAT: GSAT activity is measured by the method of Jahn et al. , ( 1991 ), which uses GSA as substrate. GSA is synthesized according to methods of Gough et al. (1989). Heat-inactivated (60°C, 10') lysates are employed as non-enzymatic

controls. ALA is quantified following chromatographic separation (Weinstein and

Beale, 1985). This approach allows the definitive detection of GSAT activity in cmde extracts.

2) ALA Synthase: To determine whether parasites contain ALA synthase,

an activity also present in mammalian host cell mitochondria, cell fractions from

purified parasites are assayed. (Weinstein and Beale, 1985) ALA produced from added glycine and succinyl CoA is quantified as for GSAT.

3) Isocitrate Lvase: The biochemical assay for isocitrate lyase activity used is the method of Kahn et al. (1977).

4) Alternative Oxidase: activity is measured in parasite lysates or purified mitochondria or plastids by oxygen uptake using an oxygen electrode described by Bass et al. (1990). Confirmation of the oxidation being due to alternative oxidase(s) is achieved by successful inhibition of oxygen uptake in the presence of 0 5mM SHAM, but not in the presence of KCN

5) Shikimate Pathway The biochemical assay for EPSP synthase,

chorismate synthase, chorismate lyase, activity in cellular lysates is conducted as

described by Mousdale and Coggins (1985) and Nichols and Green (1992)

6) Branched Amino Acids The biochemical assay for hydroxy acid synthase is as described

7) Amylopectin Synthesis The biochemical assays for starch synthase, Q

enzymes, and UDP-glucose starch glycosyl transferase are as described 8) Lipid Synthesis Assays for lipid synthases are as described

Some of the additional representative enzyme assays are precisely as described by Mousdale and Coggins(1985) and are as follows

5-Enolpyruvylshikιmate 3 -phosphate synthase is assayed in forward and reverse directions as described previously (Mousdale and Coggins 1984) Shikimate NADP oxidoreductase (shikimate dehydrogenase), shikimate kinase,

3 -Dehydroquinase (DHQase) are assayed Assay mixtures contained in a total volume of 1 ml 100 mM potassium phosphate (pH 7 0) and 0 8 mM

ammonium 3 -dehydroquinate 3 -Dehydroquinate synthase is assayed by

coupling for forward reaction to the 3 -dehydroquinase reaction, assay mixtures

contained in a total volume of 1 ml 10 mM potassium phosphate (pH 7 0), 50 μM NAD⁺ 0 1 mM CoCl₂, 0 5 nkat partially-punfied Escherichia cob DHQase and (to initiate assay) 0.4 mM DAHP. The DAHP is prepared from E. coli

strain AB2847A and DHQase from E. coli strain ATCC 14948.

Assay of DAHP synthase is by a modification of the method of Sprinson et al.. Assay mixtures contained in a total volume of 0.5 ml: 50 mM 1,3-bis [tris(hydroxymethyl)-methylamino] propane-HCl (pH 7.4), 1 mM erythrose 4- phosphate, 2 mM phosphoenolpymvate and 1 mM CoCl₂. The reaction is

initiated by the addition of a 50 to 100 μl sample containing DAHP synthase

and terminated after 10 min at 37°C by 100 μl 25% (w/v) trichloroacetic acid.

The mixture was chilled for 1 h and centrifuged to remove precipitated protein.

A 200 μl aliquot of the supernatant was mixed with 100 μl 0.2 M NalO₄ in 9 M

H₃PO₄ and incubated at 37°C for 10 min; 0.5 ml, 0.8 M NaASO₂ and 0.5 M

Na₂SO₄ in 0.1 M H₂SO₄ in 0.1 m H₂SO₄ was then added and the mixture left at

37°C for 15 min; 3 ml 0.6% (w/v) sodium thiobarbiturate and 0.5 M Na₂SO₄ in

5 mM NaOH was added and the mixture placed in a boiling-water bath for 10

min. After cooling to room temperature the solution was centrifuged (8500 xg,

2 min) and the optical density at 549 n read immediately. Appropriate controls assayed in triplicate lack substrates, sample or both."

Another representative assay is an assay for chorismate lyase which is as described by Nichols and Green, 1992:

Chorismate lyase assays are carried out in a volume of 0.5 ml containing 50 mM

Tris-HCI (pH 7.5), 5 mM EDTA, 10 mM 2-mercaptoethanol, 60 μM

chorismate, and 0.2 to 4 U of chorismate lyase. After incubation at 37°C for 30 min, 4-hydroxybenzoate is detected and quantitated by high-pressure liquid chromatography (HPLC). Fifty microliters of each reaction mixture is applied to an HPLC system (Waters 625) equipped with a Nova-Pak Ci_ϋ column

equilibrated in 5% acetic acid and monitored at 240 nM. The height of the

4-hydroxybenzoate peak is compared with those of standard curves generated by treating known amounts of 4-hydroxybenzoate in a similar manner. One unit

of chorismate lyase activity is defined as the amount of enzyme required to

produce 1 nmol of 4-hydroxybenzoate in 30 min at 37°C.

Assays for 4-aminobenzoate and 4-amino-4-deoxychorismate are performed as described previously."

E. Construction and Analysis of Gene "Knock-Outs"

In order to determine whether a gene, e.g., chorismate synthase or alternative

oxidase is essential for growth or survival of the organism, gene knockout organisms are generated by the method of Roos et al, 1996. Specifically, the strategy for creating mutants is with homologous recombination and to generate a targeted gene knock-out a sequential positive/negative selection procedure is used (Roos et al, 1996). In this

procedure positive and negative selectable markers are both introduced adjacent to, but not within the cloned and suitably mutated locus. This constmct is transfected as a

circular plasmid. Positive selection is applied to yield a single-site homologous

recombinant that is distinguished from non-homologous recombinants by molecular screening. In the resulting 'pseudodiploid,' mutant and wild-type alleles flank

selectable marker and other vector sequences. In the next step, parasites are removed from positive selection, which permits recombination between the duplicated loci This

event appears to occur at a frequency of 2 x 10^"6 per cell generation These

recombinants are isolated with negative selection Next, they are screened to distinguish those that have recombined in a manner that deletes the mutant locus and yields a wild-type revertant from those that deleted the wild-type gene to leave a perfect allelic replacement

This 'hit-and-run' approach has the disadvantage of being time-consuming Nonetheless, it offers several distinct advantages over other gene knock-out strategies

First, because gene replacement occurs by two sequential single-cross-overs instead of

one double cross-over which is a very rare event, it is more likely to be successful

Second, because selectable marker(s) are located outside of the targeted gene itself, expenments are not limited to gene knock-outs A variety of more subtle point mutations are introduced as allelic replacements Third, this strategy provides a means

of distinguishing essential genes from those which cannot be deleted for purely

technical reasons Specifically, if the hit-and-mn mutagenesis procedure yields only wild-type revertants instead of the theoretical 1 1 ratio of wild-type mutant, this provides positive evidence that the locus in question is essential

An example is a knockout created for the chorismate synthase gene It also can

be made more general to include knockout of other genes for attenuated vaccines such

as EPSP synthase and alternative oxidase The parasite with the gene of interest to be

knocked out is grown ("manufactured") in vitro in presence of product, but when used in vivo the needed product is not present The parasite functions as an attenuated vaccine as described below under vaccines A specific example follows. Specifically, the strategy of product inhibition discussed above is also useful for growing gene

knockout parasites (which lack a key gene for their survival) in vitro by providing the

essential product and thus bypassing the need for the gene during in vitro propagation

of the parasite. Such gene knockouts cultivated in vitro in this manner are useful attenuated organisms that are used as attenuated vaccines.

The chorismate synthase cDNA clones are used as hybridization probes for

recovering genomic clones from a 71 gondii genomic cosmid library. Coding regions

are mapped onto the genomic clones using the cDNA clones as a guide. Appropriate sections are sequenced to verify the gene location. Ultimately, full genomic sequences are obtained. Enough of the genomic clones are sequenced to develop a strategy for generating a putative null allele. Segments that can be deleted at the 5' end of the coding region to generate an allele that is unlikely to generate a functional gene product

are identified. A putative neutral allele is generated that can be distinguished from the wild type allele on the basis of an introduced restriction site polymorphism, but that

does not differ in encoded protein sequence. These putative chorismate synthase-null and chorismate synthase-neutral alleles are cloned into the pminiHXGPRT transfection vector plasmid.

The resulting chorismate synthase -null and chorismate synthase-neutral

plasmids are transfected into HXGPRT-negative strains of T. gondii (strains

RH(EP)ΗXGPRT [a ME49 derivative]. Numerous independent clones are selected for

survival on mycophenolic acid to select for insertion of the plasmid. These strains are screened by Southern analysis designed to detect the presence of both the normal and

modified copies of the chorismate synthase gene and for tandem location of the two

copies (with the vector HXGPRT gene between). This is the stmcture expected for insertion of the plasmid by homologous recombination at the AroC genomic locus (the "hit" needed for the hit-and-mn gene knock-out strategy). The feasibility of recovering

these strains is critically dependent upon the ratio of homologous to non-homologous integration following transfection, which will depend upon the length of homologous, genomic DNA in the clone (Donald and Roos, 1994; Roos et al, 1996) Eight kb of homology is sufficient to obtain >50% homologous integration (Roos et al, 1996)

HXGPRT clones with verified pseudodiploid stmcture of the chorismate synthase alleles are selected for loss of HXGPRT using 6-thioxanthine (the "run" part of the protocol). Numerous clones are selected. If the loss of HXGPRT is based upon random homologous exchange between the two chorismate synthase pseudodiploid

alleles, theoretically half of the events should lead to excision of the modified chorismate synthase allele along with the HXGPRT, leaving the original wild type allele

in the chromosome. The other half should excise the wild type allele, leaving the modified allele in the chromosome. During selection and grow-out of these clones, the

medium is supplemented with chorismate at the concentration determined to best rescue cells from inhibitor toxicity. The puφose of the supplementation is to enhance the chances of recovering chorismate synthase-null strains The genomic stmcture of

the selected clones is examined by Southern analysis to confirm loss of the vector

HXGPRT and of one copy of the chorismate synthase and to identify the remaining allele of chorismate synthase. The ratio of mutant to wild type is tabulated. The chorismate synthase-neutral allele is intended as a positive control to confirm that either allele (wild type or mutant) can be lost in this procedure. If chorismate synthase-

neutral strains can be recovered but chorismate synthase-null strains cannot, the conclusion is that the chorismate synthase gene is essential for growth. If it proves possible to recover chorismate synthase-null strains, they are subjected to further

phenotypic analysis, first, using immunoblotting of electrophoretically separated cell

extracts to confirm absence of chorismate synthase protein, then, determining if these strains show hypersensitivity to inhibitors of the alternative oxidase or to any of the other potential inhibitors. Sensitivity to chorismate synthase inhibitors is analyzed to

determine the relative specificity of inhibition. If chorismate synthase is the sole target

of the inhibitors, then the null mutants should be insensitive to further inhibition. Sensitivity analysis is conducted in vitro as described herein. Whether strains show

alterations in expression of the alternative oxidase or in any stage-specific antigens is of interest. These analyses are conducted by immunoblotting of electrophoretically separated cell extracts. In vivo analysis using a mouse model is conducted to determine if these strains are infective and what stages of parasites can be detected following

infection. Genetically altered 71 gondii organisms are used to infect C3H/HeJ mice by

the intraperitoneal route. Mortality is monitored and brains examined for cysts at 30

days post infection.

Knockouts with bradyzoite reporter genes are useful to determine whether these

enzymes influence stage switch. Stage switch also is characterized by quantitating relative amounts of parasite mRNA present in each stage of parasite using Northern blotting, isolation of mRNA and RT-PCR using a competitive inhibitor, and enzyme assay.

G. Reagents used for construction of "Knock-Outs"

Library

Me49 genomic libraries are used.

Plasmids

/?miniHXGPRT. Contains 71 gondii HXGPRT gene under control of DHFR-TS 5' and

3' flanking sequences. Functions as either a positive or negative selection marker (using 6-thioxanthine or mycophenolic acid, respectively) in suitable host strains.

Parasite Strains (obtained from AIDS Repository. Bethesda. Md

RH(EP). Wild-type host strain RH (highly pathogenic in mice).

RH(EP)ΗXGPRT. HXGPRT knock-out mutant of RH strain. Suitable for positive selection of HXGPRT-containing vectors.

P(LK). Wild-type host strain P, (clonal isolate of strain ME49; produces brain cysts in mice).

P(LK)HXGPRT-. HXGPRT-deficient mutant of P strain. Suitable for positive selection of HXGPRT-containing vectors. H. Antibodies

Antibodies have been raised against homologous plant enzymes by standard

techniques for both polyclonal and monoclonal antibodies (Current Protocols in Immunology, 1996)

1) Heme Synthesis

Antibody to soybean, barley and synechococcus GSAT are polyclonal antibodies with preimmune sera the control for the barley and synechococcus antibodies

2) Glyoxylate Cycle T. gondii contains a glyoxylate cycle that allows growth using lipids as a carbon source, thus the lipid mobilization pathway of T. gondii is similar to the pathway of

plants (Tolbert, 1980) A similar approach using polyclonal antibodies to isocitrate lyase and to malate synthase and preimmune control sera are used.

3) Alternative Energy Generation Monoclonal and polyclonal antibodies to alternative oxidases in plants

(Mclntosh et al, 1994) and Trypanosomes (Hill, 1976) are used.

4) Shikimate Pathway

To demonstrate that T. gondii has the same unique enzymes that permit interconversion of shikimate to chorismate as plants do, the antibody to shikimate

pathway plant EPSP synthase is used 5) Synthesis of Branched Chain Amino Acids

Antibodies to acetohydroxy acid synthase are used.

6) Amylose and Amylopectin Synthesis and Degradation

Antibodies to starch synthesis, branching (Q) enzymes and UDP glucose starch

glycosyl transferase are used.

1. Complementation of Enzyme Deficient E. coli Demonstrates Functional

Product

The E. coli AroC mutant which lacks chorismate synthase (AroC) was obtained from the E. coli genetic stock center. AroC bacteria is made competent to take up DNA by transformation with CaCl₂ treatment. Alternatively, the cells are electroporated to take up DNA. The presence of the plasmid is demonstrated in this

system by growth on media which contains ampicillin, as the plasmid contains an ampicillin resistance gene. Complementation is confirmed by demonstrating growth on media lacking the product catalyzed by (i.e., chorismate). Thus, this transformation/ complementation is used with the 71 gondii cDNA library system or a constmct which

contains some or all of the chorismate synthase gene to transform the AroC mutant. Functional enzyme is then demonstrated.

J. Immunizations Of Mice For Polyclonal Antibody Production:

As an alternative approach if complementation studies are unsuccessful and the

monoclonal antibodies to a plant protein are not cross reactive, purified plant protein is used to immunize mice to raise polyclonal antibodies to each enzyme. Where

necessary, antibodies to the pertinent enzymes are generated in mice, ND4 outbred mice are immunized with 20 μg of enzyme emulsified in Titermax complete adjuvant

injected intramuscularly into their gluteal muscle. Two weeks later mice are immunized with a further 20 μg of enzyme emulsified in Titermax. After a further 2 weeks mice receive a further boost of enzyme alone in PBS by the intraperitoneal route. Mice are bled and the semm tested for specificity by the standard Western blotting technique.

K. Immunofluorescence

Antibodies used to identify enzymes in the Apicomplexan metabolic pathways disclosed here are used for immunofluorescence studies. Examples are demonstration of alternative oxidase in 71 gondii by immunofluorescence assay (IFA). 71 gondii alternative oxidase is immunolocalized to mitochondria. L. ELISAs

ELISAs are used for documenting the presence and quantitating the amounts of

alternative oxidase.

M. Reporter Constructs To Demonstrate Organelle Targeting Are Made And

Characterized As Described Using β Glucoronidase Or Other Chimeric

Constructs

Importance of the targeting sequence for localization of the enzyme to an

organelle is demonstrated with immunoelectronmicroscopy. Organelle targeting sequences in proteins expressed in bacteria which lack the organelle cause misfolding of

proteins and thereby impair protein function.

A useful reporter protein for a chimeric constmct is β glucoronidase, expressed in E. coli under control of the 355 promoter of cauliflower mosaic vims. The glucoronidase alone without the transit sequence is expressed in parallel. The transit peptide constmct is found in the plastid. The control glucoronidase is found in the

cytoplasm. Antibodies to the chorismate synthase protein are also used to detect the

presence of the product of the gene (with the transit sequence) in the plastid and the product of a constmct (in which the transit sequence is not present) in the cytoplasm only. Further mutations and deletions are made which identify the minimal transit sequence using the same techniques as described above for the entire peptide.

Antisense, ribozyme or intracellular antibodies directed against the transit sequence

nucleic acid or translated protein are useful as medicines. The amino acid or nucleic acid which encodes the transit sequences are the bases for development of diagnostic reagents and vaccines.

N. Modifications of Inhibitory Compounds to Improve Oral Absorption Tissue Distribution (especially to brain and eve). Tissue distribution is characterized using radiolabeled inhibitor administered to

mice with its disposition to tissues measured. Compounds are modified to improve oral absoφtion and tissue distribution.

O. Methods to Demonstrate Protection Against Conjoint Infections

Infections are established and influence of an inhibitor or combination of inhibitors on outcomes are as outlined below.

Infections: Infections with Toxoplasma gondii, Pneumocystis carinii, Mycobacterium

tuberculosis, Mycobacterium avium intracellular and Cryptosporidium parvum are established alone and together using an immunosuppressed rodent model Endpoints

in these infections are

Survival Ability of an inhibitor to protect, measured as prolonged survival

Parasitemia This is measured using isolation of mRNA and RT-PCR with a competitive inhibitor for quantitation

Tissue Parasite Burden This is determined by quantitating brain and eye cyst numbers Inflammatory Response This is noted in histopathologic preparations Representative combinations of inhibitors are NPMG and sulfadiazine, SHAM and atovaquone, NPMG and pyrimethamine, NPMG and SHAM

P. Testing of Antimicrobial Compounds

Presence of inhibitory activity of new antimicrobial compounds is tested in

enzymatic assays, in vitro, and in vivo assays as described above and in the literature

Q. Efficacy, Safety, Pharmakokinetics, and Therapeutic Toxic Index

The testing in murine models includes standard Thompson tests Testing of antimicrobial agents for efficacy and safety in primate models for malaria is performed Dosages are selected based on safety information available from data bases of

information concerning herbicides and the literature Measurements of semm and

tissue levels of antimicrobial compounds are performed using assays which detect inhibitor concentrations and concentrations of their metabolites Representative assays

are high performance liquid chromatography, and assaying tissues for percentage of

radiolabeled compounds administered using liquid scintillation and other assays also

are used R. Carcinogenicity and Teratogenicity

Standard assays to evaluate carcinogenicity include administration of medicines

as described above to rodents and observation of offspring for teratogenic effects and carcinogenicity Observation includes general physical examination, autopsy and

histopathologic studies which detect any teratogenic or carcinogenic effects of

medicines S. Constructs to Measure Parasitemia

Portions of genes are deleted and the shorter gene is used as an internal standard in RT PCR assays to measure amount of parasites present (Kirisits, Mui,

Mack, McLeod, 1996)

T. Vaccine Constructs and Proteins and their Administration

These are prepared, and sensitivity and specificity are established as is standard in the literature and as described above. Tests and reagents include DNA constmcts

(Tine et al, 1996) with the appropriate gene or portions of the gene alone or together, with adjuvants Representative adjuvants include ISCOMS, nonionicsurfactant vesicles, cytokine genes in the constmcts and other commonly used adjuvants. Native and recombinant proteins also are used in studies of vaccines Protection is measured

using immunologic in vitro assays, and by assessing survival and reduction of

parasitemia and tissue parasite burden and prevention of congenital infection (McLeod et al, 1988). U. Preparation of Diagnostic Test Reagents and Diagnostic Tests:

These assays are as described (McLeod and Boyer, 1996) They include

ELISAs in which antibodies to the proteins or peptides and recombinant proteins are

used and PCR methodology in which primers to amplify DNA which encodes the enzymes or parts of this DNA are used A test useful in an outpatient setting is based on conjugation of a monoclonal antibody to human red blood cells with antibody to

peptides or proteins The red cells are cross linked if the antibody to the parasite

component interacts with the parasite component and agglutinates the red cells in the blood sample ELISA and PCR can be utilized with samples collected on filter paper as is standard in Newborn Screening Programs and also facilitates outpatient and field

use V. Antisense

Antisense oligonucleotides are short synthetic stretches of DNA and RNA

designed to block the action of the specific genes described above, for example,

chorismate synthase of 71 gondii or P. falciparum, by binding to their RNA transcript

They turn off the genes by binding to stretches of their messenger RNA so that there is breakdown of the mRNA and no translation into protein Antisense reagents have

been found to be active against neoplasms, inflammatory disease of the bowel (Crohn's

Disease) and HIV in early trials Antisense oligonucleotides directed against the nucleic acids which encode the essential parasite metabolic process described herein

are effective medicines to treat these infections Antisense oligonucleotides also are

directed against transit sequences in the genes Antisense will not contain cytosine nucleotides followed by guanines as this generates extreme immune responses (Roush,

1997) Antisense oligonucleotides with sequence for thymidine kinase also is used for regulatable gene therapy

W. Ribozymes and Other Toxic Compounds

Ribozymes are RNA enzymes (Mack, McLeod, 1996) and they and toxic compounds such as ricins (Mahal et al, 1997) are conjugated to antisense oligonucleotides (see V, DNA), or intracellular antibodies (see X, for proteins), and these constmcts destroy the enzyme

X. Intracellular Antibodies

Intracellular antibodies are the Fab portions of monoclonal antibodies directed against the enzymes or portions of them (e.g., anti-transit sequence antibodies) which

can be delivered either as proteins or as DNA constmcts, as described under vaccines

Y. Development of New Antimicrobial Compounds Based on Lead Compounds

The herbicide inhibitors comprise lead compounds and are modified as is standard For example, side chain modifications or substitutions of groups are made to make more active inhibitors Their mode of action and stmcture as well as the enzyme

and substrate stmctures are useful in designing related compounds which better

abrogate the function of the enzymes Examples of such substrate or active site targeting are described above

Native or recombinant protein is used in enzymatic assays and in vitro assays described above are used to test activity of the designed newly synthesized compounds Subsequently, they will be tested in animals

Z. Trials to Demonstrate Efficacy for Human Disease

Trials to demonstrate efficacy for human disease are performed when in vitro

and murine and primate studies indicate highly likely efficacy and safety They are standard Phase I (Safety), Phase II (small efficacy) and Phase III (larger efficacy with

outcomes data) trials For medicines effective against 71 gondii tachyzoites, resolution

of intracerebral Toxoplasma brain abscess in HIV-infected individuals with no other

therapeutic options available due to major intolerance to available medicines is the

initial strategy for Phase II trials For medications effective against T. gondii bradyzoites, absence of development of toxoplasmic encephalitis in individuals with

HIV infection and individuals who are seropositive for T. gondu infection followed

after a one-month treatment for a 2 year period when their CD4 counts are low Effective medicines demonstrate efficacy, as 50% of such individuals otherwise

develop toxoplasmic encephalitis. When medications efficacious against bradyzoites and recmdescent toxoplasmic encephalitis in patients with AIDS are discovered and

found to be safe, similar trials of efficacy and safety for individuals with recurrent

toxoplasmic chorioretinitis are performed.

DEFINITIONS

3-deoxy-d-arabino-heptuloonate 7 phosphate synthase: An enzyme which

functions in chorismate synthesis

3-enolpyruvyshikimate phosphate synthase (3-phosphoshikimate-l-

carboxyvinyltransferase): An enzyme which functions in chorismate synthesis

3-NPA: An inhibitor of isocitrate lyase in the glyoxylate pathway and also of succinate

dehydrogenase

3-oxtaprenyl-4-hydroxybenzoate carboxylyase: An enzyme which functions in ubiquinone synthesis

4-hydroxybenzoate octaprenyltransferase: An enzyme which functions in ubiquinone synthesis

8-OH-quinoline: An inhibitor of the alternative oxidase

Abscissic Acid Metabolism in Plants: A 15-carbon sequiteφenoid synthesized

partly in plastids by the mevalonic acid pathway Abscissic acid protects plants against stress and is a marker of the plant's maturation and activation of transcription, and causes dormancy Inhibits protein synthesis and leads to specific activation and

deactivation of genes

Acetohydroxy acid synthase: Enzyme which catalyzes production of acetohydroxy acids (the branched chain amino acids valine, leucine and isoleucine in plants)

Alternative oxidase: An enzyme important in the alternative pathway of respiration

There are examples of alternative oxidases in plants and trypanosomes. (Pollakis et al, 1995, Rhoads & Mclntosh, 1992, Clarkson et al, 1989) Alternative respiration or energy generation: A different pathway for energy generation utilizing the alternative oxidase and election flow in the electron transport chain which is not dependent on conventional cytochromes or heme

Altered gene includes knockouts

Amide: The R portion of the amino group has an amino group connected to a carbonyl carbon Glutamine and asparagine are amides Important for nitrogen transport and storage

Amylopectin: A branched starch of plants Also found in T gondii bradyzoites

Amyloplast: Storage granule for starch in plants Derived from chloroplasts

Amylose: An unbranched starch of plants

Anabolism: Formation of large molecules such as starch, cellulose, proteins, fats and

nucleic acids from small molecules Requires input of energy

Anthranilate phosporibolsyltransferase: An enzyme which functions in tryptophan synthesis

Anthranilate synthase component I: An enzyme which functions in tryptophan synthesis

Anthranilate synthase component II: An enzyme which functions in tryptophan synthesis

Antimicrobial agent: A chemical, for example a protein or antisense nucleic acid

which effectively inhibits or kills a pathogenic microbe There are examples (Schwab et al, 1994, Strath et al, 1993; Beckers et al, 1995, Blais et al. 1993, Fichera et al, 1995, Pfefferkorn & Borotz, 1994, Pfefferkorn et al, 1992, Pukivittaykamee et al,

1994).

Apicomplex: The common feature of Apicomplexan parasites including a conoid and rhoptry organelles and micronemes at the apical end of the parasite

Apicomplexan parasite: A microorganism that belongs to the Apicomplexan group of

parasites These parasites share a number of moφhologic features, including a conoid and rhoptry which are organelles in the cytoplasm at the apical end of the organism and plastids which are multilamellar stmctures Representative examples of Apicomplexan

parasites include Toxoplasma gondu, Plasmodium, Cryptosporidia and Eimeria

Aromatic acid aminotransferase (aromatic transaminase): An enzyme which functions in tyrosine synthesis

Aspartate, glutamate and glutamine synthesis: Involve glutamine synthase and

glutamate synthetase and are plastid associated in plants Glutamine synthase in plants is inhibited by the herbicide glufosinate (2 amino-4-[hydroxymethylphosphinyl) butanoic acid. Glutamine synthase also is present in animals

ATP-phosphofructokinase: (ATP-PFK) May exert control over glycolytic pathway

because a step when hexoses phosphate cannot also be used to form sucrose or starch

Nearly all animals lack PPi-PFK with plant-like substrate specificity (i e PPi, not

ATP)

Auxins Growth regulators in plants, which are tryptophan derivatives Herbicides

modeled on auxins are stmctural mimics of these compounds rather than inhibitors of auxin function Biochemical pathways: Biochemical pathways include metabolic pathways Any

chemical reaction in life Herein "biochemical pathways" and "metabolic pathways" are

used interchangeably

Bradyzoite: The slowly replicating life cycle stage of the Apicomplexan parasite

Toxoplasma gondii This stage is responsible for latent and recmdescent infection due

to this parasite The morphologic features which characterize this parasite stage are

electron dense rhoptries and amylopectin granules Bradyzoites contain a plastid organelle as do other life cycle stages of this parasite This parasite stage also has specific antigens which other life cycle stages do not have, including bradyzoite surface

antigen 4 and bradyzoite antigen 5 (lactate dehydrogenase), which is an intracellular and cyst matrix antigen Bradyzoites exist together in a stmcture called a cyst which

has a cyst wall and matrix Cysts contain a few to thousands of bradyzoites The cyst containing bradyzoites is a major means of transmission of the organism Toxoplasma

gondii when it is ingested in meat which is not cooked to well done It is also a form of the organism responsible for recmdescent eye and brain disease in infants and children who are congenitally infected with the parasite and also in patients whose immune

system is not normal

Branched chain amino acid synthesis (valine, leucine and isoleucine) involving acetohydroxy acid synthase, is the first of the series of reactions, is another metabolic pathway present in plants but not in animals

Branched chain amino acids: Amino acids (valine, leucine and isoleucine), the

synthesis of which can be inhibited by sulfonylurea and imidazolinone herbicides There are examples in plants (Kuriki el al, 1996; Morell et al, 1997; Kortostee et al,

1996; Gmla et al, 1995; Khoshnoodi et al, 1996).

Branching or Q enzyme: Forms branches in amylopectins between C6 of the main chain and Cl of the branch chain.

Cataboiism: Degradation or breakdown of large molecules to small molecules, often releasing energy.

Calmodulin: is a calcium binding protein (Robson et al, 1993)

Catechol 1,2-deoxygenase (phenol hydroxylase): An enzyme which functions in phenylalanine synthesis.

Chloroplast: A DNA-containing multilamellar organelle of plants and algae

associated with metabolic pathways important for photosynthesis and other energy production. Chloroplasts utilize proteins encoded in their own DNA and also proteins encoded by nuclear DNA.

Chorismate: The product of the action of the enzyme EPSP synthase on shikimate.

Chorismate lyase: An enzyme responsible for the conversion of chorismate to

3 ,4-dihydroxybenzoate.

Chorismate mutase (7-phospho-2-dehydro-3-deoxy-arabino-heptulate-aIdolase):

An enzyme which functions in chorismate synthesis.

Chorismate synthase: An enzyme responsible for the conversion of 3-phospho 5- enolpyruvyl shikimate to chorismate.

Chorismate: The product of the action of the enzyme EPSP synthase on shikimate. Competitive inhibitors: Stmctures sufficiently similar to the substrate that they

compete for the active site of the enzyme Addition of more natural substrate overcomes effect of the inhibitor.

Components: includes nucleic acids, proteins, peptides, enzymes, peptide targeting sequences, transit peptides, carbohydrates, starch, lipids, hormones, for example those

listed in Table 1 and other constituents of metabolic pathways or products derived from these components

Conventional energy generation: Usual pathways of generation of energy in

mitochondria utilizing cytochromes for the transfer of electrons

Conversion of Fats to Sugars in Plants: Occurs by oxidation and the glyoxylate

cycle

Cryptosporidiosis: The disease due to the Apicomplexan parasite Cryptosporidium

parvum It causes self-limited diarrhea or no symptoms in immunologically normal

individuals In individuals who have immunocompromising illnesses, such as the acquired immune deficiency syndrome, Cryptosporidiosis causes life-threatening, persistent, copious, watery diarrhea.

Cryptosporidium parvum: Cryptosporidium parvum is an Apicomplexan parasite

which causes cryptosporidiosis

Cyanide-insensitive, non-he e "alternative" oxidase is a metabolic activity that is found in most eukaryotic plants and algae and is absent from multicellular animals The

alternative oxidase is a single polypeptide enzyme that lacks heme and can serve as the terminal electron acceptor to support respiratory growth of E. cob in the absence of heme The coupling efficiency of this oxidase is lower than that of the cyanide-sensitive cytochrome oxidase. That is, not as many protons are pumped across the

mitochondrial inner membrane in parallel with electron transfer through the alternative

oxidase as they are through the cytochrome oxidase The alternative oxidase appears to be used by plants and algae only under certain conditions The alternative oxidase also is used during different life-cycle stages or under different environmental

conditions Thus, inhibitors of the alternative oxidase may act cooperatively or synergistically with GSAT inhibitors.

Cyclohexadienyl dehydratase: An enzyme which functions in phenylalanine synthesis

Cyclohexadienyl dehydrogenase: An enzyme which functions in tyrosine synthesis

Cytochrome oxidase: An enzyme utilized in the conventional pathway of energy generation

Dehydroquinate dehydratase: An enzyme which functions in chorismate synthesis

Deoxyribonucleases: Enzymes which are hydrolases which hydrolyze DNA

(phosphate esters)

Eimeria bovis: Causes bovine eimeriosis

Eimeria maxima and Eimeria tenella: Cause eimeriosis in chickens.

Eimeria: A group of Apicomplexan parasites which cause gastrointestinal disease in agriculturally important animals including poultry and cattle These economically important parasites include Eimeria tenella, E. maxima and E. bovis Endosymbiont: An organism which is taken up by another organism and then lives

within it

Enzyme: A protein which catalyzes (makes more rapid) the conversion of a substrate

into a product Enzymes are catalysts which speed reaction rates generally by factors between 10⁸ and 10²⁰ They may require ion or protein cofactors Control is by

products and environmental changes There are more than 5000 enzymes in living systems Enzymes are named with common or trivial names, and the suffix-ase which characterizes the substrate acted upon (e g , cytochrome oxidase removes an electron

from a cytochrome) Sequential series of steps in a metabolic pathway Enzymes that

govern the steps in a metabolic pathway are sometimes arranged so that a kind of assembly-line production process occurs

EPSP synthase: An enzyme important in the conversion of shikimate to chorismate

EST: Expressed sequence tag, a short, single pass cDNA sequence generated from

randomly selected library clones Eukaryote: Microorganism or phylogenetically higher organism, the cells of which have a nucleus with a limiting membrane

Fatty Acid Synthesis in Plants: Occurs only in chloroplasts of leaves and proplastids of seeds and roots Mainly palmitic acid and oleic acid AcetylCo A carboxylases

differ in plants and animals Linoleic acid synthase and linoleneic acid synthase are lipid synthases present in plants and not animals

Glycolysis -> pyruvate →-acetyl CoA Example

8 acetyl CoA +7 ATP + 14 NADPH + \+W^→ palmityl CoA + 7 CoA + 7 ADP^2' +

7H₂PO4 + 14 NADP" + 7H₂O

Fragment: Refers to a sequence of nucleic acids or aminoacids, where a fragment is sufficient to function as a component of or product derived from an Apicomplexan as defined herein

Gabaculine: An inhibitor of the enzyme GSAT in the heme synthesis pathway Gene: Nucleotide sequence which encodes an amino acid sequence or another nucleotide sequence Giberellin Metabolism in Plants: Plant hormones which promote plant growth,

overcome dormancy, stimulate Gl to S transition and shorten S phase of cell cycle, increase hydrolysis of starch and sucrose into glucose and fructose They are

derivatives of ent-gibberellane skeleton synthesized from a 2acetyl CoA to mevalonic acid to isopentemyl pyrophosphate to 4 isopentenyl pyrophosphate to geranylgeranyl

pyrophosphate to copalylpyrophosphate to kaurene to kaurenol to keaurenal to kaurenoic acid to GAι₂ aldehyde to other giberellins These functions are not clearly

established but it is hypothesized that hydrolysis of starch to sugar occurs by inducing

formation of amylase enzymes Isoprenoid compounds, diteφenes synthesized from

acetate units of acetyl coenzyme A by mevalonic acid pathway stimulate growth Inhibitors of giberellin synthesis include phosphon D, Amo 1618 (blocks conversion of

geranyl pyrophosphate to CO palylpyrophosphate), phosphon D, which also inhibits

conversion of (oxidation) formation of Kaurene, CCC or cycocel, ancymidol, and pactobutrazol (blocks oxidation of karene and kaurenoic acid) Young leaves are

major sites for giberellin synthesis These plant hormones which induce hydrolysis of polysaccharide into hexoses are used in glycolysis When hexoses are abundant,

glycolysis is more rapid

Glutamyl-tRNA reductase: An enzyme which functions in heme synthesis

Glutamyl-tRNA synthetase: An enzyme which functions in heme synthesis

Glycolysis in Plants: Several reactions of glycolysis also occur in plastids Glycolysis = lysis of sugar, degradation of hexosis to pymvic acid in plants In animals,

degradation of glycogen (animal starch) to pymvate. Plants form no glycogen Glyoxylate pathway: The pathway important for lipid degradation which takes acetyl CoA and converts it to CoA-SH through the conversion of isocitrate to C4 acids

including succinate This pathway utilizes isocitrate lyase and also converts glyoxylate

to malate, a reaction catalyzed by the enzyme malate synthase The glyoxysome or Glyoxylate pathway which is cytoplasmic in certain algae involves isocitrate lyase and malate synthase to metabolize lipids and provide C4 acids A metabolic distinction between autotrophic eukaryotes and heterotrophs is the presence of a glyoxylate cycle

This cycle employs two enzymes, isocitrate lyase and malate synthase, to bypass the

two decarboxylation steps of the TCA cycle and enables the utilization of carbon stored

in fatty acids for growth In plants, the enzymes of the glyoxylate cycle are compartmentalized within a unique single-membrane-bound organelle, the glyoxysome

In certain algae, the cycle is entirely cytoplasmic In plants, these enzymes are most abundant during germination and senescence In animals, the glyoxylate cycle enzymes have been described as being present only during starvation

Glyoxysome: An organelle which in some instances contains enzymes important in the glyoxylate cycle

GSAT: Glutamate-1 semialdehyde aminotransferase is the enzyme important in heme

synthesis for the conversion of glutamate semialdehyde to ALA (δ-aminolevulinic acid)

Heme synthesis pathway: A metabolic pathway important for generation of heme, poφhyrins and other iron sulfated proteins used in mitochondria in the conventional pathway of energy generation This pathway occurs in plant chloroplasts and uses the nuclear encoded enzyme GSAT A metabolic distinction between plants and animals

occurs in the heme biosynthesis pathway Non-photosynthetic eukaryotes, including

animals, yeast, fungi and protists, produce δ-aminolevulinic acid (ALA), the common

precursor of heme biosynthesis, by condensation of glycine and succinate In contrast,

photosynthetic organisms, including plants, algae and cyanobacteria, E. cob and some other bacteria synthesize ALA from glutamate (a 5-carbon pathway) Euglena utilize

both condensation of glycine and succinate and the 5 carbon pathway to produce δ-

aminolevulinic acid T. gondii also has the ALA synthase which results in formation of

heme by condensation of glycine and succinate, as does P. falciparum (Surolia and

Padmanaban, 1992) Expression of this enzyme is developmentally regulated For example, in plants, GSAT is most abundant in the leaves There are examples in plants (Matters & Beale, 1995, Elich et al, 1988) Herbicide: A compound which kills plants or algae

Hydrolases: Enzymes which break chemical bonds (e g , amides, esters, glycosides) by

adding the elements of water

Imidazolinones: Inhibitor of acetohydroxy acid synthase (an enzyme involved in the synthesis of branched chain amino acids, a pathway not in or rarely present in animals,

Indole-3-glycerol phosphate synthase (anthranilateisomerase), (indoleglycerol

phosphate synthase): An enzyme which functions in tryptophan synthesis

Inhibitor: A compound which abrogates the effect of another compound A compound which inhibits the replication or survival of a microorganism or the

function of an enzyme or key component of a metabolic pathway or otherwise

abrogates the function of another key molecule in a microorganism or other organisms

or plant

Isocitrate lyase: An enzyme which functions in glyoxylate cycle

Isomerases: Enzymes which rearrange atoms of a molecule to form a stmctural

isomer

Isoprenoid Metabolism in Plants: Teφenes are isoprenoids that lack oxygen and are

pure hydrocarbons, 5 carbon units with some of the general properties of lipids

Giberellins and abscidic acid are others of this vast complex of compounds not found

in animals

Isoprene units (head) are CH₂ - CH3C = CH - CH₂ (tail) and are synthesized entirely from acetate of acetyl CoA and restricted to plants Synthesized by mevalonic acid

pathway because mevalonate is an important intermediate Kinases: A subclass of transferases which transfer phosphate groups, especially from

ATP

Latency: The dormant form of the parasitic infection One example is with

Toxoplasma gondii in which the infection is not active and the parasite is primarily

within cysts in the bradyzoite phase of the life cycle Another example is the hypnozoite phase of Plasmodium falciparum

Ligases or Synthetases: Enzymes which join two molecules coupled with hydrolysis

of ATP or other nucleoside triphosphate

Lipases: Enzymes which are hydrolases which hydrolyze fats (esters) Lipid and terpene synthesis associated with plant plastids Also see fatty acid synthesis and teφenes

Lysases: Enzymes which form double bonds by elimination of a chemical group

Malaria'. Disease due to pathogenic Plasmodia Examples are Plasmodium falciparum, Plasmodium virax, Plasmodium ovale, Plasmodium malaria, in humans

and Plasmodium knowlesn in monkeys

Malate synthase: An enzyme which functions in glyoxylate cycle

Metabolic pathways: Both anabolism and cataboiism consist of metabolic pathways in which an initial Compound A is converted to another B, then B is converted to C, C

to D and so on until a final product is formed In respiration, glucose is the initial

compound, and CO₂ and H₂O are the final products There are approximately 50 distinct reactions in respiration but other metabolic pathways have fewer reactions Herein the phrases "metabolic pathways" and "biochemical pathways" are used

interchangeably.

Metabolism: Chemical reactions that make life possible. Thousands of such reactions

occur constantly in each cell.

Microbes: Organisms which are visible only with use of a microscope. Some cause disease (are pathogenic).

Microbicidal: An agent (e.g., an antibiotic or antimicrobial compound) which kills

microbes.

Mitochondria: An organelle responsible for the generation of energy.

Multilamellar: An adjective which refers to the multiple membranes within an

organelle.

Noncompetitive inhibitors: Combine with enzymes at sites other than active site.

"Not involve": Are not a starting point, a component, or a product of the metabolic pathways described in relation to this invention.

NPMG: An inhibitor of EPSP synthase in the shikimate pathway.

Nucleic Acid: Deoxyribonucleic acid and ribonucleic acid molecules are constmcted of a sugar phosphate backbone and nitrogen bases; important in the encoding, transcription and synthesis of proteins.

Oocyst: A life cycle stage of a parasite, e.g., Toxoplasma gondii that contains sporozoites. T. gondii sporozoites and oocysts form only in the cat intestine. This form of the parasite is able to persist in nature in warm, moist soil for up to a year and

is highly infectious. Spomlation occurs several days after excretion of oocysts by members of the cat family (e.g., domestic cats or wild cats such as lions or tigers)

Spomlation must occur before the oocyst becomes infectious

Organelle: A stmcture within a cell Examples are plastids, mitochondria, rhoptries,

dense granules and micronemes

Oxidoreductases (oxidases, reductases, dehydrogenases): Remove and add electrons or electrons and hydrogen Oxidases transfer electrons or hydrogen to O₂ only

Paraminobenzoic acid (PABA): A product of the shikimate pathway in plants

Parasite: An organism which lives in or on a host for a period of time during at least one life-cycle stage

Phagemid: Plasmid packaged within a filamentous phage particle

Phosphoribosyl anthranilate isomerase: An enzyme which functions in tryptophan

synthesis

Plant-like: Present in algae and higher plants, but not or only rarely, or in unusual

circumstances in animals

Plasmodium falciparum: One species of Plasmodium which causes substantial human

disease.

Plasmodium knowlesii: A species of Plasmodium which causes malaria in monkeys

Plastid: A multilamellar organelle of plants, algae and Apicomplexan parasites which contains its own DNA separate from nuclear DNA Plastids have been described in

studies of Apicomplexan parasites which used electron micrographs (Siddall, 1992, Williamson et al, 1994, Wilson et al, 1991, Wilson et al, 1994, Wilson et al, 1996,

Hackstein et al. , 1995, McFadden et al , 1996)

Polymerases: Enzymes which link subunits (monomers) into a polymer such as RNA or DNA

PPi phosphofructokinase Type I : An enzyme present in plants that functions in glycolysis and in a number of organisms regulates glycolysis In plants and protozoans

PPi, not ATP (as in animals) is utilized to synthesize Fru-1-6P₂ from Fm 6P Activity is not stimulated in protozoa by Fru-2-6-P₂ (Peng & Mansour, 1992, Denton et al ,

1996a,b)

Prephenate dehydratase (phenol 2-monoxygenase): An enzyme which functions in

phenylalanine synthesis

Prephenate dehydrogenase: An enzyme which functions in tyrosine synthesis

Product: The end result of the action of an enzyme on a substrate

Prosthetic group: Smaller organic nonprotein portion of an enzyme essential for

catalytic activity Flavin is an example

Proteinases: Enzymes which are hydrolases which hydrolyze proteins (peptide bonds)

PS H: Important alternative means for producing energy within chloroplasts and apparently also described as being present in Apicomplexans

Pyrimethamine: An inhibitor of the conversion of folate to folinic acid and thus an inhibitor of nucleic acids production effective against Toxoplasma gondu.

Recrudescence: Reactivation of the parasite Toxoplasma gondii from its latent phase Respiration: Major catabolic process that releases energy in all cells It involves breakdown of sugars to CO₂ and H₂O

Ribonucleases: Enzymes which are hydrolases which hydrolyze RNA (phosphate

esters)

Salicylic Acid Metabolism in Plants: Salicylic acid is a plant hormone which promotes activity of cyanide resistant respiration

SHAM: An inhibitor of the alternative oxidase

Shikimate dehydrogenase: An enzyme which functions in chorismate synthesis

Shikimate kinase: (shikimate 3-phosphotransferase) An enzyme which functions in chorismate synthesis

Shikimate pathway A pathway that involves the conversion of shikimate to

chorismate and subsequently the production of folate, aromatic amino acids, and ubiquinone This pathway contains enzymes which lead to production of folic acid, ubiquinone, and aromatic amino acids Folate, ubiquinone, and aromatic amino acids

are products derived from this pathway in plants There is sequential use of products of these pathways as reactants in subsequent enzymatically catalyzed reactions For

example, ubiquinone is an essential coenzyme for both conventional and alternative

respiration There are examples in plants, bacteria and fungi (Bornemann e/ α/., 1995,

Marzabadi et al, 1996, Ozenberger et al, 1989, Shah et al.of 1997, Gilchrist &

Kosuge, 1980, Walsh et al , 1990, Weische & Leistemer, 1985, Green et al , 1992, Young et al, 1971) Shikimate: The substrate for EPSP synthase.

Sporozoite: Another phase of the life cycle of Toxoplasma gondii which forms within the oocyst which is produced only within the cat's intestine. A highly infectious form

of the parasite.

Stage specific: A characteristic of the parasite which is expressed or present only in a single life cycle stage or in some but not all life cycle stages.

Starch Degradation in Plants: 3 enzymes: α amylase (attack 1, 4 bonds of

amylopectin (to maltose) and amylase (to dextrin). Many activated by Ca++ Located

in chloroplasts. β amylase hydrolyzes starch to maltose; starch phosphorylase

degrades starch beginning at nonreducing end. (Starch + H2PO4 *= glucose + - Phosphate) Only partially degrades amylopectin debranching enzymes hydroxy 1.6

branch linkage in amylopectin. Hexoses cannot move out of chloroplasts or

amyloplasts thus must be converted to triose phosphate (3-PG aldehyde and dehydroxyacetone P), sucrose + UDP *— fructose + UDP-glucose, *= sucrose

synthase

Starch Formation in Plants: Animals store starch as glycogen and plants store starch

as amylose and amylopectin. Starch synthesis is dependent on starch synthase and

branching Q enzymes. Mutations in genes encoding these enzymes lead to diminished production of starch. In addition, amylopectin synthesis predominates in plant mutants

without UDP-glucose-starch glycosyl transferase whereas wild type plants with this enzyme make predominantly amylose and a smaller amount of amylopectin. In the

mutant UDP-glucose-starch glycosyl transferase appears to be transcriptionally regulated Amino acid motifs that target proteins to plant plastid organelles have been

identified in UDP-glucose starch glycosyl transferase, as have other motifs that

determine transit into plastids and mitochondria and these have been used to target the transported proteins in plants Reactions include ADPG + small amylose (in glucose) *>→larger amylose (N+l glucose units)+ADP,*= starch synthase K+ Branching or Q

enzymes form branches in amylopectins between C6 of the main chain and Cl of the

branch chain There are examples in plants (Abel et al, 1996, Van der Leif et al, 1991, Van der Steege et al, 1992)

Starch synthase: catalyzes reaction ADPG + small amylose (n-glucose units) ->

larger amylose n+l glucose units + ADP and is activated by K+ Thus, sugars not starch accumulate in plants deficient in K+

Starch: Major storage carbohydrate of plants, used for energy regeneration Two

types composed of D glucose connected by 1, 4 bonds which cause starch chains to

coil into helices. The two types are amylose and amylopectin Amylopectin is highly branched with the branches occurring between C-6 of a glucose in the main chain and C-l of the first glucose in the branch chain (-1,6 bonds) Amyloses are smaller and

have fewer branches Amylopectin becomes puφle or blue when stained with iodine-

potassium-iodine solution Amylopectin exhibits a puφle red color Control of starch formation is by K+ and a light activated sucrose phosphate synthase enzyme, invertase

enzymes and the allosteric effect of fructose 2, 6 phiphosphate adenosine

diphosphoglucose (ADPG) donates glucoses to form starch Starch in amyloplasts is a

principal respiratory substrate for storage organs Substrate reactant: Enzyme substrates have virtually identical functional groups that are capable of reacting Specificity results from enzyme substrate combinations similar to a lock and key arrangement

Substrate: The protein on which an enzyme acts that leads to the generation of a

product

Sucrose Formation Reactions in Plants: UTP+glucose 1 phosphate ^UDPG+PPi

PPi+H₂0 +2 Pi

UDPG+fructose 6 phosphate^sucrose-6-phosphate+UDP

Sucrose-6-PHOSPHATE+H₂0-sucrose+Pi UDP+ATP-*UTP+ADP

^•. glucose- 1 -phosphate+fmctose 6 phosphate+2 H₂0+ATP^sucrose 3Pi+ADP

Sulfadiazine: An antimicrobial agent effective against Toxoplasma gondii which

competes with para-aminobenzoic acid important in folate synthesis

Sulfonylureas: Inhibitors of acetohydroxy acid synthase (an enzyme involved in the

synthesis of branched chain amino acids, a pathway not or rarely present in animals),

Synergy: The effect of a plurality of inhibitors or antimicrobial agents which is greater than the additive effect would be combining effects of either used alone Synergy occurs particularly when the action of an enzyme (which is inhibited) on a substrate

leads to a product which is then the substrate for another enzyme which also is

inhibited, that is, when the enzymes are in series or follow one another in a pathway

This effect occurs because the production of the first enzymatic reaction provides less substrate for the second reaction and thus amplifies the effect of the second inhibitor or antimicrobial agent In contrast, an additive effect is when the effect of the compounds

used together is simply the sum of the effects of each inhibitory compound used alone

This most often occurs when the pathways are in parallel, for example, when the effect on the first enzyme does not modify the effect of the second enzyme

Tachyzoite: The rapidly replicating form of the parasite Toxoplasma gondii

Theileria: An Apicomplexan parasite infecting cattle

Toxoplasma gondii: A 3-5 micron, obligate, intracellular, protozoan parasite which is an Apicomplexan

Toxoplasmosis: Disease due to Toxoplasma gondii

Transit (translocation) peptide sequence: Amino acid sequence which results in transit into or out of an organelle These have been described in plants (Volkner &

Schatz, 1997, Theg & Scott, 1993) Herein we also call it a "metabolic pathway," although it is part of a component of a metabolic pathway or may function

independently of a metabolic pathway

Triazine: An inhibitor of PS II complex

Tryptophan synthase alpha subunit: An enzyme which functions in tryptophan

synthesis

Tryptophan synthase beta subunit: An enzyme which functions in tryptophan

synthesis

Type I PPi phosphofructokinase is another enzyme present in plants and there is

different substrate utilization by phosphofructokinases of animals UDP glucose starch glycosyl transferase: An enzyme involved in production of amylose in plants. The absence of this enzyme leads to starch formation as amylopectin rather than amylose.

USPA: Gene which encodes a universal stress protein. This has been described in E. Co// (Nystrom & Neidhardt, 1992).

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Claims

WE CLAIM:

1 The use of a component of a plant-like metabolic pathway in an Apicomplexan parasite, wherein the pathway does not involve the psbA gene or PPi

phosphofructokinase, is not encoded by the plastid genome, and is not generally

operative in animals, to produce a composition that interferes with the growth or

survival of the parasite

2 The use of claim 1, wherein the plant-like metabolic pathway is selected

from the group consisting of the plant-like metabolic pathway for

a) synthesis of heme from glutamate and tRN A glu by the plantlike, heme synthesis (5 carbon) pathway, b) synthesis of C4 acids by the breakdown of lipids into fatty acids

and then acetyl CoA, and their use in the glyoxylate cycle, c) synthesis of chorismate from phosphoenolpyruvate and erythrose 4 phosphate by the shikimate pathway, d) synthesis of tetrahydrofolate from chorismate by the shikimate

pathway, e) synthesis of ubiquinone from chorismate by the shikimate

pathway, f) electron transport through the alternative pathway with use of

the alternative oxidase; and g) transport of proteins into or out of an organelle through the use of a transit peptide sequence.

3. The use of claim 1, wherein the plant-like metabolic pathway is selected from the group consisting of the plant-like metabolic pathway for

a) synthesis of aromatic amino acids (phenylalanine, tyrosine and tryptophan) from chorismate by the shikimate pathway;

b) synthesis of the menaquinone, enterobactin and vitamin Kl from chorismate by the shikimate pathway; c) synthesis of the branched chain amino acids from pyruvate and

α-ketobutyrate by the plant-like branched chain amino acid synthesis pathway;

d) synthesis of the essential amino acids, not synthesized by animals and including histidine, threonine, lysine and methionine by the use of plant¬

like amino acid synthases; e) synthesis of linoleneic and linoleic acid;

f) synthesis of amylose and amylopectin with starch synthases and branching enzymes and their degradation;

g) synthesis of auxin growth regulators from indoleacetic acid

derived from chorismate; and h) synthesis of isoprenoids such as giberellins and abscidic acid by the mevalonic acid to giberellin pathway.

4 The use of claim 1 , wherein the component is selected from the group consisting of enzymes, substrates, transition states of substrates, reaction products, transit peptides, and nucleotide sequences encoding the enzymes or peptides, and

promoters

5 The use of claim 1, wherein the interfering composition is selected from the group consisting of enzyme inhibitors including enzyme competitors, substrate

inhibitors , substrate competitors, toxic analogues of substrates, transition state

analogues, products, antibodies to components of the pathway, toxin conjugated antibodies, toxin-conjugated components, antisense molecules, and inhibitors of a

transit peptide in an enzyme

6 The use of claim 1, wherein the interfering composition comprises a plurality of inhibitors

7 The use of claim 6, wherein the plurality of inhibitors exhibits a synergistic effect

8 The use of claim 6, wherein the interfering composition is selected from

the group consisting of gabaculine, 3-NPA, SHAM, 8-OH-quinoline, NPMG, gabaculine and sulfadiazine, NPMG and gabaculine, SHAM and gabaculine. pyrimethamine and NPMG, sulfadiazine and NPMG, cycloguanil and NPMG, 8-OH- quinoline and NPMG, SHAM and NPMG

9 The use of claim 1 , wherein the interfering composition acts on a latent bradyzoite form of the parasite

10 The use of claim 1, wherein the composition interferes with more than one component of the pathway

11 The use of claim 2, wherein the transit peptide sequence is

SCSFSESAASTΓKHERDGCSAATLSRERASDGRTTSRHEEEVERG or a fragment thereof

12 The use of claim 1, wherein the component of the pathway is selected

from the group consisting of an isolated nucleotide sequence or fragment thereof as shown on the top line of each row of the following a. CT CAT CTT CTC GGT TTC 17

ACT TTT CTT TGA GTG CCT GTG TGA GAG ACG GTC GTC GCA ACA AGA ATC 65

TCC TCC GCT CAC GCC TTT CCT CAC AGT CCT GTT TTT CCT CCA GCT GTC 113 ACA CAT CCC GCT CGT TCC GCT GCA TCT CCT CAC ATT TCT TGC AGT CAG 161

ATG TCT TCC TAT GGA GCC GCT CTG CGC ATA CAC ACT TTC GGT GAA TCT 209

M S S Y G A A L R I H T F G E S 16

CAC GGC TCA GCC GTT GGG TGT ATA ATC GAC GGG CTG CCT CCT CGC CTC 257

H G S A V G C I I D G L P P R L 32 CCT CTT TCT GTC GAA GAT GTT CAG CCT CAA TTA AAT CGC AGA AGA CCC 305

P L S V E D V Q P Q L N R R R P 48

GGC CAA GGG CCT CTC TCG ACG CAG CGG AGA GAG AAA GAT CGA GTC AAC 353

G Q G P L S T Q R R E K D R V 64 ATA CTC TCC GGT GTT GAA GAC GGA TAT ACA CTC GGT ACT CCC CTG GCG 401

I L S G V E D G Y T L G T P L A 80

ATG CTC GTC TGG AAT GAA GAC CGG CGG CCC CAG GAA TAC CAC GCC CTC 449

M L V N E D R R P Q E Y H A 96

GCG ACA GTC CCG CGT CCA GGT CAC GGG GAT TTC ACC TAC CAT GCA AAG 497 A T V P R P G H G D F T Y H A K 112

TAC CAC ATT CAC GCG AAA AGC GGG GGC GGT CGG AGC AGC GCG CGG GAG 545

Y H I H A K S G G G R Ξ S A R E 128

ACT TTG GCG CGC GTC GCC GCT GGA GCA GTC GTT GAG AAG TGG CTA GGC 593

T L A R V A A G A V V E K L G 144 ATG CAC TAC GGC ACC AGC TTC ACA GCT TGG GTC TGT CAG GTT GGT GAT 641

M H Y G T S F T A V C Q V G D 160

GTC TCT GTG CCC CGA TCG CTC CGA AGA AAG TGG GAG CGG CAG CCG CCA 689

V S V P R S L R R K W E R Q P P 176

ACT CGC CAA GAC GTC GAT CGC CTT GGC GTG GTC CGC GTG AGC CCA GAT 737 T R Q D V D R L G V V R V S P D 192

GGA ACC ACA TTT CTC GAC GCG AAC AAC CGC CTT TAC GAC GAG CGA GGA 785

G T T F L D A N N R L Y D E R G 208

GAG GAA CTC GTC GAG GAG GAA GAC AAA GCC AGG CGT CGG CTT CTT TTC 833

E E L V E E E D K A R R R L L F 224 GGA GTC GAC AAC CCG ACG CCA GGA GAA ACA GTG ATT GAG ACC AGG TGC 881

G V D N P T P G E T V I E T R C 240

CCG TGC CCC TCC ACA GCT GTT CGC ATG GCT GTG AAA ATC AAC CAG ACC 929

P C P S T A V R M A V K I N Q T 256

CGA TCT CTG GGC GAT TCG ATT GGC GGA TGC ATC TCC GGT GCA ATC GTG 977 R S L G D S I G G C I S G A I V 272

CGG CCA CCG CTG GGC CTC GGC GAG CCG TGT TTC GAC AAA GTG GAG GCG 1025

R P P L G L G E P C F D K V E A 288 GAG CTG GCG AAG GCG ATG ATG TCG CTC CCT GCT ACG AAA GGG TTT GAG 1073

E L A K A M M S L P A T K G F E 304

ATT GGC CAG GGC TTT GCG AGT GTC ACG TTG CGA GGC AGC GAG CAC AAC 1121

I G Q G F A S V T L R G S E H N 320 GAC CGC TTC ATT CCC TTC GAG AGA GCG TCG TGT TCA TTC TCG GAA TCA 1169

D R F I P F E R A S C S F S E S 336

GCC GCG AGC ACG ATC AAG CAT GAA AGA GAT GGG TGT TCA GCT GCT ACA 1217

A A S T I K H E R D G C S A A T 352

CTC TCA CGG GAG CGA GCG AGT GAC GGT AGA ACA ACT TCT CGA CAT GAA 1265 L S R E R A S D G R T T S R H E 368

GAG GAG GTG GAA AGG GGG CGG GAG CGC ATA CAG CGC GAT ACC CTC CAT 1313

E E V E R G R E R I Q R D T L H 384

GTT ACT GGT GTA GAT CAG CAA AAC GGC AAC TCC GAA GAT TCA GTT CGA 1361

V T G V D Q Q N G N S E D S V R 396 TAC ACT TCC AAA TCA GAG GCG TCC ATC ACA AGG CTG TCG GGA AAT GCT 1409

Y T S K S E A S I T R L S G N A 416

GCC TCT GGA GGT GCT CCA GTC TGC CGC ATT CCA CTA GGC GAG GGA GTA 1457

A S G G A P V C R I P L G E G V 432

CGG ATC AGG TGT GGA AGC AAC AAC GCT GGT GGA ACG CTC GCA GGC ATT 1505 R I R C G S N N A G G T L A G I 48

ACA TCA GGA GAG AAC ATT TTT TTT CGG GTG GCC TTC AAG CCT GTT TCT 1553

T S G E N I F F R V A F K P V S 464

TCC ATC GGC TTG GAA CAA GAA ACT GCA GAC TTT GCT GGT GAA ATG AAC 1601

S I G L E Q E T A D F A G E M N 480 CAG CTA GCT GTG AAA GGC CGC CAC GAT CCC TGC GTC CTT CCG CGA GCC 1649

Q L A V K G R H D P C V L P R A 496

CCT CCT CTG GTT GAG AGC ATG GCT GCC CTT GTG ATT GGC GAT CTG TGC 1697

P P L V E S M A A L V I G D C 512

CTC CGC CAG CGC GCC CGG GAA GGG CCG CAC CCC CTT CTC GTC CTT CCT 1745 L R Q R A R E G P H P L V L P 528

CAA CAC AGT GGT TGC CCA TCT TGC TGA GCT CTA CCT TGT TCC AAA AAC 1793

Q H S G C P S C * 536 TTG TGC ATA CGG GGT ACA CCA GGT TCC TCA CAA GGA GAA TCG TGA GGC 1841

GGT GAC TGG CCA GCG CCA CAG ATT GCT GTT CAT GCA CAA GAA AGA AAA 1889

CAG CGC ATT TCC GCC ACA ACC CAG CTG CAT GAA GTT GCT GGA TAT CGT 1937

TCC GGC GGT GCT CGG CCT TCT TCT CTA CGC TCG CGA TGA TAC GTC GCG 1985 AGC TTC ATC AAG CTC CTT TTG CAT TGT TAG TGG CTC CCA ACA GAA CCC 2033

TTT GTG GAA GGG AAT CTG GTC TCA CGC TTG CAG GAG AGA GTT CGC CTT 2081

TGT TCA CGA AAT AAC GAA GCC AAG CAG CTC AGT TGC ATT CAG CCT GCA 2129

CAC AGT TGC ATT CAG CCT GCA CAC TAA ACA CGG GCG AAA TCG TCG CGT 2177

GAT ATG TAG TTC TTC GGT TGT CAC GGT AAT TGT CGT CGT GTT TGA ACA 2225 ACT AAA CGT TTC TAA TGC TGG ATC TTA AAA AAA AAA AAA AAA AAA AAA 2273

AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 2312 and b. CT CGA GTT 8 _{τττ τττ τττ τττ τττ τττ TTG ATA CAT AAT AAT CAA GAG ττc τττ ATA 5}g CTA ACA GAC TTA TTT AAT GTA TTA TTT TTG GTA AAC AAA AAA AAC ATT 104

ATG AGC ACA TAT GGG ACT TTA TTA AAA GTA ACA TCC TAC GGA GAA AGT 152

M S T Y G T L L K V T S Y G E S 16

CAT GGG AAA GCT ATT GGG TGT GTG ATC GAT GGG TTT TTA TCC AAT ATA 200

H G K A I G C V I D G F L S N I 32 GAA ATA AAT TTT GAT TTA ATA CAA AAA CAA TTA GAT AGA CGA AGA CCA 248

E I N F D L I Q K Q D R R R P 48

AAT CAA TCA AAA CTA ACT AGT AAT AGA AAC GAA AAA GAT AAA CTT GTT 296

N Q S K L T S N R N E K D K L V 64

ATA CTT TCA GGA TTT GAT GAA AAT AAA ACA TTA GGT ACA CCT ATT ACA 344 I L S G F D E N K T G T P I T 80

TTT TTA ATA TAT AAT GAA GAT ATT AAA AAA GAA GAT TAT AAT TCT TTT 392

F L I Y N E D I K K E D Y N S F 96

ATA AAT ATT CCT AGA CCA GGA CAT GGA GAT TAT ACC TAT TTT ATG AAA 440

I N I P R P G H G D Y T Y F M K 112 TAT CAT GTT AAA AAT AAA AGT GGA AGT AGT AGA TTT TCT GGA AGA GAA 488

Y H V K N K S G S S R F S G R E 128 ACA GCC ACA AGA GTT GCT GCT GGG GCG TGC ATT GAA CAA TGG CTT AT 536

T A T R V A A G A C I E Q L Y 144

AAA TCT TAT AAT TGT TCT ATT GTT AGT TAT GTA CAT TCA GTT GGG AAT 584

K S Y N C S I V S Y V H S V G N 160 ATA AAG ATA CCT GAA CAA GTC AGC AAA GAA TTG GAA AAT AAA AAT CCA 632

I K I P E Q V S K E L E N K N P 176

CCC TCA AGA GAT TTA GTA GAT TCT TAT GGA ACC GTT AGA TAT AAT GAA 680

P S R D L V D S Y G T V R Y N E 192

AAA GAA AAA ATA TTT ATG GAT TGT TTT AAT AGA ATA TAT GAT ATG AAT 728 K E K I F M D C F N R I Y D M N 208

GCT TCT ATG TTA AAA ACT GAT GAA TAT AAT AAA AAC ACA TTG ACT ATT 776

A S M L K T D E Y N K N T L T I 224

CCT TCA ATA GAT AAC ACG TAT ATA AAT GTA AAA ACT AAT GAA TGT AAT 824

P Ξ I D N T Y I N V K T N E C N 240 ATA AAT CAG GTT GAT AAT AAT CAT AAC AAT TAT ATT AAT GAT AAG GAT 872

I N Q V D N N H N N Y I N D K D 256

AAC ACT TTT AAT AAT TCT GAA AAA TCG GAT GAA TGG ATT TAT TTA CAA 920

N T F N N S E K S D E I Y L Q 272

ACA AGA TGT CCA CAT CCA TAT ACT GCT GTA CAA ATT TGT TCT TAT ATT 968 T R C P H P Y T A V Q I C S Y I 288

TTG AAA CTA AAA AAT AAA GGA GAT AGT GTT GGG GGT ATT GCT ACA TGC 1016

L L K N K G D S V G G I A T C 304

ATT ATA CAA AAT CCT CCT ATA GGT ATT GGA GAA CCT ATT TTT GAC AAA 1064

I I Q N P P I G I G E P I F D K 320 TTG GAA GCT GAG CTA GCC AAA ATG ATT TTA TCT ATT CCA CCC GTG AAA 1112

L E A E L A K M I L S I P P V K 336

GGA ATA GAA TTC GGG AGT GGA TTT AAT GGT ACA TAT ATG TTT GGC TCA 1160

G I E F G S G F N G T Y M F G S 352

ATG CAT AAT GAT ATC TTC ATA CCT GTA GAA AAT ATG TCT ACA AAA AAA 1208 M H N D I F I P V E N M S T K K 368

GAA AGT GAT TTA TTA TAT GAT GAT AAA GGT GAA TGT AAA AAT ATG TCT 1256

E S D L Y D D K G E C K N M S 384 TAT CAT TCA ACG ATT CAA AAT AAT GAG GAT CAA ATA TTA AAT TCA ACT 1304

Y H Ξ T I Q N N E D Q I L N S T 400

AAA GGA TTT ATG CCT CCT AAA AAT GAC AAG AAT TTT AAT AAT ATT GAT 1352

K G F M P P K N D K N F N N I D 416 GAT TAC AAT GTT ACG TTT AAT AAT AAT GAA GAA AAA TTA TTA ATT ACA 1400

D Y N V T F N N N E E K L L I T 432

AAA ACA AAT AAT TGT GGT GGG ATT TTA GCT GGC ATT TCA ACA GGA AAC 1448

K T N N C G G I L A G I S T G N 448

AAT ATT GTT TTT AGA TCA GCA ATC AAA CCT GTA TCA TCA ATA CAA ATA 1496 N I V F R S A I K P V S S I Q I 464

GAA AAA GAA ACA AGT GAT TTT TAT GGA AAT ATG TGT AAC TTG AAA GTT 1544

E K E T S D F Y G N M C N L K V 480

CAA- GGG AGA CAT GAT AGC TGT ATT TTA CCA AGA TTA CCA CCC ATT ATT 1592

Q G R H D S C I L P R L P P I I 496 GAA GCA TCT TCT TCA ATG GTT ATA GGA GAT TTA ATA TTA CGA CAA ATA 1640

E A S S S M V I G D L I L R Q I 512

TCA AAG TAT GGA GAT AAA AAG TTG CCA ACA TTG TTT AGG AAT ATG TAA 1688

S K Y G D K K L P T L F R N M * 527

CAT AAT GAT TTT GTA ATC CTC AAT TAA AAT GAA AAA TTA TAA AAT ATA 1736 TAT TTT ATA TAT ATA TAT AAA ATA TAT ATA TAT ATA TAT AAA ATA TAA 1784

ATA TAT GTA TAA TAA TTC AAT TTG CGC AAT CGA TCA AAA TAC ATT TCG 1832

TCT AC 1837

13. The use of claim 1, wherein the component of the pathway is an amino

acid sequence selected from the group consisting of sequences or a fragment thereof as shown on the bottom line of each row of the following: a. CT CAT CTT CTC GGT TTC 17

ACT TTT CTT TGA GTG CCT GTG TGA GAG ACG GTC GTC GCA ACA AGA ATC 65

TCC TCC GCT CAC GCC TTT CCT CAC AGT CCT GTT TTT CCT CCA GCT GTC 113 ACA CAT CCC GCT CGT TCC GCT GCA TCT CCT CAC ATT TCT TGC AGT CAG 161 ATG TCT TCC TAT GGA GCC GCT CTG CGC ATA CAC ACT TTC GGT GAA TCT 209

M S S Y G A A R I H T F G E S 16

CAC GGC TCA GCC GTT GGG TGT ATA ATC GAC GGG CTG CCT CCT CGC CTC 257

H G S A V G C I I D G P P R L 32 CCT CTT TCT GTC GAA GAT GTT CAG CCT CAA TTA AAT CGC AGA AGA CCC 305

P L S V E D V Q P Q L N R R R P 48

GGC CAA GGG CCT CTC TCG ACG CAG CGG AGA GAG AAA GAT CGA GTC AAC 353

G Q G P L S T Q R R E K D R V N 64

ATA CTC TCC GGT GTT GAA GAC GGA TAT ACA CTC GGT ACT CCC CTG GCG 401 I L S G V E D G Y T L G T P L A 80

ATG CTC GTC TGG AAT GAA GAC CGG CGG CCC CAG GAA TAC CAC GCC CTC 449

M L V N E D R R P Q E Y H A L 96

GCG ACA GTC CCG CGT CCA GGT CAC GGG GAT TTC ACC TAC CAT GCA AAG 497

A T V P R P G H G D F T Y H A K 112 TAC CAC ATT CAC GCG AAA AGC GGG GGC GGT CGG AGC AGC GCG CGG GAG 545

Y H I H A K S G G G R S S A R E 128

ACT TTG GCG CGC GTC GCC GCT GGA GCA GTC GTT GAG AAG TGG CTA GGC 593

T L A R V A A G A V V E K W G 144

ATG CAC TAC GGC ACC AGC TTC ACA GCT TGG GTC TGT CAG GTT GGT GAT 641 M H Y G T S F T A V C Q V G D 160

GTC TCT GTG CCC CGA TCG CTC CGA AGA AAG TGG GAG CGG CAG CCG CCA 689

V S V P R S L R R K E R Q P P 176

ACT CGC CAA GAC GTC GAT CGC CTT GGC GTG GTC CGC GTG AGC CCA GAT 737

T R Q D V D R L G V V R V S P D 192 GGA ACC ACA TTT CTC GAC GCG AAC AAC CGC CTT TAC GAC GAG CGA GGA 785

G T T F L D A N K R L Y D E R G 208

GAG GAA CTC GTC GAG GAG GAA GAC AAA GCC AGG CGT CGG CTT CTT TTC 833

E E L V E E E D K A R R R L L F 224

GGA GTC GAC AAC CCG ACG CCA GGA GAA ACA GTG ATT GAG ACC AGG TGC 881 G V D N P T P G E T V I E T R C 240

CCG TGC CCC TCC ACA GCT GTT CGC ATG GCT GTG AAA ATC AAC CAG ACC 929

P C P S T A V R M A V K I N Q T 256 CGA TCT CTG GGC GAT TCG ATT GGC GGA TGC ATC TCC GGT GCA ATC GTG 977 R S L G D S I G G C I S G A I V 272 CGG CCA CCG CTG GGC CTC GGC GAG CCG TGT TTC GAC AAA GTG GAG GCG 1025 R P P L G G E P C F D K V E A 288 GAG CTG GCG AAG GCG ATG ATG TCG CTC CCT GCT ACG AAA GGG TTT GAG 1073 E L A K A M M S L P A T K G F E 304 ATT GGC CAG GGC TTT GCG AGT GTC ACG TTG CGA GGC AGC GAG CAC AAC 1121 I G Q G F A S V T L R G S E H N 320 GAC CGC TTC ATT CCC TTC GAG AGA GCG TCG TGT TCA TTC TCG GAA TCA 1169 D R F I P F E R A S C S F S E S 336 GCC GCG AGC ACG ATC AAG CAT GAA AGA GAT GGG TGT TCA GCT GCT ACA 1217

A A S T I K H E R D G C S A A T 352

CTC TCA CGG GAG CGA GCG AGT GAC GGT AGA ACA ACT TCT CGA CAT GAA 1265

L S R E R A S D G R T T S R H E 368 GAG GAG GTG GAA AGG GGG CGG GAG CGC ATA CAG CGC GAT ACC CTC CAT 1313

E E V E R G R E R I Q R D T L H 384

GTT ACT GGT GTA GAT CAG CAA AAC GGC AAC TCC GAA GAT TCA GTT CGA 1361

V T G V D Q Q N G N S E D S V R 396 TAC ACT TCC AAA TCA GAG GCG TCC ATC ACA AGG CTG TCG GGA AAT GCT 1409 Y T S K S E A S I T R L S G N A 416 GCC TCT GGA GGT GCT CCA GTC TGC CGC ATT CCA CTA GGC GAG GGA GTA 1457

A S G G A P V C R I P L G E G V 432

CGG ATC AGG TGT GGA AGC AAC AAC GCT GGT GGA ACG CTC GCA GGC ATT 1505

R I R C G S N N A G G T L A G I 448 ACA TCA GGA GAG AAC ATT TTT TTT CGG GTG GCC TTC AAG CCT GTT TCT 1553

T S G E N I F F R V A F K P V S 464

TCC ATC GGC TTG GAA CAA GAA ACT GCA GAC TTT GCT GGT GAA ATG AAC 1601

S I G E Q E T A D F A G E M N 480

CAG CTA GCT GTG AAA GGC CGC CAC GAT CCC TGC GTC CTT CCG CGA GCC 1649 Q L A V .K G R H D P C V L P R A 496

CCT CCT CTG GTT GAG AGC ATG GCT GCC CTT GTG ATT GGC GAT CTG TGC 1697

P P L V E S M A A L V I G D L C 512 CTC CGC CAG CGC GCC CGG GAA GGG CCG CAC CCC CTT CTC GTC CTT CCT 1745

L R Q R A R E G P H P L L V P 528

CAA CAC AGT GGT TGC CCA TCT TGC TGA GCT CTA CCT TGT TCC AAA AAC 1793

Q H S G C P S C * 536 TTG TGC ATA CGG GGT ACA CCA GGT TCC TCA CAA GGA GAA TCG TGA GGC 18 1

GGT GAC TGG CCA GCG CCA CAG ATT GCT GTT CAT GCA CAA GAA AGA AAA 1889

CAG CGC ATT TCC GCC ACA ACC CAG CTG CAT GAA GTT GCT GGA TAT CGT 1937

TCC GGC GGT GCT CGG CCT TCT TCT CTA CGC TCG CGA TGA TAC GTC GCG 198S

AGC TTC ATC AAG CTC CTT TTG CAT TGT TAG TGG CTC CCA ACA GAA CCC 2033 TTT GTG GAA GGG AAT CTG GTC TCA CGC TTG CAG GAG AGA GTT CGC CTT 2081

TGT TCA CGA AAT AAC GAA GCC AAG CAG CTC AGT TGC ATT CAG CCT GCA 2129

CAC AGT TGC ATT CAG CCT GCA CAC TAA ACA CGG GCG AAA TCG TCG CGT 2177

GAT ATG TAG TTC TTC GGT TGT CAC GGT AAT TGT CGT CGT GTT TGA ACA 2225

ACT AAA CGT TTC TAA TGC TGG ATC TTA AAA AAA AAA AAA AAA AAA AAA 2273 AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 2312 and b. CT CGA GTT 8 _{τττ τττ τττ τττ τττ τττ TTG ATA CAT AAT AAT CAA GAG ττc τττ ATA 5g}

CTA ACA GAC TTA TTT AAT GTA TTA TTT TTG GTA AAC AAA AAA AAC ATT 104 ATG AGC ACA TAT GGG ACT TTA TTA AAA GTA ACA TCC TAC GGA GAA AGT 152

M S T Y G T L L K V T Ξ Y G E S 16

CAT GGG AAA GCT ATT GGG TGT GTG ATC GAT GGG TTT TTA TCC AAT ATA 200

H G K A I G C V I D G F L S N I 32

GAA ATA AAT TTT GAT TTA ATA CAA AAA CAA TTA GAT AGA CGA AGA CCA 248 E I N F D I Q K Q L D R R R P 8

AAT CAA TCA AAA CTA ACT AGT AAT AGA AAC GAA AAA GAT AAA CTT GTT 296

N Q Ξ K L T S N R N E K D K L V 64

ATA CTT TCA GGA TTT GAT GAA AAT AAA ACA TTA GGT ACA CCT ATT ACA 344

I L S G F D E N K T L G T P I T 80 TTT TTA ATA TAT AAT GAA GAT ATT AAA AAA GAA GAT TAT AAT TCT TTT 392

F L I Y N E D I K K E D Y N S F 96 ATA AAT ATT CCT AGA CCA GGA CAT GGA GAT TAT ACC TAT TTT ATG AAA 440

I N I P R P G H G D Y T Y F M K 112

TAT CAT GTT AAA AAT AAA AGT GGA AGT AGT AGA TTT TCT GGA AGA GAA 488

Y H V K N K S G S S R F S G R E 128 ACA GCC ACA AGA GTT GCT GCT GGG GCG TGC ATT GAA CAA TGG CTT AT 536

T A T R V A A G A C I E Q W L Y 144

AAA TCT TAT AAT TGT TCT ATT GTT AGT TAT GTA CAT TCA GTT GGG AAT 584

K S Y N C S I V S Y V H S V G N 160

ATA AAG ATA CCT GAA CAA GTC AGC AAA GAA TTG GAA AAT AAA AAT CCA 632 I K I P E Q V S K E L E N K N P 176

CCC TCA AGA GAT TTA GTA GAT TCT TAT GGA ACC GTT AGA TAT AAT GAA 680

P S R D L V D Ξ Y G T V R Y N E 192

AAA GAA AAA ATA TTT ATG GAT TGT TTT AAT AGA ATA TAT GAT ATG AAT 728

K E K I F M D C F N R I Y D M N 208 GCT TCT ATG TTA AAA ACT GAT GAA TAT AAT AAA AAC ACA TTG ACT ATT 776

A S M L K T D E Y N K N T L T I 224

CCT TCA ATA GAT AAC ACG TAT ATA AAT GTA AAA ACT AAT GAA TGT AAT 824

P S I D N T Y I N V K T N E C N 240

ATA AAT CAG GTT GAT AAT AAT CAT AAC AAT TAT ATT AAT GAT AAG GAT 872 I N Q V D N N H N N Y I N D K D 256

AAC ACT TTT AAT AAT TCT GAA AAA TCG GAT GAA TGG ATT TAT TTA CAA 920

N T F N N S E K S D E I Y L Q 272

ACA AGA TGT CCA CAT CCA TAT ACT GCT GTA CAA ATT TGT TCT TAT ATT 968

T R C P H P Y T A V Q I C S Y I 288 TTG AAA CTA AAA AAT AAA GGA GAT AGT GTT GGG GGT ATT GCT ACA TGC 1016 K L K N K G D S V G G I A T C 304

ATT ATA CAA AAT CCT CCT ATA GGT ATT GGA GAA CCT ATT TTT GAC AAA 1064

I I Q N P P I G I G E P I F D K 320

TTG GAA GCT GAG CTA GCC AAA ATG ATT TTA TCT ATT CCA CCC GTG AAA 1112 L E A E L A K M I L S I P P V K 336

GGA ATA GAA TTC GGG AGT GGA TTT AAT GGT ACA TAT ATG TTT GGC TCA 1160

G I E F G S G F K G T Y M F G S 352 ATG CAT AAT GAT ATC TTC ATA CCT GTA GAA AAT ATG TCT ACA AAA AAA 1208

M H N D I F I P V E N M S T K K 368

GAA AGT GAT TTA TTA TAT GAT GAT AAA GGT GAA TGT AAA AAT ATG TCT 1256

E S D L Y D D K G E C K N M S 384 TAT CAT TCA ACG ATT CAA AAT AAT GAG GAT CAA ATA TTA AAT TCA ACT 1304

Y H S T I Q N N E D Q I N Ξ T 400

AAA GGA TTT ATG CCT CCT AAA AAT GAC AAG AAT TTT AAT AAT ATT GAT 1352

K G F M P P K N D K N F N N I D 416

GAT TAC AAT GTT ACG TTT AAT AAT AAT GAA GAA AAA TTA TTA ATT ACA 1400 D Y N V T F N N N E E K L L I T 432

AAA ACA AAT AAT TGT GGT GGG ATT TTA GCT GGC ATT TCA ACA GGA AAC 1448

K T N N C G G I L A G I S T G N 448

AAT ATT GTT TTT AGA TCA GCA ATC AAA CCT GTA TCA TCA ATA CAA ATA 1496

N I V F R S A I K P V S S I Q I 464 GAA AAA GAA ACA AGT GAT TTT TAT GGA AAT ATG TGT AAC TTG AAA GTT 1544

E K E T S D F Y G N M C N K V 480

CAA GGG AGA CAT GAT AGC TGT ATT TTA CCA AGA TTA CCA CCC ATT ATT 1592

Q G R H D S C I L P R L P P I I 496

GAA GCA TCT TCT TCA ATG GTT ATA GGA GAT TTA ATA TTA CGA CAA ATA 1640 E A S S S M V I G D L I L R Q 1 512

TCA AAG TAT GGA GAT AAA AAG TTG CCA ACA TTG TTT AGG AAT ATG TAA 1688

S K Y G D K K L P T L F R N M * 527

CAT AAT GAT TTT GTA ATC CTC AAT TAA AAT GAA AAA TTA TAA AAT ATA 1736

TAT TTT ATA TAT ATA TAT AAA ATA TAT ATA TAT ATA TAT AAA ATA TAA 1784 ATA TAT GTA TAA TAA TTC AAT TTG CGC AAT CGA TCA AAA TAC ATT TCG 1832

TCT AC 1837

14. A composition capable of interfering with a component of a plant-like

metabolic pathway of an Apicomplexan parasite, said component selected from the group consisting of an isolated nucleotide sequence or a fragment thereof as shown on the top line of each row of the following: a. CT CAT CTT CTC GGT TTC 17

ACT TTT CTT TGA GTG CCT GTG TGA GAG ACG GTC GTC GCA ACA AGA ATC 65 TCC TCC GCT CAC GCC TTT CCT CAC AGT CCT GTT TTT CCT CCA GCT GTC 113

ACA CAT CCC GCT CGT TCC GCT GCA TCT CCT CAC ATT TCT TGC AGT CAG 161

ATG TCT TCC TAT GGA GCC GCT CTG CGC ATA CAC ACT TTC GGT GAA TCT 209

M Ξ S Y G A A L R I H T F G E S 16

CAC GGC TCA GCC GTT GGG TGT ATA ATC GAC GGG CTG CCT CCT CGC CTC 257 H G S A V G C I I D G P P R 32

CCT CTT TCT GTC GAA GAT GTT CAG CCT CAA TTA AAT CGC AGA AGA CCC 305

P L S V E D V Q P Q N R R R P 8

GGC CAA GGG CCT CTC TCG ACG CAG CGG AGA GAG AAA GAT CGA GTC AAC 353

G Q G P S T Q R R E K D R V 64 ATA CTC TCC GGT GTT GAA GAC GGA TAT ACA CTC GGT ACT CCC CTG GCG 401

I L S G V E D G Y T L G T P A 80

ATG CTC GTC TGG AAT GAA GAC CGG CGG CCC CAG GAA TAC CAC GCC CTC 449

M L V N E D R R P Q E Y H A L 96

GCG ACA GTC CCG CGT CCA GGT CAC GGG GAT TTC ACC TAC CAT GCA AAG 497 A T V P R P G H G D F T Y H A K 112

TAC CAC ATT CAC GCG AAA AGC GGG GGC GGT CGG AGC AGC GCG CGG GAG 545

Y H I H A K S G G G R S S A R E 128

ACT TTG GCG CGC GTC GCC GCT GGA GCA GTC GTT GAG AAG TGG CTA GGC 593

T L A R V A A G A V V E K L G 144 ATG CAC TAC GGC ACC AGC TTC ACA GCT TGG GTC TGT CAG GTT GGT GAT 641

M H Y G T S F T A W V C Q V G D 160

GTC TCT GTG CCC CGA TCG CTC CGA AGA AAG TGG GAG CGG CAG CCG CCA 689

V S V P R S L R R K W E R Q P P 176

ACT CGC CAA GAC GTC GAT CGC CTT GGC GTG GTC CGC GTG AGC CCA GAT 737 T R Q D V D R L G V V R V S P D 192

GGA ACC ACA TTT CTC GAC GCG AAC AAC CGC CTT TAC GAC GAG CGA GGA 785 G T T F L D A N N R L Y D E R G 208

GAG GAA CTC GTC GAG GAG GAA GAC AAA GCC AGG CGT CGG CTT CTT TTC 833

E E L V E E E D K A R R R L L F 224

GGA GTC GAC AAC CCG ACG CCA GGA GAA ACA GTG ATT GAG ACC AGG TGC 881 G V D N P T P G E T V I E T R C 240

CCG TGC CCC TCC ACA GCT GTT CGC ATG GCT GTG AAA ATC AAC CAG ACC 929

P C P S T A V R M A V K I N Q T 256

CGA TCT CTG GGC GAT TCG ATT GGC GGA TGC ATC TCC GGT GCA ATC GTG 977

R S L G D S I G G C I S G A I V 272 CGG CCA CCG CTG GGC CTC GGC GAG CCG TGT TTC GAC AAA GTG GAG GCG 1025

R P P L G L G E P C F D K V E A 288

GAG CTG GCG AAG GCG ATG ATG TCG CTC CCT GCT ACG AAA GGG TTT GAG 1073

E L A K A M M S L P A T K G F E 304

ATT GGC CAG GGC TTT GCG AGT GTC ACG TTG CGA GGC AGC GAG CAC AAC 1121 I G Q G F A S V T L R G S E H N 320

GAC CGC TTC ATT CCC TTC GAG AGA GCG TCG TGT TCA TTC TCG GAA TCA 1169

D R F I P F E R A S C S F S E S 336

GCC GCG AGC ACG ATC AAG CAT GAA AGA GAT GGG TGT TCA GCT GCT ACA 1217

A A S T I K H E R D G C S A A T 352 CTC TCA CGG GAG CGA GCG AGT GAC GGT AGA ACA ACT TCT CGA CAT GAA 1265

L S R E R A S D G R T T S R H E 368

GAG GAG GTG GAA AGG GGG CGG GAG CGC ATA CAG CGC GAT ACC CTC CAT 1313

E E V E R G R E R I Q R D T L H 384

GTT ACT GGT GTA GAT CAG CAA AAC GGC AAC TCC GAA GAT TCA GTT CGA 1361 V T G V D Q Q N G N S E D S V R 396

TAC ACT TCC AAA TCA GAG GCG TCC ATC ACA AGG CTG TCG GGA AAT GCT 1409

Y T S K S E A S I T R S G N A 416

GCC TCT GGA GGT GCT CCA GTC TGC CGC ATT CCA CTA GGC GAG GGA GTA 1457

A S G G A P V C R I P G E G V 432 CGG ATC AGG TGT GGA AGC AAC AAC GCT GGT GGA ACG CTC GCA GGC ATT 1505

R I R C G S N N A G G T L A G I 448

ACA TCA GGA GAG AAC ATT TTT TTT CGG GTG GCC TTC AAG CCT GTT TCT 1553 T S G E N I F F R V A F K P V S 464

TCC ATC GGC TTG GAA CAA GAA ACT GCA GAC TTT GCT GGT GAA ATG AAC 1601

S I G L E Q E T A D F A G E M N 480

CAG CTA GCT GTG AAA GGC CGC CAC GAT CCC TGC GTC CTT CCG CGA GCC 1649 Q L A V K G R H D P C V L P R A 496

CCT CCT CTG GTT GAG AGC ATG GCT GCC CTT GTG ATT GGC GAT CTG TGC 1697

P P L V E S M A A L V I G D L C 512

CTC CGC CAG CGC GCC CGG GAA GGG CCG CAC CCC CTT CTC GTC CTT CCT 1745

L R Q R A R E G P H P L L V L P 528 CAA CAC AGT GGT TGC CCA TCT TGC TGA GCT CTA CCT TGT TCC AAA AAC 1793

Q H S G C P S C * 536

TTG TGC ATA CGG GGT ACA CCA GGT TCC TCA CAA GGA GAA TCG TGA GGC 1841

GGT GAC TGG CCA GCG CCA CAG ATT GCT GTT CAT GCA CAA GAA AGA AAA 1889

CAG CGC ATT TCC GCC ACA ACC CAG CTG CAT GAA GTT GCT GGA TAT CGT 1937 TCC GGC GGT GCT CGG CCT TCT TCT CTA CGC TCG CGA TGA TAC GTC GCG 1985

AGC TTC ATC AAG CTC CTT TTG CAT TGT TAG TGG CTC CCA ACA GAA CCC 2033

TTT GTG GAA GGG AAT CTG GTC TCA CGC TTG CAG GAG AGA GTT CGC CTT 2081

TGT TCA CGA AAT AAC GAA GCC AAG CAG CTC AGT TGC ATT CAG CCT GCA 2129

CAC AGT TGC ATT CAG CCT GCA CAC TAA ACA CGG GCG AAA TCG TCG CGT 2177 GAT ATG TAG TTC TTC GGT TGT CAC GGT AAT TGT CGT CGT GTT TGA ACA 2225

ACT AAA CGT TTC TAA TGC TGG ATC TTA AAA AAA AAA AAA AAA AAA AAA 2273

AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 2312 and

b. CT CGA GTT 8 _{τττ τττ τττ τττ τττ τττ TTG ATA CAT AAT AAT CAA GAG ττc τττ ATA 56}

CTA ACA GAC TTA TTT AAT GTA TTA TTT TTG GTA AAC AAA AAA AAC ATT 104

ATG AGC ACA TAT GGG ACT TTA TTA AAA GTA ACA TCC TAC GGA GAA AGT 152

M S T Y G T L K V T S Y G E S 16 CAT GGG AAA GCT ATT GGG TGT GTG ATC GAT GGG TTT TTA TCC AAT ATA 200

H G K A I G C V I D G F L S N I 32 GAA ATA AAT TTT GAT TTA ATA CAA AAA CAA TTA GAT AGA CGA AGA CCA 248

E I N F D I Q K Q L D R R R P 48

AAT CAA TCA AAA CTA ACT AGT AAT AGA AAC GAA AAA GAT AAA CTT GTT 296

N Q S K T S N R N E K D K L V 64 ATA CTT TCA GGA TTT GAT GAA AAT AAA ACA TTA GGT ACA CCT ATT ACA 344

I S G F D E N K T G T P I T 80

TTT TTA ATA TAT AAT GAA GAT ATT AAA AAA GAA GAT TAT AAT TCT TTT 392

F L I Y N E D I K K E D Y N S F 96

ATA AAT ATT CCT AGA CCA GGA CAT GGA GAT TAT ACC TAT TTT ATG AAA 440 I N I P R P G H G D Y T Y F M K 112

TAT CAT GTT AAA AAT AAA AGT GGA AGT AGT AGA TTT TCT GGA AGA GAA 488

Y H V K N K S G S Ξ R F S G R E 128

ACA GCC ACA AGA GTT GCT GCT GGG GCG TGC ATT GAA CAA TGG CTT AT 536

T A T R V A A G A C I E Q Y 144 AAA TCT TAT AAT TGT TCT ATT GTT AGT TAT GTA CAT TCA GTT GGG AAT 584

K S Y N C S I V S Y V H S V G N 160

ATA AAG ATA CCT GAA CAA GTC AGC AAA GAA TTG GAA AAT AAA AAT CCA 632

I K I P E Q V S K E L E N K N P 176

CCC TCA AGA GAT TTA GTA GAT TCT TAT GGA ACC GTT AGA TAT AAT GAA 680 P S R D L V D S Y G T V R Y N E 192

AAA GAA AAA ATA TTT ATG GAT TGT TTT AAT AGA ATA TAT GAT ATG AAT 728

K E K I F M D C F N R I Y D M N 208

GCT TCT ATG TTA AAA ACT GAT GAA TAT AAT AAA AAC ACA TTG ACT ATT 776

A S M L K T D E Y N K N T L T I 224 CCT TCA ATA GAT AAC ACG TAT ATA AAT GTA AAA ACT AAT GAA TGT AAT 824

P S I D N T Y I N V K T N E C N 240

ATA AAT CAG GTT GAT AAT AAT CAT AAC AAT TAT ATT AAT GAT AAG GAT 872

I N Q V D N N H N N Y I N D K D 256

AAC ACT TTT AAT AAT TCT GAA AAA TCG GAT GAA TGG ATT TAT TTA CAA 920 N T F N N S E K S D E W I Y L Q 272

ACA AGA TGT CCA CAT CCA TAT ACT GCT GTA CAA ATT TGT TCT TAT ATT 968

T R C P H P Y T A V Q I C S Y I 288 TTG AAA CTA AAA AAT AAA GGA GAT AGT GTT GGG GGT ATT GCT ACA TGC 1016

L K L K N K G D S V G G I A T C 304

ATT ATA CAA AAT CCT CCT ATA GGT ATT GGA GAA CCT ATT TTT GAC AAA 1064

I I Q N P P I G I G E P I F D K 320 TTG GAA GCT GAG CTA GCC AAA ATG ATT TTA TCT ATT CCA CCC GTG AAA 1112

L E A E A K M I S I P P V K 336

GGA ATA GAA TTC GGG AGT GGA TTT AAT GGT ACA TAT ATG TTT GGC TCA 1160

G I E F G S G F N G T Y M F G S 352

ATG CAT AAT GAT ATC TTC ATA CCT GTA GAA AAT ATG TCT ACA AAA AAA 1208 M H N D I F I P V E N M S T K K 368

GAA AGT GAT TTA TTA TAT GAT GAT AAA GGT GAA TGT AAA AAT ATG TCT 1256

E Ξ D L Y D D K G E C K N M S 384

TAT CAT TCA ACG ATT CAA AAT AAT GAG GAT CAA ATA TTA AAT TCA ACT 1304

Y H S T I Q N N E D Q I N S T 400 AAA GGA TTT ATG CCT CCT AAA AAT GAC AAG AAT TTT AAT AAT ATT GAT 1352

K G F M P P K N D K N F N N I D 416

GAT TAC AAT GTT ACG TTT AAT AAT AAT GAA GAA AAA TTA TTA ATT ACA 1400

D Y N V T F N N N E E K L L I T 432

AAA ACA AAT AAT TGT GGT GGG ATT TTA GCT GGC ATT TCA ACA GGA AAC 1448 K T N N C G G I L A G I S T G N 448

AAT ATT GTT TTT AGA TCA GCA ATC AAA CCT GTA TCA TCA ATA CAA ATA 1496

N I V F R S A I K P V S S I Q I 464

GAA AAA GAA ACA AGT GAT TTT TAT GGA AAT ATG TGT AAC TTG AAA GTT 1544

E K E T S D F Y G N M C N L K V 480 CAA GGG AGA CAT GAT AGC TGT ATT TTA CCA AGA TTA CCA CCC ATT ATT 1592

Q G R H D S C I L P R L P P I I 496

GAA GCA TCT TCT TCA ATG GTT ATA GGA GAT TTA ATA TTA CGA CAA ATA 1640

E A S S S M V I G D L I R Q I 512

TCA AAG TAT GGA GAT AAA AAG TTG CCA ACA TTG TTT AGG AAT ATG TAA 1688 S K Y G D K K P T L F R N M * 527

CAT AAT GAT TTT GTA ATC CTC AAT TAA AAT GAA AAA TTA TAA AAT ATA 1736

TAT TTT ATA TAT ATA TAT AAA ATA TAT ATA TAT ATA TAT AAA ATA TAA 1784 ATA TAT GTA TAA TAA TTC AAT TTG CGC AAT CGA TCA AAA TAC ATT TCG 1832 TCT AC 1837

15. A composition capable of interfering with a component of a plant-like

metabolic pathway of an Apicomplexan parasite, said component selected from an isolated amino acid sequence or a fragment thereof as shown in the bottom row of each of the following: a. CT CAT CTT CTC GGT TTC 17

ACT TTT CTT TGA GTG CCT GTG TGA GAG ACG GTC GTC GCA ACA AGA ATC 65 TCC TCC GCT CAC GCC TTT CCT CAC AGT CCT GTT TTT CCT CCA GCT GTC 113

ACA CAT CCC GCT CGT TCC GCT GCA TCT CCT CAC ATT TCT TGC AGT CAG 161

ATG TCT TCC TAT GGA GCC GCT CTG CGC ATA CAC ACT TTC GGT GAA TCT 209

M S S Y G A A L R I H T F G E S 16

CAC GGC TCA GCC GTT GGG TGT ATA ATC GAC GGG CTG CCT CCT CGC CTC 257 H G S A V G C I I D G L P P R L 32

CCT CTT TCT GTC GAA GAT GTT CAG CCT CAA TTA AAT CGC AGA AGA CCC 305

P L S V E D V Q P Q L N R R R P 48

GGC CAA GGG CCT CTC TCG ACG CAG CGG AGA GAG AAA GAT CGA GTC AAC 353

G Q G P S T Q R R E K D R V N 64 ATA CTC TCC GGT GTT GAA GAC GGA TAT ACA CTC GGT ACT CCC CTG GCG 401

I L S G V E D G Y T L G T P L A 80

ATG CTC GTC TGG AAT GAA GAC CGG CGG CCC CAG GAA TAC CAC GCC CTC 449

M L V N E D R R P Q E Y H A L 96

GCG ACA GTC CCG CGT CCA GGT CAC GGG GAT TTC ACC TAC CAT GCA AAG 497 A T V P R P G H G D F T Y H A K 112

TAC CAC ATT CAC GCG AAA AGC GGG GGC GGT CGG AGC AGC GCG CGG GAG 545

Y H I H A K S G G G R S S A R E 128

ACT TTG GCG CGC GTC GCC GCT GGA GCA GTC GTT GAG AAG TGG CTA GGC 593

T L A R V A A G A V V E K L G 144 ATG CAC TAC GGC ACC AGC TTC ACA GCT TGG GTC TGT CAG GTT GGT GAT 6 1

M H Y G T S F T A V C Q V G D 160

GTC TCT GTG CCC CGA TCG CTC CGA AGA AAG TGG GAG CGG CAG CCG CCA 689

V S V P R S L R R K E R Q P P 176 ACT CGC CAA GAC GTC GAT CGC CTT GGC GTG GTC CGC GTG AGC CCA GAT 737

T R Q D V D R L G V V R V S P D 192

GGA ACC ACA TTT CTC GAC GCG AAC AAC CGC CTT TAC GAC GAG CGA GGA 785

G T T F L D A N N R L Y D E R G 208

GAG GAA CTC GTC GAG GAG GAA GAC AAA GCC AGG CGT CGG CTT CTT TTC 833 E E L V E E E D K A R R R L F 224

GGA GTC GAC AAC CCG ACG CCA GGA GAA ACA GTG ATT GAG ACC AGG TGC 881

G V D N P T P G E T V I E T R C 240

CCG TGC CCC TCC ACA GCT GTT CGC ATG GCT GTG AAA ATC AAC CAG ACC 929

P C P S T A V R M A V K I N Q T 256 CGA TCT CTG GGC GAT TCG ATT GGC GGA TGC ATC TCC GGT GCA ATC GTG 977

R S L G D S I G G C I S G A I V 272

CGG CCA CCG CTG GGC CTC GGC GAG CCG TGT TTC GAC AAA GTG GAG GCG 1025

R P P G G E P C F D K V E A 288

GAG CTG GCG AAG GCG ATG ATG TCG CTC CCT GCT ACG AAA GGG TTT GAG 1073 E L A K A M M S L P A T K G F E 304

ATT GGC CAG GGC TTT GCG AGT GTC ACG TTG CGA GGC AGC GAG CAC AAC 1121

I G Q G F A S V T L R G S E H N 320

GAC CGC TTC ATT CCC TTC GAG AGA GCG TCG TGT TCA TTC TCG GAA TCA 1169

D R F I P F E R A S C S F S E S 336 GCC GCG AGC ACG ATC AAG CAT GAA AGA GAT GGG TGT TCA GCT GCT ACA 1217

A A S T I K H E R D G C S A A T 352

CTC TCA CGG GAG CGA GCG AGT GAC GGT AGA ACA ACT TCT CGA CAT GAA 1265

L S R E R A S D G R T T S R H E 368

GAG GAG GTG GAA AGG GGG CGG GAG CGC ATA CAG CGC GAT ACC CTC CAT 1313 E E V E R G R E R I Q R D T L H 384

GTT ACT GGT GTA GAT CAG CAA AAC GGC AAC TCC GAA GAT TCA GTT CGA 1361

V T G V D Q Q N G N S E D S V R 396 TAC ACT TCC AAA TCA GAG GCG TCC ATC ACA AGG CTG TCG GGA AAT GCT 1409

Y T S K S E A S I T R L S G N A 416

GCC TCT GGA GGT GCT CCA GTC TGC CGC ATT CCA CTA GGC GAG GGA GTA 1457

A S G G A P V C R I P L G E G V 432 CGG ATC AGG TGT GGA AGC AAC AAC GCT GGT GGA ACG CTC GCA GGC ATT 1505

R I R C G S N N A G G T L A G I 448

ACA TCA GGA GAG AAC ATT TTT TTT CGG GTG GCC TTC AAG CCT GTT TCT 1553

T S G E N I F F R V A F K P V S 464

TCC ATC GGC TTG GAA CAA GAA ACT GCA GAC TTT GCT GGT GAA ATG AAC 1601 Ξ I G L E Q E T A D F A G E M N 480

CAG CTA GCT GTG AAA GGC CGC CAC GAT CCC TGC GTC CTT CCG CGA GCC 1649

Q L A V K G R H D P C V P R A 496

CCT CCT CTG GTT GAG AGC ATG GCT GCC CTT GTG ATT GGC GAT CTG TGC 1697

P P V E S M A A V I G D L C 512 CTC CGC CAG CGC GCC CGG GAA GGG CCG CAC CCC CTT CTC GTC CTT CCT 1745 R Q R A R E G P H P L L V L P 528

CAA CAC AGT GGT TGC CCA TCT TGC TGA GCT CTA CCT TGT TCC AAA AAC 1793

Q H Ξ G C P S C ⁺ 536

TTG TGC ATA CGG GGT ACA CCA GGT TCC TCA CAA GGA GAA TCG TGA GGC 1841 GGT GAC TGG CCA GCG CCA CAG ATT GCT GTT CAT GCA CAA GAA AGA AAA 1889

CAG CGC ATT TCC GCC ACA ACC CAG CTG CAT GAA GTT GCT GGA TAT CGT 1937

TCC GGC GGT GCT CGG CCT TCT TCT CTA CGC TCG CGA TGA TAC GTC GCG 1985

AGC TTC ATC AAG CTC CTT TTG CAT TGT TAG TGG CTC CCA ACA GAA CCC 2033

TTT GTG GAA GGG AAT CTG GTC TCA CGC TTG CAG GAG AGA GTT CGC CTT 2081 TGT TCA CGA AAT AAC GAA GCC AAG CAG CTC AGT TGC ATT CAG CCT GCA 2129

CAC AGT TGC ATT CAG CCT GCA CAC TAA ACA CGG GCG AAA TCG TCG CGT 2177

GAT ATG TAG TTC TTC GGT TGT CAC GGT AAT TGT CGT CGT GTT TGA ACA 2225

ACT AAA CGT TTC TAA TGC TGG ATC TTA AAA AAA AAA AAA AAA AAA AAA 2273

AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 2312 and b. CT CGA GTT 8 _{τττ τττ τττ τττ τττ τττ TTG ATA CAT AAT AAT CAA GAG ττc τττ ATA 56}

CTA ACA GAC TTA TTT AAT GTA TTA TTT TTG GTA AAC AAA AAA AAC ATT 104

ATG AGC ACA TAT GGG ACT TTA TTA AAA GTA ACA TCC TAC GGA GAA AGT 152

M S T Y G T L K V T S Y G E S 16 CAT GGG AAA GCT ATT GGG TGT GTG ATC GAT GGG TTT TTA TCC AAT ATA 200

H G K A I G C V I D G F L S N I 32

GAA ATA AAT TTT GAT TTA ATA CAA AAA CAA TTA GAT AGA CGA AGA CCA 248

E I N F D L I Q K Q L D R R R P 48

AAT CAA TCA AAA CTA ACT AGT AAT AGA AAC GAA AAA GAT AAA CTT GTT 296 N Q S K L T S N R N E K D K L V 64

ATA CTT TCA GGA TTT GAT GAA AAT AAA ACA TTA GGT ACA CCT ATT ACA 344

I L S G F D E N K T L G T P I T 80

TTT TTA ATA TAT AAT GAA GAT ATT AAA AAA GAA GAT TAT AAT TCT TTT 392

F L I Y N E D I K K E D Y N S F 96 ATA AAT ATT CCT AGA CCA GGA CAT GGA GAT TAT ACC TAT TTT ATG AAA 440

I N I P R P G H G D Y T Y F M K 112

TAT CAT GTT AAA AAT AAA AGT GGA AGT AGT AGA TTT TCT GGA AGA GAA 488

Y H V K N K S G S S R F S G R E 128

ACA GCC ACA AGA GTT GCT GCT GGG GCG TGC ATT GAA CAA TGG CTT AT 536 T A T R V A A G A C I E Q L Y 144

AAA TCT TAT AAT TGT TCT ATT GTT AGT TAT GTA CAT TCA GTT GGG AAT 584

K S Y N C S I V S Y V H S V G N 160

ATA AAG ATA CCT GAA CAA GTC AGC AAA GAA TTG GAA AAT AAA AAT CCA 632

I K I P E Q V S K E L E N K N P 176 CCC TCA AGA GAT TTA GTA GAT TCT TAT GGA ACC GTT AGA TAT AAT GAA 680

P S R D L V D S Y G T V R Y N E 192

AAA GAA AAA ATA TTT ATG GAT TGT TTT AAT AGA ATA TAT GAT ATG AAT 728

K E K I F M D C F N R I Y D M N 208

GCT TCT ATG TTA AAA ACT GAT GAA TAT AAT AAA AAC ACA TTG ACT ATT 776 A S M K T D E Y N K N T L T I 224

CCT TCA ATA GAT AAC ACG TAT ATA AAT GTA AAA ACT AAT GAA TGT AAT 824

P S I D N T Y I N V K T N E C N 240 ATA AAT CAG GTT GAT AAT AAT CAT AAC AAT TAT ATT AAT GAT AAG GAT 872

I N Q V D N N H N N Y I N D K D 256

AAC ACT TTT AAT AAT TCT GAA AAA TCG GAT GAA TGG ATT TAT TTA CAA 920

N T F N N S E K S D E I Y L Q 272 ACA AGA TGT CCA CAT CCA TAT ACT GCT GTA CAA ATT TGT TCT TAT ATT 968

T R C P H P Y T A V Q I C S Y I 288

TTG AAA CTA AAA AAT AAA GGA GAT AGT GTT GGG GGT ATT GCT ACA TGC 1016

L K K N K G D S V G G I A T C 304

ATT ATA CAA AAT CCT CCT ATA GGT ATT GGA GAA CCT ATT TTT GAC AAA 1064 I I Q N P P I G I G E P I F D K 320

TTG GAA GCT GAG CTA GCC AAA ATG ATT TTA TCT ATT CCA CCC GTG AAA 1112

L E A E L A K M I L S I P P V K 336

GGA ATA GAA TTC GGG AGT GGA TTT AAT GGT ACA TAT ATG TTT GGC TCA 1160

G. I E F G S G F N G T Y M F G S 352 ATG CAT AAT GAT ATC TTC ATA CCT GTA GAA AAT ATG TCT ACA AAA AAA 1208

M H N D I F I P V E N M S T K K 368

GAA AGT GAT TTA TTA TAT GAT GAT AAA GGT GAA TGT AAA AAT ATG TCT 1256

E S D L L Y D D K G E C K N M S 384

TAT CAT TCA ACG ATT CAA AAT AAT GAG GAT CAA ATA TTA AAT TCA ACT 1304 Y H S T I Q N N E D Q I L N Ξ T 400

AAA GGA TTT ATG CCT CCT AAA AAT GAC AAG AAT TTT AAT AAT ATT GAT 1352

K G F M P P K N D K N F N N I D 416

GAT TAC AAT GTT ACG TTT AAT AAT AAT GAA GAA AAA TTA TTA ATT ACA 1400

D Y N V T F N N N E E K L L I T 432 AAA ACA AAT AAT TGT GGT GGG ATT TTA GCT GGC ATT TCA ACA GGA AAC 1448

K T N N C G G I L A G I S T G N 448

AAT ATT GTT TTT AGA TCA GCA ATC AAA CCT GTA TCA TCA ATA CAA ATA 1496

N I V F R S A I K P V S S I Q I 464

GAA AAA GAA ACA AGT GAT TTT TAT GGA AAT ATG TGT AAC TTG AAA GTT 1544 E K E T S D F Y G N M C N K V 480

CAA GGG AGA CAT GAT AGC TGT ATT TTA CCA AGA TTA CCA CCC ATT ATT 1592

Q G R H D S C I L P R L P P I I 496 GAA GCA TCT TCT TCA ATG GTT ATA GGA GAT TTA ATA TTA CGA CAA ATA 1640

E A S S S M V I G D L I L R Q I 512

TCA AAG TAT GGA GAT AAA AAG TTG CCA ACA TTG TTT AGG AAT ATG TAA 1688

S K Y G D K K L P T L F R N M * 527 CAT AAT GAT TTT GTA ATC CTC AAT TAA AAT GAA AAA TTA TAA AAT ATA 1736

TAT TTT ATA TAT ATA TAT AAA ATA TAT ATA TAT ATA TAT AAA ATA TAA 1784

ATA TAT GTA TAA TAA TTC AAT TTG CGC AAT CGA TCA AAA TAC ATT TCG 1832

TCT AC 1837

16 A diagnostic reagent for identifying the presence of an Apicomplexan parasite in a subject, said reagent selected from the group consisting of a component of a plant-like metabolic pathway in Apicomplexan, an antibody specific for an enzyme

that is a component of the plant-like metabolic pathway, and a nucleotide sequence that hybridizes to a nucleic acid encoding a component of the pathway

17 A diagnostic assay that detects the presence of an Apicomplexan

parasite in a biological sample, said assay comprising a selecting a diagnostic reagent from claim 16, b applying the reagent to the sample, and

c determining from the reaction between the reagent and the

sample whether the parasite is present in the sample

18 A vaccine for protecting an animal against infection by an

Apicomplexan parasite, said vaccine comprising an Apicomplexan parasite in which a gene encoding a component of a metabolic pathway in the parasite is altered, and wherein said metabolic pathway is plant-like, does not involve t e psbA gene or PPi

phosphofructokinase, is not generally operative present in animals, is not encoded in

the plastid genome and operates for the growth and survival of the parasite

19 The vaccine of claim 18, wherein the component of the pathway is operative at a particular life stage of the parasite

20 The vaccine of claim 18, wherein the altered gene is the AroC gene of an Apicomplexan.

21. The vaccine of claim 18, wherein the Apicomplexan parasite is cultivated in the presence of media that supplies a deficiency due to the altered gene

22 The vaccine of claim 21 in which products including chorismate, paba, ubiquinone and aromatic amino acids are present in the media to supply a deficiency

due to an altered AroC gene.

23 A vaccine for protecting an animal against infection by an

Apicomplexan parasite, said vaccine comprising a component of a metabolic pathway

that is plant-like and an immunogen

24. The vaccine of claim 23, wherein the component of the pathway is

operative at a particular life stage of the parasite.

25. The vaccine of claim 23, wherein the gene is the AroC gene of an Apicomplexan.

26. A method to identify a component of a plant-like pathway in an

Apicomplexan parasite said method comprising: a. selecting a metabolic pathway in a plant that i. does not include the psbA gene,

ii. does not include PPi phosphofructokinase; iii. is not encoded by the plastid genome; iv. is not generally operative in animals; and

b. determining whether the selected metabolic pathway is operative

in an Apicomplexan parasite and is necessary for the growth or survival of the parasite

27 An assay for a candidate inhibitor of a plant-like Apicomplexan

metabolic pathway, said assay comprising:

a. selecting an Apicomplexan plant-like metabolic pathway; b contacting the pathway with the candidate inhibitor; and c. determing whether the candidate inhibitor interferes with a

component of the metabolic pathway.

28. An antibody to a component of a plant-like metabolic pathway in Apicomplexan.

29. An antisense molecule directed to a component of a plant-like metabolic pathway in Apicomplexan.

30. A method for developing a lead compound that interferes with the

growth and survival of an Apicomplexan parasite, said method comprising: a. identifying a component of a plant-like metabolic pathway in an

Apicomplexan; and b. developing an inhibitior to the component.