WO2015033344A1

WO2015033344A1 - Methods and kits for inhibiting pathogenicity of group a streptococcus (gas) or group g streptococcus (ggs)

Info

Publication number: WO2015033344A1
Application number: PCT/IL2014/050795
Authority: WO
Inventors: Emanuel Hanski; Miriam RAVINS; Baruch HERTZOG; Moshe Baruch
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
Priority date: 2013-09-05
Filing date: 2014-09-04
Publication date: 2015-03-12

Abstract

A method of inhibiting pathogenicity of Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteriain a subject in need thereof is provided. The method comprising administering to the subject a therapeutically effective amount of an asparagine-reducing agent, thereby inhibiting pathogenicity of the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria.

Description

METHODS AND KITS FOR INHIBITING PATHOGENICITY OF GROUP A STREPTOCOCCUS (GAS) OR GROUP G STREPTOCOCCUS (GGS)

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to methods and kits for inhibiting pathogenicity of group A Streptococcus (GAS) or group G Streptococcus (GGS).

The group A Streptococcus (GAS) is a strict human pathogen typically infecting the throat and skin of the host, causing mild infections such as pharyngitis and impetigo. GAS can also cause highly invasive life-threatening conditions including bacteremia, necrotizing fasciitis (NF), and streptococcal toxic shock syndrome 1 ' 2. In addition, repeated infection with GAS may result in the non-suppurative sequelae of acute rheumatic fever, and acute glomerulonephritis . Annually, GAS causes an estimated 700 million cases of mild noninvasive infections worldwide, of which about 650,000 progress to severe invasive infections with an associated mortality of approximately 25% ¹. While GAS remains sensitive to penicillins, severe invasive GAS infections are often complicated to treat and may require supportive care and surgical intervention⁴.

An underexploited therapeutic opportunity in bacterial pathogenesis is the premise that the pathogen has to acquire nutrients from the host. To achieve this, bacteria adapt and respond to different nutritional cues within the various hosts' niches it faces during the infectious process. Indeed, studies from several laboratories have demonstrated that GAS regulation of metabolic genes is strongly linked to the regulation of its virulence functions [for example see 5-^"13 ] . Yet, the fact that GAS is able to directly alter host metabolism for its own benefit has not been previously reported.

Additional background art includes:

1. Cywes Bentley, C, Hakansson, A., Christianson, J. & Wessels, M.R.

Extracellular group A Streptococcus induces keratinocyte apoptosis by dysregulating calcium signalling. Cellular microbiology 7, 945-955 (2005).

2. Fontaine, M.C., Lee, J.J. & Kehoe, M.A. Combined contributions of streptolysin O and streptolysin S to virulence of serotype M5 Streptococcus pyogenes strain

Manfredo. Infection and immunity 71, 3857-3865 (2003). 3. Hakansson, A., Bentley, C.C., Shakhnovic, E.A. & Wessels, M.R. Cytolysin- dependent evasion of lysosomal killing. Proceedings of the National Academy of Sciences of the United States of America 102, 5192-5197 (2005).

4. Magassa, N., Chandrasekaran, S. & Caparon, M.G. Streptococcus pyogenes cytolysin-mediated translocation does not require pore formation by streptolysin O. EMBO Rep 11, 400-405 (2010).

5. Nizet, V. Streptococcal beta-hemolysins: genetics and role in disease pathogenesis. Trends in microbiology 10, 575-580 (2002).

6. Palmer, M. The family of thiol-activated, cholesterol-binding cytolysins. Toxicon

39, 1681-1689 (2001).

7. Datta, V., et al. Mutational analysis of the group A streptococcal operon encoding streptolysin S and its virulence role in invasive infection. Molecular microbiology 56, 681-695 (2005).

8. Melby, J.O., Nard, N.J. & Mitchell, D.A. Thiazole/oxazole-modified microcins: complex natural products from ribosomal templates. Curr Opin Chem Biol 15, 369-378 (2011).

9. Molloy, E.M., Cotter, P.D., Hill, C, Mitchell, D.A. & Ross, R.P. Streptolysin S- like virulence factors: the continuing sagA. Nat Rev Microbiol 9, 670-681 (2011).

10. Nizet, V., et al. Genetic locus for streptolysin S production by group A streptococcus. Infection and immunity 68, 4245-4254 (2000).

11. Leday, T.V., et al. TrxR, a new CovR-repressed response regulator that activates the Mga virulence regulon in group A Streptococcus. Infection and immunity 76, 4659-4668 (2008).

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of inhibiting pathogenicity of Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteriain a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an asparagine-reducing agent, thereby inhibiting pathogenicity of the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria. According to an aspect of some embodiments of the present invention there is provided a method of reducing infection of Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an asparagine-reducing agent, thereby reducing infection of the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria in the subject.

According to an aspect of some embodiments of the present invention there is provided a method of arresting growth of Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria, the method comprising reducing availability of asparagine to the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria, thereby arresting growth of the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria.

According to some embodiments of the invention, the reducing availability of asparagine to the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria is performed by contacting the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria or a host infected therewith with an effective amount of an asparagine-reducing agent.

According to some embodiments of the invention, the administering comprises topical administering.

According to some embodiments of the invention, the method is an ex vivo or in vitro method.

According to some embodiments of the invention, the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria are in a biological sample.

According to an aspect of some embodiments of the present invention there is provided an article-of-manufacture comprising in separate packaging an asparagine- reducing agent and an antibiotic agent, wherein the article-of-manufacture further comprises instructions for use in reducing infection of Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria.

According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition formulated for local administration comprising as an active ingredient an asparagine-reducing agent. According to some embodiments of the invention, the pharmaceutical composition is a topical formulation.

According to some embodiments of the invention, the pharmaceutical composition is formulated as lotion, cream, gel, ointment or spray.

According to some embodiments of the invention, the asparagine-reducing agent is selected from the group consisting of an agent which increases asparagine degradation, an agent which reduces asparagine synthesis, an agent which reduces uptake by the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria, an agent which reduces asparagine excretion, an agent which sequesters free asparagine.

According to some embodiments of the invention, the agent which increases asparagine degradation comprises an asparaginase (EC 3.5.1.1).

According to some embodiments of the invention, the subject is inflicted with- or is at risk of septic sore throat (pharyngitis), tonsillitis, impetigo, cellulitis, erysipelas, necrotizing fasciitis, sinusitis, otitis, pneumonia, meningitis, septic arthritis, osteomyelitis, vaginitis, endocarditis, myositis, bacteremia, toxic shock syndrome, scarlet fever, rheumatic fever, post streptococcal glomerulonephritis and PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders).

According to some embodiments of the invention, the subject is not inflicted with cancer.

According to some embodiments of the invention, the method further comprises administering to the subject a therapeutically effective amount of an antibiotic agent or an anti-fungal agent.

According to some embodiments of the invention, the antibiotic agent is a cytotoxic antibiotic.

According to some embodiments of the invention, the contacting is performed in vivo.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIGs. 1A-G show that sil is activated in vivo. (A) Schematic representation of the sil genes, sil core contains 3 polycistronic units: silA/B - TCS SilA/B, silE/D/CR - ABC transporter system (SilD/E), plus the autoinducer peptide SilCR, and blp bacteriocin-like peptides including BlpM. Their transcription is initiated from PI, P3, and P4 promoters, respectively. The transcript of silC is initiated from the P2 promoter. Promoters induced and non-induced by SilCR are illustrated by filled and empty flags, respectively. (B,C) sil is activated in vivo. Biopsies were taken 6 hours after subcutaneous inoculation of mice, with

or JS95 TGpP4-gfp. Tissue sections were stained and analyzed by confocal microscopy (x63), for (B) and xlO for (C). Brackets-hair follicles; empty arrows-bacteria; filled arrows -bacteria expressing GFP; triangles-muscle layer; bars=100 μιη. (D) Quantification of sil activation. Mice were inoculated with

Punch biopsies were homogenized, and relative luminescence units (RLU) were normalized to the CFUs. Each value represents the mean of two determinations conducted for each punch biopsy. The highest mean value designated as 100 % was obtained for a mouse challenged with

Horizontal lines-medians. The probabilities were calculated using Mann-Whitney U test. Two independent experiments were performed yielding similar results. (E) A change of ATA to ATG in the start codon of SilCR is sufficient for its production. The "Jump Start" assays were performed as previously described (Belotserkovsky et al. (2009). Functional analysis of the quorum- sensing streptococcal invasion locus (sil). PLoS Pathog 5, el000651). The values shown are the mean + the S.D. results of two independent experiments each performed in triplicates. (F) Schematic representation of the two assays performed to assess sil activation during mammalian cells infection with GAS and GGS. For the first one (left), GAS and GGS strains and mutants are transformed with pP4-gfp and GFP-labeled bacteria are quantified by FACS analysis (see "Experimental Procedures"). The second assay (right) is based on quantification of SilCR production using the reporter strain JS95ATApP4-gfp or its Erm-resistant derivative JS95 p_i p_isilE^~pP4-gfp . It includes culturing of the reporter strains with the tested supernatants for 2 hours in THY, which strongly amplifies the amount of GFP-labeled bacteria. In this case, the fluorescence measurements are conducted using a fluorometer and readings are normalized to the number of bacteria. This assay was mainly used when mammalian cells were infected with mutants of JS95A_TG containing 2 antibiotic resistance genes or the Erm-resistant gene. This either prevented the transformation with pP4-gfp plasmid or affected the rate of bacterial growth. (The Erm resistant reporter was used in cases where the tested strain had to be grown with Erm). (G) Two-dimensional flow cytometric FSC-SSC density-plot performed on supernatant of HeLa cells infected with JS95 TGpP4-gfp. The density plot is displayed according to the relative abundance of events, ranging from low (red), to medium (yellow), to high (green/blue). The black circle represents the gate set to detect non- aggregated GAS. Encircled are the 50,000 gated events which are above 90% of the overall number of events of the reading.

FIGs. 2A-M show that interaction of GAS with eukaryotic cells is required for sil activation. (A,B) FACS analyses of sil activation. HeLa cells were infected (MOI -1.0) with JS95 _ATGpP4-gfp (A) or JS95_ATAP^-^ (B). FACS analyses of GFP-labeled bacteria were performed at the indicated times as explained in the Examples section below and Figures 1E-G. (C) Quantification of sil activation. Infection of HeLa cells was conducted with the indicated strains (Table 2). The mean fluorescence intensities (MFI) were computed from the FACS analyses for each time point. All values represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5). (D) Contact between GAS and HeLa cells facilitates sil activation. JS95ATGpP4-gfp was added to Transwell™ chambers as indicated in the figure, sil activation was determined by FACS analyses. The values represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5). (E) Both SLS and SLO mediate sil activation. JS95A_TG and the specified mutants (Table 2) were incubated with MEF cells for the indicated time periods, sil activation was determined by quantifying the level of SilCR as explained in the Examples section below and Figure IF. The fluorescence of the GFP-labeled reporter strain was normalized to the number of the bacteria (Relative fluorescence). The values represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5). (F) Both SLS and SLO mediate sil activation in vivo. Mice were inoculated with

or JS95 ATG^ slo,sagipP4-luc. Punch biopsies were taken 6 hours after injection, homogenized, and relative luminescence units (RLU) were normalized to the CFUs. Each value represents the mean of two determinations conducted for each punch biopsy. The highest mean value designated as 100% was obtained for a mouse challenged with JS95A_TG- Horizontal lines-medians. The probabilities were calculated using Mann- Whitney U test. Two independent experiments were performed yielding similar results. (G) Intact but not lysed HeLa cells promote sil activation. Intact HeLa cells, lysed HeLa cells (see Examples section below) or DMEM medium were infected with JS95A_TGP^4- gfp. The level of sil activation at the indicated time points was determined by FACS analyses, computing the mean fluorescence of GFP-labeled bacteria. The values shown represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5). (H) The activation of sil in vitro is blocked by anti-SilCR serum. HeLa cells were infected with JS95_ATGpP4-gfp. Anti-SilCR serum (Belotserkovsky et al., 2009, supra) or control serum were added at the indicated dilutions and the level of sil activation after 8 hours of incubation was determined by FACS analyses. All values represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5). (I) Infection of MEF cells with streptococcal strains possessing naturally intact sil leads to its activation. GAS and GGS possessing naturally intact sil (Belotserkovsky et al., 2009, supra) were transformed with pP4-gfp. MEF cells were infected with the resulting mutants and incubated for 8 hours in the presence of either anti-SilCR serum or control serum (1: 100). sil activation was determined by FACS analyses, computing the mean fluorescence of GFP-labeled bacteria. The values shown are the mean of three determinations + S.D. Results are representative of two independent experiments. (J) sil activation occurs in vivo in a strain containing naturally active sil. Punch biopsies of mice challenged with WT NS 144 and its isogenic silE mutant (as a control) were taken 6 hours after injection, homogenized, and relative luminescence units (RLU) were normalized to the CFUs. Each value represents the mean of two determinations conducted for each punch biopsy. The highest mean value designated as 100 % was obtained for a mouse challenged with ^~NS l44pP4-luc. Horizontal lines-medians. The probabilities were calculated using Mann-Whitney U test. Two independent experiments were performed yielding similar results. (K) Internalization of GAS into mammalian cells is not required for sil activation. (Left panel) HeLa cells were untreated or treated with cytochalasin D (Ozeri et al., 2001, Mol Microbiol 41 , 561-573) for an hour prior to infection with JS95 TGpP4-gfp. sil activation was determined by FACS analyses, computing the mean fluorescence intensities of GFP labeled bacteria. All values represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5). (Right panel) Entry of bacteria into cells after 6 hours of incubation was quantified by the standard gentamicin protection assay as described previously (Ozeri et al., 2001). The values shown are the mean + S.D. of 2 independent experiments each performed in triplicate. (L) Either expression of SLS or SLO is sufficient to trigger sil activation. For complementation of either SLO or SLS activity, the double mutant lacking both SLO and as transformed with a shuttle vector expressing either SLS

or SLO QS95_ATG&slo,sagrpLZslo) (Table 2). MEF cells were infected with the JS95_ATG, and the indicated mutants and production of SilCR was quantified using the reporter assay (Figure IF). The fluorescence of GFP-labeled reporter was normalized to the number of the bacteria. The values shown represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5). (M) Complementation of hemolysis in the SLS and SLO double mutant by expressing SLS from a plasmid. The hemolysis of JS95ATG, JS95ATG slo,sagr, or

pLZsagl was determined as explained in the EXTENDED EXPERIMENTAL PROCEDURES. Under non-reducing conditions SLO is not active and trypan blue specifically inhibits SLS-mediated hemolysis. The values shown are the mean + S.D. of 2 independent experiments each performed in triplicate. FIGs. 3A-G show that triggering of ER stress in mammalian cells produces a conditioned medium capable of activating sil. (A) Treatment of MEF cells with staurosporine (STS). MEF cells were incubated with or without 0.1 μΜ STS. Supernatants were collected and added to

and the mixture was further incubated for 7 hours, sil activation was determined by FACS analyses, computing the mean fluorescence intensities of GFP-labeled bacteria. The values represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5). (B) Treatment of MEF cells with DTT or TG. MEF cells were incubated with 0.5 mM DTT or 1.0 μΜ TG. Then, supernatants were collected and mixed with

and sil activation was determined as in (A). The values shown represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5).

(C) STS and TG mediated activation of sil bypasses the requirement for SLO and SLS. MEF cells were infected with JS95A_TG or with the indicated mutants in the presence and absence of STS (0.1 uM) (left panel) or TG (1.0 uM) (right panel). At the indicated time points sil activation was determined by quantifying the production of SilCR as conducted in Figure 2E. The values shown represent the mean of 3 determinations + S.D. Four independent experiments (left panel) and 2 independent experiments (right panel) were performed yielding similar results (Table 5). (D) Autophagy is not involved in sil activation. Atg5^_/~ MEF cells support sil activation. Atg5^+/+ and Atg5^_/" MEF cells were infected with

AS a control

was added into DMEM medium without MEF cells. At the indicated time points, samples from the culture medium were subjected to FACS analyses. The values shown represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5). (E) Inhibitors of autophagy do not inhibit sil activation. Prior to infection MEF cells were treated for 3 hours with chloroquine (5 μΜ), or wortmannin (1 μΜ) then infected with JS95A_TGP^4- gfp. As a control,

was added to DMEM medium. At the indicated time points samples from the culture medium were subjected to FACS analyses. The values shown represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5). (F) Apoptosis is not involved in sil activation. MEF cells were pretreated with the pan-caspase inhibitor Z-VAD (100 μΜ) for 3 hours before infection with JS95_ATGpP4-gfp. AS a control JS95_ATGpP4-gfp was added to DMEM medium. At the indicated time points samples from the culture medium were subjected to FACS analyses. The values shown represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5). (G) Inhibition of TNF-a-induced necroptosis in L929 cells supports sil activation.

L929 cells were treated with TNF-a (10 ng/ml) and Z-VAD (100 μΜ) or with TNF-a plus Z-VAD (100 μΜ) and necrostatin-1 (Nec-1, 100 μΜ). Representative phase-contrast images of untreated L929 cells and cells treated with the indicated reagents (taken after 2 hours of incubation) are shown in the upper panel (40x lens ;bar = 50 μΜ). In the lower panel the culture media of L929 cells, (treated as indicated in the upper panel), were collected in the indicated times and added to JS95A_TGPP4-,? P- The mixtures were further incubated for 7 hours and sil activation was determined by FACS analyses. The values shown represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5).

FIGs. 4A-D show that GAS up-regulates asns transcription in host Cells. (A) Infection of MEF cells with GAS upregulates asns transcription. MEF cells in DMEM supplemented with 5% FCS were infected with the indicated GAS strains, or TG (1.0 μΜ) was added. At the indicated time points asns transcript level was determined by RT-RT-PCR and normalized to the transcript level of β-actin using the primers described in Table 4. The RT-RT-PCR for each sample was performed in duplicates, and the values shown represent the means + S.D. Four independent experiments were performed yielding similar results (Table 5). Inset, a zoomed image of the results obtained for MEF, MEF + JS95 TG&slo,sagr and MEF + JS95A_TG- (B) Visualization of sil activation. MEF cells infected with JS95_ATGpP4-gfp for the indicated time periods and stained with rhodamine-phalloidin (red), NucBlue™ (blue), anti-group A carbohydrate antibody (orange) and GFP-labeled bacteria (green) . Overlays are presented in the lower right panels. Bar-50 μιη. (C) TG enhances sil activation. MEF cells were incubated with TG (1.0 μΜ) and after 0.5 hour were infected with JS95 _ATGpP4-gfp. At the indicated time points staining and visualization was performed as in (B). (D) Prior to infection the DMEM medium was exchanged to DMEM supplemented either with 5 or 10% FCS. MEF cells incubated in the indicated media were infected with JS95ATGpP4-gfp. (Upper panel). At the indicated time points samples from the culture media were subjected to FACS analyses (solid line) computing the mean fluorescence intensity of GFP-labeled bacteria. The values shown represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results. Cell viability assay was performed by quantifying LDH release; maximal release represents 100% cytotoxicity (dotted line) (Examples section below). The values shown represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results. (Lower panel). The growth of

was determined by measuring OD₆oo at the indicated time points. The values shown are the mean of three determinations + S.D. Results are representative of two independent experiments.

FIGs. 5A-L show that ASN is essential for sil activation and promotes bacterial proliferation. (A) F-12 (HAM) medium supports sil activation.

was incubated for 6 hours in DMEM medium, DMEM medium supplemented with SilCR (5 ng/ml) or in F-12 (HAM) medium, sil activation was determined by FACS analyses. The values shown are the mean of three determinations + S.D. Results are representative of two independent experiments. (B) Five amino acids (5AA) are responsible for sil activation. The indicated reagents were added to DMEM medium creating final concentrations equal to those present in the F-12 (HAM) medium.

was added and the mixtures were incubated for 6 hours, sil activation was determined by FACS analyses. The values shown are the mean of three determinations + S.D. Results are representative of two independent experiments. (C) ASN is essential for sil activation, sil activation in DMEM medium, DMEM medium supplemented with: 4 amino acids (4AA) [proline (35 mg/L), aspartic acid (13 mg/L), glutamic acid (15 mg/L) and alanine (9 mg/L)]; with ASN (15 mg/L) or with 4AA plus ASN was determined by FACS analyses. All values represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5). (See Figure 5G for the growth curves under these conditions). (D) Inactivation of TrxR leads to constitutive sil activation.

and the parental JS95A_TG strain were incubated in DMEM medium or in DMEM medium supplemented with 15 mg/L of ASN. SilCR production was quantified at the indicated time points as in Figure 3C. All values represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5). (E) Plasmid curing of trxK mutants restores the dependence of sil activation on ASN. The trxK mutant was cured of the insertion inactivation plasmid (Table 3). JS95A_TG, the trxK mutant and 3 cured clones were incubated for 6 hours in DMEM medium or in DMEM medium supplemented with 15 mg/L of ASN. SilCR production was quantified as in Figure 2E. The values shown are the mean of three determinations + S.D. Results are representative of two independent experiments. (F) Contribution of ASN to JS95A_TG growth in a medium totally depleted of ASN. DMEM medium depleted of ASN (see Examples section) was supplemented with 4 AA [(see (C)] and with the indicated concentrations of ASN. OD₆oo was measured at the indicated time points. The values shown are the mean of three determinations + S.D. Results are representative of three independent experiments. (G) Growth curves of JS95A_TG in DMEM medium, DMEM medium supplemented with: 4 amino acids (4AA) [proline (35 mg/L), aspartic acid (13 mg/L), glutamic acid (15 mg/L) and alanine (9 mg/L)]; with ASN (15 mg/L) or with 4AA plus ASN were determined by measuring OD₆oo at the indicated time points. The values are the mean triplicate determinations + S.D. Results are representative of three independent experiments. (H) ASN is required for sil activation. (Left panel). DMEM medium containing 4AA (see above) was supplemented with the indicated concentrations of ASN and seeded with

At the indicated time points samples from the culture medium were subjected to FACS analyses. The values represent the mean of 3 determinations + S.D. Two independent experiments were performed yielding similar results (Table 5). (Right panel). Growth of

in DMEM medium supplemented with 0.6 and 0.45 mg/L of ASN was determined by enumerating bacteria at the indicated time points. The values shown are the mean of three determinations + S.D. Results are representative of two independent experiments. (I) The predicted structure of the surface exposed domain of TrxS resembles that of McpB. The upper PAS domain of McpB of B. subtilis (Glekas et al., 2011 Microbiology 157, 56-65) stretching from AA 35 to AA 279 was subjected to structure modeling using LOMETS, I-TASSER and Phyre servers. The predicted surface exposed domain of TrxS stretching from AA 50 to AA 289 was modeled by the same servers. The predicted structures were overlaid using the Cealign algorithm (Shindyalov and Bourne, 1998 Protein Eng 11, 739-747). (J) In vivo sil activation requires ASN. Mice were inoculated with either JS95 _A1GpP4-luc + PBS, JS95 _ATApP4-luc + PBS or JS95 _ATGpP4-luc + 200 I.U Kidrolase®. Punch biopsies were taken 6 hours after injection, homogenized, and relative luminescence units (RLU) were normalized to the CFUs. Each value represents the mean of two determinations conducted for each punch biopsy. The highest mean value designated as 100% was obtained for a mouse challenged with JS95 ATGpP4-luc. Horizontal lines-medians. The probabilities were calculated using Mann-Whitney U test. Two independent experiments were performed yielding similar results. (K) Contribution of ASN to M1T1 growth in a medium depleted of ASN. M1T1 growth in a medium depleted of ASN by ASNase treatment (see Examples section) was supplemented with 4AA and the indicated concentrations of ASN. OD₆oo readings were recorded. The values shown are the mean of three determinations + S.D. Results are representative of two independent experiments. (L) ASNase arrests GAS growth. JS95A_TG and M1T1 strains were cultured in DMEM medium depleted of ASN and supplemented with the 4AA. The ASN-depleted media was seeded with either JS95A_TG or M1T1 strains and GAS was grown in the absence or the presence of the indicated concentrations of ASNase (units/ml) for 24 hours. Control represents the growth of GAS in the presence of 15 mg/L of ASN. The amount of bacteria was determined by recording OD₆oo- The values shown are the mean of three determinations + S.D. Results are representative of two independent experiments.

FIGs. 6A-H show that depletion of ASN induces upregulation in the transcription of SLS and SLO encoding genes. (A-D) Total RNA was isolated from cultures of JS95A_TG and MGAS5005 strains grown in the absence or presence of Kidrolase^® as explained in EXPERIMENTAL PROCEDURES. For JS95_ATG, the transcription level of sagA (A), sagB (B), sagD (C), and slo (D) were determined by RT-RT-PCR. For each gene, the amount of cDNA was normalized to that of gyrA in each RNA sample. The values shown are the mean + the standard deviation of at least two independently isolated RNA preparations analyzed in duplicates. For MGAS5005 the data is derived from RNA-seq (Table 6). (E) Hypothetical model showing how GAS exploits host ASN metabolism for its own benefit. (F) Heatmap of RNA-Seq results presented in Table 6. Heatmap of RNA-seq differential expression (P<0.05) of WT MGAS5005 and its isogenic TrxR mutant. These strains were grown without (-) or with (+) Kidrolase^® and total RNA was prepared as explained above. Transcripts overexpressed (yellow) and under expressed (blue) in the absence of Kidrolase^® compared to its presence, are shown. Map was generated using Genesis v 1.7.1 software (Sturn et al., 2002, Bioinformatics 18, 207-208) and is ordered based on WT MGAS5005. (G, H) Transcription of sil genes. Total RNA from JS95A_TG was prepared as indicated in A. The amounts of silE (G) and blpM (H) transcripts were determined by RT-RT-PCR. For each gene, the amount of cDNA was normalized to that of gyrA in each RNA sample. The values shown are the mean + the standard deviation of at least two independently isolated RNA preparations analyzed in duplicates.

FIGs. 7A-D show the therapeutic effects of ASNase. (A) ASNase arrests growth in human blood. Ability of JS95A_TG to grow in non-immune human blood was quantified in the absence and presence of ASNase (Kidrolase^® 4.0 I.U ml). Bacterial growth (multiplication factor, MF) represents the increase in titer during 3 hours of incubation. The values shown are the mean + S.D. of two independent experiments, performed on blood withdrawn from two donors; each experiment was performed in duplicates. (B) ASNase protects mice against GAS bacteremia. Survival rate of mice (n= 7 per group) after intravenous inoculation with GAS strain JS95A_TG only or with additional 1 or 2 intravenous injections of ASNase (Kidrolase^® 200 I.U per mouse). The Kaplan-Meier analysis showed a significant difference in the rate of death of the group receiving GAS only compared to that receiving GAS and 2 consecutive injections of ASNase. p=0.0283, log rank (Mantel-Cox) test. P=0.015 log rank (Mantel-Cox) test was obtained for an additional separate experiment. (C) The ability of M1T1 MGAS5005 strain to grow in non-immune human blood was quantified in the absence and presence of ASNase (Kidrolase^® 4.0 I.U ml). Kidrolase^® was added to the blood together with the bacteria or at 0.5 and 1.0 hour after bacterial addition. Bacterial growth (multiplication factor, MF) represents the increase in titer during a 3 hours period. The values shown are the mean + S.D. results of two independent experiments, performed on blood withdrawn from two donors; each experiment was performed in duplicate. (D) (Left panel). S. epidermidis ATCC 12228 (Zhang et al., 2003) and GAS JS95_ATG were grown in DMEM medium supplemented with 4 AA with or without Kidrolase® (40 I.U/ml). The OD₆oo was measured at the indicated time points. The values shown are the mean of two determinations + S.D. Results are representative of three independent experiments. (Right panel). The Ability of S. epidrmidis to grow in human blood was quantified in the absence and presence of ASNase (Kidrolase® 40 I.U/ml). MF represents the decrease in titer during 2 hours of incubation. The values shown are the mean + S.D. of two independent experiments, performed on blood withdrawn from two donors; each experiment was performed in duplicates. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Therapies for GAS infection, an important public health concern are needed. An underexploited therapeutic opportunity in bacterial pathogenesis is the premise that the pathogen has to acquire nutrients from the host. To achieve this, bacteria adapt and respond to different nutritional cues within the various hosts' niches it faces during the infectious process. Indeed, studies from several laboratories have demonstrated that GAS regulation of metabolic genes is strongly linked to the regulation of its virulence functions. Yet, the fact that GAS is able to directly alter host metabolism for its own benefit has not been previously reported.

While investigating the conditions under which the quorum sensing (QS) locus sil is activated, the present inventors have found that upon adherence to mammalian cells, GAS delivers into these cells streptolysin O (SLO) and streptolysin S (SLS). The delivered toxins generate unfolded protein response (UPR) that up-regulates the expression of asparagine synthetase (ASNS) and increases the production of asparagine (ASN) in the host cell. ASN is used by GAS for sensing. ASN sensing requires the

GAS two-component system (TCS) TrxSR 24 , which consequently triggers the activation of sil. In addition, asparagine significantly increases the growth rate of GAS.

Based on these findings, and whilst further reducing the present invention to practice, the present inventors envisaged that inhibiting asparagine availability to GAS or GGS can be used as a novel therapeutic modality for inhibiting bacterial pathogenicity. Accordingly, asparginase, which is used as a chemotherapeutic agent, was found to arrest GAS growth in human blood and to block GAS proliferation in a mouse model of human bacteremia.

Collectively, these results reveal a previously unrecognized pathway through which GAS alters host metabolism for its own benefit, which can be targeted for development of effective anti-GAS treatments.

Thus, according to an aspect of the invention there is provided a method of arresting growth of Group A Streptococcus bacteria or Group G Streptococcus bacteria, the method comprising reducing availability of asparagine to the Group A Streptococcus bacteria or Group G Streptococcus bacteria, thereby arresting growth of the Group A Streptococcus bacteria or Group G Streptococcus bacteria.

Streptococcus pyogenes, or Group A streptococcus (GAS), is a facultative, Gram-positive coccus which grows in chains and causes numerous infections in humans such as pharyngitis, tonsillitis, scarlet fever, cellulitis, erysipelas, rheumatic fever, poststreptococcal glomerulonephritis, necrotizing fasciitis, myonecrosis and lymphangitis. The clinical diseases produced by GAS are well described. Sequencing of the gene encoding M-protein provides a rapid definitive way of comparing M-typeable and M- non-typeable strains of GAS.

Hence GAS strains contemplated according to the present teachings can be M- type of non-M-type.

Group G Streptococcus (GGS) are usually, but not exclusively, beta-hemolytic.

S. canis is an example of a GGS which is typically found on animals, but can cause infection in humans.

Hence the present teachings refer to human as well as veterinary applications. According to a specific embodiment the Gram-positive bacteria are Group A Streptococcus (GAS) type bacteria, also referred to as Streptococcus pyogenes.

As used herein, the phrase "arresting growth" refers to reproduction inhibition under conditions wherein asparagine is not available for the bacteria, as compared to the reproduction of the same bacterial species under the same conditions with the exception that asparagine is available (control). Growth inhibition or growth arrest is also known as a bacteristatic or cytostatic effect.

Asparagine is not available means at least one of: insufficient asparagine concentration to induce sil activation or bacterial proliferation. Growth (or proliferation) refers to growth in a host subject or ex- vivo, e.g., in a medium, corresponding to in vivo or in vitro (ex vivo) conditions, respectively.

Growth arrest can be manifested by at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or even 100% growth inhibition as compared to the same bacterial species under the same conditions with the exception that asparagine is available to allow sil activation or proliferation (control).

Depletion or deprivation of asparagine from the Group A Streptococcus bacteria or Group G Streptococcus bacterial cells can be partial or substantially complete, so long as the desired therapeutic benefit is achieved. In certain embodiments, more than about 50 % of asparagine in the serum is depleted, preferably greater than about 75 %, with depletion of more than 95% being most preferably achieved.

As mentioned the method according to this aspect of the invention, is effected by reducing the availability of asparagine to the Group A Streptococcus bacteria or Group G Streptococcus bacteria.

As the present inventors have found that asparagine is a limiting factor in vivo, withdrawal of this factor has a cytostatic effect on the bacteria.

According to a specific embodiment, reducing the availability of asparagine to the bacteria is effected by contacting the Group A Streptococcus bacteria or Group G Streptococcus bacteria or a host infected or at risk of being infected therewith with an effective amount of an asparagine-reducing agent, as further described hereinbelow.

As used herein, the term "contacting" refers to exposing the bacteria or a host infected therewith or at risk of being infected therewith, to an asparagine-reducing agent such that the agent inhibits bacterial growth.

As used herein "a host" refers to a eukaryotic host cell infected or at risk of being infected with the Group A Streptococcus bacteria or Group G Streptococcus bacteria. The host can be isolated cell(s) or tissue or a whole organism (e.g., human, animal), as further described hereinbelow.

Thus, contacting the bacteria with an asparagine-reducing agent can occur in vitro, for example, by adding the agent to a cell culture, or contacting a bacterially contaminated surface with the agent.

Alternatively contacting can occur ex vivo such as in a biological sample which may comprise eukaryotic cells. Such biological samples include, but are not limited to, body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk as well as white blood cells, malignant tissues, amniotic fluid and chorionic villi. Alternatively, the biological sample may comprise tissues (biopsies) or organs.

According to a specific embodiment, the culture is a eukaryotic cell culture which is contaminated or at risk of being contaminated with the Group A Streptococcus bacteria or Group G Streptococcus bacteria.

Alternatively, contacting can occur in vivo by administering the agent to the host. The present findings have a profound clinical significance in the treatment of bacterial infections.

Thus, according to an aspect of the invention there is provided a method of inhibiting pathogenicity of Group A Streptococcus bacteria or Group G Streptococcus bacteria in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an asparagine-reducing agent, thereby inhibiting pathogenicity of the Group A Streptococcus bacteria or Group G Streptococcus bacteria.

Alternatively or additionally, there is provided a method of reducing infection of Group A Streptococcus bacteria or Group G Streptococcus bacteria in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an asparagine-reducing agent, thereby reducing infection of the Group A Streptococcus bacteria or Group G Streptococcus bacteria in the subject.

Alternatively or additionally there is provided a method of treating a disease (e.g., medical condition, syndrome) associated with infection of Group A Streptococcus bacteria or Group G Streptococcus bacteria in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an asparagine-reducing agent, thereby treating the disease associated with infection of Group A Streptococcus bacteria or Group G Streptococcus bacteria in the subject.

As used herein, the phrase "inhibiting pathogenicity of Group A Streptococcus bacteria or Group G Streptococcus bacteria" refers to amelioration or prevention of clinical symptoms, also referred to as disease or medical condition, associated with infection by the Group A Streptococcus bacteria or Group G Streptococcus bacteria. As used herein the term "subject" or "subject in need thereof refers to an organism to be treated by the methods of the present invention. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like often referred to as veterinary use), and most preferably includes humans. In the context of the invention, the term "subject" generally refers to an individual who will receive or who has received treatment (e.g., administration of a compound of the present invention and optionally one or more other agents) for a condition characterized by bacterial infection. The subject can be of any age including preterm infants, new born, infants, children, adolescents, adults and elderly.

According to specific embodiments, the subject is inflicted with- or is at risk of septic sore throat (pharyngitis), tonsillitis, impetigo, cellulitis, erysipelas, necrotizing fasciitis, sinusitis, otitis, pneumonia, meningitis, septic arthritis, osteomyelitis, vaginitis, endocarditis, myositis, bacteremia, toxic shock syndrome, scarlet fever, rheumatic fever, post streptococcal glomerulonephritis and PANDAS (Pediatric Autoimmune Neuropsychiatry Disorders).

According to a specific embodiment, the agents are used in settings such as foreign-body, catheter or endovascular infections, chronic osteomyelitis, hospital acquired or postoperative infections, recurrent skin infections, or for bacterial infections in the immunocompromised host.

According to a specific embodiment the subject has a life threatening condition.

According to a specific embodiment, the subject is not inflicted with cancer such as malignant hematologic diseases, including lymphomas, leukemias and myelomas, e.g., acute lymphyblastic leukemia (ALL), acute non-lymphocytic leukemias, B-cell and T-cell leukemias, chronic leukemias, and acute undifferentiated leukemia.

Alternatively or additionally, the subject is not inflicted with an immune system- mediated blood diseases, e.g., infectious diseases such as those caused by HIV infection (i.e., AIDS), rheumatoid arthritis, SLE, autoimmune, collagen vascular diseases, AIDS, osteoarthritis, Issac's syndrome, psoriasis, insulin dependent diabetes mellitus, multiple sclerosis, sclerosing panencephalitis, systemic lupus erythematosus, rheumatic fever, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), primary billiary cirrhosis, chronic active hepatitis, glomerulonephritis, myasthenia gravis, pemphigus vulgaris, or Graves' disease.

As used herein the phrase "therapeutically effective amount" refers to the amount of the agent that is sufficient to cause, for example, a bacteristatic effect.

As used herein "an asparagine reducing agent" refers to an agent that reduces the amount of asparagine available to the bacteria by: increasing its degradation, reducing its production (i.e., asparagine synthesis inhibiting agent), reducing its uptake by the bacteria, reducing its excretion or by binding to it and making it less available, i.e by reducing the amount of free asparagine. The agent may also be an agent which reduces the amount of asparagine precursors such as reducing the amount of aspartate ATP, or amine source as further described hereinbelow as part of the asparagine synthesis pathway. The agent may also affect bacterial components which bind asparagine and mediate a downstream effect e.g., TrxSA. Further exemplary targets are provided hereinbelow.

The agent may be a molecule such as a small molecule, a nucleic acid (e.g., an siRNA, dsRNA, microRNA, ribozyme or antisense molecule and others as further described hereinbelow), a polypeptide (e.g., an enzyme or an antibody), a peptide, a carbohydrate or a combination of same. The agent may target a bacterial gene or a host cell gene.

An agent which increases asparagine degradation

According to a specific embodiment, the agent is asparaginase i.e. an enzyme that catalyzes the hydrolysis of asparagine to aspartic acid (EC 3.5.1.1), also referred to herein as L-ASP.

Any suitable natural or artificially constructed or modified L-ASP can be employed in the methods and materials of the present application. References to L-ASP herein refer to L-ASPs in general unless otherwise specified. Bacterial L-ASPs can be used in accordance with the materials and methods of the invention. In some embodiments, the bacterial L-ASP is E. coli L-ASP, e.g., Merck's Elspar^R™, Kidrolase^R™. Other suitable L-ASPs include those obtained from Erwinia chrysanthemi, e.g., Erwinase, Serratia marcescens, guinea pig, and Caviodea. In some embodiments, the L-ASP contains alternative or additional groups. Such groups can be selected for increasing the stability of the L-ASP. In some embodiments, the L-ASP is pegylated. Suitable L-ASP enzymes are described in Chabner et al., Cancer Chemotherapy and Biotherapy: Principles and Practice, XV, p. 879 (Philadelphia: Lippincott, Williams & Wilkins 2006). In some embodiments, the L-ASP contains alternative or additional groups. Pegylated-asparaginase is described in Hak et al., Leukemia 18: 1072-1077 (2004). Oncospar^R™ is an example of a pegylated L-ASP. A L-ASP can be employed in the invention even though it has been modified in sequence or otherwise. The L-ASP employed should retain at least partial enzymatic activity in regards to the degradation of asparagine. U.S. Patent Applications 20140099401, 20130330316, 20130209608, 20120100249, 20120100121, 20100284982, 20100183765, 20030186840 and 20030186380 provide examples of asparaginases which can be used in accordance with the present teachings, as well as methods of producing same.

Asparagine synthesis inhibitor

The precursor to asparagine is oxaloacetate. Oxaloacetate is converted to aspartate using a transaminase enzyme (EC 2.6.1). The enzyme transfers the amino group from glutamate to oxaloacetate producing a-ketoglutarate and aspartate. The enzyme asparagine synthetase (EC 6.3.5.4) produces asparagine, AMP, glutamate, and pyrophosphate from aspartate, glutamine, and ATP. In the asparagine synthetase reaction, ATP is used to activate aspartate, forming β-aspartyl-AMP. Glutamine donates an ammonium group, which reacts with β-aspartyl-AMP to form asparagine and free AMP.

Thus, any of the above mentioned enzymes or precursors can be targeted according to the teachings of some embodiments of the invention. Platform technologies for silencing expression or neutralizing the function of any of the above- mentioned enzymes/proteins/metabolites are further described hereinbelow.

U.S. Patent Application 20110229984 teaches numerous agents targeting asparagine synthesis.

Agents which reduce uptake of asparagine by the bacteria

According to some embodiments of the invention such an agent (e.g., nucleic acid or antibody) reduces expression or activity of an asparagine binding molecule such as TrxS (having an asparagine binding domain) expressed on the bacterial cell. Such platform technologies are described hereinunder. According to a specific embodiment, the agent targets a bacterial target such as TrxS, SUA, SilB, SilCR, M5005_s/ry_1359, M5005_s/ry_0743 or M5005_¾ry_0745.

Alternatively or additionally, the agent targets as eukaryotic target (i.e., of the host) e.g., Transaminase, Asparagine synthetase, Asparagine, asparaginyl-tRNA or nutrient sensing-response elements (NSRE1/2).

Thus, the below lists a number of platform technologies in line with some embodiment of the present invention that can be used to downregulate activity or expression of genes (DNA) or products of same (RNA or protein) which participate in asparagine synthesis, excretion, uptake and bacterial signaling such as described above .

One example, of an agent capable of downregulating a target (e.g., a gene or gene product of the asparagine synthesis pathway) is an antibody or antibody fragment capable of specifically binding thereto. Preferably, the antibody specifically binds at least one epitope of the target. As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.

The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages. These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Downregulation of a target (e.g., a gene or gene product of the asparagine synthesis pathway) can be also achieved by DNA or RNA silencing. As used herein, the phrase "RNA silencing" refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi. Further below described are DNA silencing agents, such as those which cleave the DNA.

As used herein, the term "RNA silencing agent" refers to an RNA which is capable of specifically inhibiting or "silencing" the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and/or expression) of an mRNA molecule through a post- transcriptional silencing mechanism. RNA silencing agents include noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated.

Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs. In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational repression.

According to an embodiment of the invention, the RNA silencing agent is specific to the target RNA (e.g., a gene product of the asparagine synthesis pathway) and does not cross inhibit or silence a gene or a splice variant which exhibits 99% or less global homology to the target gene, e.g., less than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% global homology to the target gene.

RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla. Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA.

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.

Accordingly, some embodiments of the invention contemplates use of dsRNA to downregulate protein expression from mRNA.

According to one embodiment, the dsRNA is greater than 30 bp. The use of long dsRNAs (i.e. dsRNA greater than 30 bp) has been very limited owing to the belief that these longer regions of double stranded RNA will result in the induction of the interferon and PKR response. However, the use of long dsRNAs can provide numerous advantages in that the cell can select the optimal silencing sequence alleviating the need to test numerous siRNAs; long dsRNAs will allow for silencing libraries to have less complexity than would be necessary for siRNAs; and, perhaps most importantly, long dsRNA could prevent viral escape mutations when used as therapeutics.

Various studies demonstrate that long dsRNAs can be used to silence gene expression without inducing the stress response or causing significant off-target effects - see for example [Strat et al., Nucleic Acids Research, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al. Brain Res. Protoc. 2004;13: 115-125; Diallo M., et al., Oligonucleotides. 2003;13:381-392; Paddison P.J., et al., Proc. Natl Acad. Sci. USA. 2002;99: 1443-1448; Tran N., et al., FEBS Lett. 2004;573: 127-134].

In particular, the invention according to some embodiments thereof contemplates introduction of long dsRNA (over 30 base transcripts) for gene silencing in cells where the interferon pathway is not activated (e.g. embryonic cells and oocytes) see for example Billy et al., PNAS 2001, Vol 98, pages 14428-14433. and Diallo et al, Oligonucleotides, October 1, 2003, 13(5): 381-392. doi: 10.1089/154545703322617069.

The invention according to some embodiments thereof also contemplates introduction of long dsRNA specifically designed not to induce the interferon and PKR pathways for down-regulating gene expression. For example, Shinagwa and Ishii [Genes & Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP, to express long double-strand RNA from an RNA polymerase II (Pol II) promoter. Because the transcripts from pDECAP lack both the 5'-cap structure and the 3'-poly(A) tail that facilitate ds-RNA export to the cytoplasm, long ds-RNA from pDECAP does not induce the interferon response.

Another method of evading the interferon and PKR pathways in mammalian systems is by introduction of small inhibitory RNAs (siRNAs) either via transfection or endogenous expression.

The term "siRNA" refers to small inhibitory RNA duplexes (generally between

18-30 basepairs) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21mers with a central 19 bp duplex region and symmetric 2-base 3'-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100- fold increase in potency compared with 21mers at the same location. The observed increased potency obtained using longer RNAs in triggering RNAi is theorized to result from providing Dicer with a substrate (27mer) instead of a product (21mer) and that this improves the rate or efficiency of entry of the siRNA duplex into RISC.

It has been found that position of the 3'-overhang influences potency of an siRNA and asymmetric duplexes having a 3'-overhang on the antisense strand are generally more potent than those with the 3'-overhang on the sense strand (Rose et al., 2005). This can be attributed to asymmetrical strand loading into RISC, as the opposite efficacy patterns are observed when targeting the antisense transcript.

The strands of a double-stranded interfering RNA (e.g., an siRNA) may be connected to form a hairpin or stem-loop structure (e.g., an shRNA). Thus, as mentioned the RNA silencing agent of some embodiments of the invention may also be a short hairpin RNA (shRNA). The term "shRNA", as used herein, refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11.

Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop. It will be recognized by one of skill in the art that the resulting single chain oligonucleotide forms a stem-loop or hairpin structure comprising a double-stranded region capable of interacting with the RNAi machinery.

According to another embodiment the RNA silencing agent may be a miRNA. miRNAs are small RNAs made from genes encoding primary transcripts of various sizes. They have been identified in both animals and plants. The primary transcript (termed the "pri-miRNA") is processed through various nucleolytic steps to a shorter precursor miRNA, or "pre-miRNA." The pre-miRNA is present in a folded form so that the final (mature) miRNA is present in a duplex, the two strands being referred to as the miRNA (the strand that will eventually basepair with the target) The pre-miRNA is a substrate for a form of dicer that removes the miRNA duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex. It has been demonstrated that miRNAs can be transgenically expressed and be effective through expression of a precursor form, rather than the entire primary form (Parizotto et al. (2004) Genes & Development 18:2237-2242 and Guo et al. (2005) Plant Cell 17: 1376- 1386).

Unlike, siRNAs, miRNAs bind to transcript sequences with only partial complementarity (Zeng et al., 2002, Molec. Cell 9: 1327-1333) and repress translation without affecting steady-state RNA levels (Lee et al., 1993, Cell 75:843-854; Wightman et al., 1993, Cell 75:855-862). Both miRNAs and siRNAs are processed by Dicer and associate with components of the RNA-induced silencing complex (Hutvagner et al., 2001, Science 293:834-838; Grishok et al., 2001, Cell 106: 23-34; Ketting et al., 2001, Genes Dev. 15:2654-2659; Williams et al., 2002, Proc. Natl. Acad. Sci. USA 99:6889- 6894; Hammond et al., 2001, Science 293: 1146-1150; Mourlatos et al., 2002, Genes Dev. 16:720-728). A recent report (Hutvagner et al., 2002, Sciencexpress 297:2056- 2060) hypothesizes that gene regulation through the miRNA pathway versus the siRNA pathway is determined solely by the degree of complementarity to the target transcript. It is speculated that siRNAs with only partial identity to the mRNA target will function in translational repression, similar to an miRNA, rather than triggering RNA degradation.

Synthesis of RNA silencing agents suitable for use with some embodiments of the invention can be effected as follows. First, the selected mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (wwwdotambiondotcom/techlib/tn/91/912dothtml).

Second, potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server

(wwwdotncbidotnlmdotnihdotgov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.

Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene. It will be appreciated that the RNA silencing agent of some embodiments of the invention need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.

In some embodiments, the RNA silencing agent provided herein can be functionally associated with a cell-penetrating peptide." As used herein, a "cell- penetrating peptide" is a peptide that comprises a short (about 12-30 residues) amino acid sequence or functional motif that confers the energy-independent (i.e., non- endocytotic) translocation properties associated with transport of the membrane- permeable complex across the plasma and/or nuclear membranes of a cell. The cell- penetrating peptide used in the membrane-permeable complex of some embodiments of the invention preferably comprises at least one non-functional cysteine residue, which is either free or derivatized to form a disulfide link with a double-stranded ribonucleic acid that has been modified for such linkage. Representative amino acid motifs conferring such properties are listed in U.S. Pat. No. 6,348,185, the contents of which are expressly incorporated herein by reference. The cell-penetrating peptides of some embodiments of the invention preferably include, but are not limited to, penetratin, transportan, plsl, TAT(48-60), pVEC, MTS, and MAP.

Another agent capable of downregulating a target (e.g., a gene or gene product of the asparagine synthesis pathway) is a DNAzyme molecule capable of specifically cleaving an mRNA transcript or DNA sequence of the a target (e.g., a gene or gene product of the asparagine synthesis pathway).

Downregulation of a target (e.g., a gene or gene product of the asparagine synthesis pathway) can also be performed by using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding a target (e.g., a gene or gene product of the asparagine synthesis pathway).

Another agent capable of downregulating a target (e.g., a gene or gene product of the asparagine synthesis pathway) is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding a target (e.g., a gene or gene product of the asparagine synthesis pathway). Ribozymes are being increasingly used for the sequence- specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)]. The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications.

Site-directed nucleases can be used to down-regulate bacterial gene expression (e.g., of TrxS, TrxR etc.). Such agents include that CRISPR, TALEN, zinc finger nucleases, meganucleases and the like as further described hereinbelow and find a specific use in down-regulating expression of genes in bacteria (though these systems can be applied to the silencing of gene expression in the eukaryotic host as well).

According to some embodiments, described herein is a zinc-finger protein (ZFP) that binds to target site in a region of interest (e.g., of TrxS, TrxR) in a genome, wherein the ZFP comprises one or more engineered zinc-finger binding domains. In one embodiment, the ZFP is a zinc-finger nuclease (ZFN) that cleaves a target genomic region of interest, wherein the ZFN comprises one or more engineered zinc-finger binding domains and a nuclease cleavage domain or cleavage half-domain. Cleavage domains and cleavage half domains can be obtained, for example, from various restriction endonucleases and/or homing endo nucleases. In one embodiment, the cleavage half-domains are derived from a Type IIS restriction endonuclease (e.g., Fok I). In certain embodiments, the zinc finger domain recognizes a target site e.g., of TrxS, TrxR. In certain embodiments, the zinc finger domain comprises 5 or 6 zinc finger domains and recognizes a target site in a bacterial target.

According to other embodiments, described herein is a TALE protein

(Transcription activator like) that binds to target site in a region of interest (e.g., e.g., of TrxS, TrxR) in a genome, wherein the TALE comprises one or more engineered TALE binding domains. In one embodiment, the TALE is a nuclease (TALEN) that cleaves a target genomic region of interest, wherein the TALEN comprises one or more engineered TALE DNA binding domains and a nuclease cleavage domain or cleavage half-domain. Cleavage domains and cleavage half domains can be obtained, for example, from various restriction endonucleases and/or homing endonucleases. In one embodiment, the cleavage half-domains are derived from a Type IIS restriction endonuclease (e.g., Fok I). In certain embodiments, the TALE DNA binding domain recognizes a target site in a target e.g., of TrxS, TrxR.

According to other embodiments, described herein is a CRISPR/Cas system that binds to target site in a region of interest (e.g., a highly expressed gene, a disease associated gene or a safe harbor gene) in a genome, wherein the CRISPR/Cas system comprises a CRIPSR/Cas nuclease and an engineered crRNA/tracrRNA (or single guide RNA).

The ZFNs, TALENs and/or CRISPR/Cas system as described herein may bind to and/or cleave the region of interest in a coding or non-coding region within or adjacent to the gene, such as, for example, a leader sequence, trailer sequence or intron, or within a non-transcribed region, either upstream or downstream of the coding region. In certain embodiments, the ZFNs, TALENs and/or CRISPR/Cas system binds to and/or cleave a bacterial target.

Another agent capable of downregulating a protein of interest would be any molecule which binds to and/or cleaves the protein of interest. Such molecules can be enzymes (e.g., asparaginase) antagonists, or inhibitory peptides.

It will be appreciated that a non-functional analogue of at least a catalytic or binding portion of an asparagine synthesis pathway can be also used as an asparagine - reducing agent.

The nucleic acid agents or proteins (e.g., peptide, antibody) can be provided per se. In other cases a nucleic acid sequence encoding same is ligated into a nucleic acid construct suitable for mammalian/bacterial cell expression. Such a nucleic acid construct includes a promoter sequence for directing transcription of the nucleic acid sequence in the cell in a constitutive or inducible manner.

The asparagine-reducing agent can be administered alone or simultaneously, or sequentially, with another (different) antibiotic agent.

According to a specific embodiment, the asparagine-reducing agent is administered prior to the antibiotic agent.

According to a specific embodiment, the antibiotic agent is a bactericidal agent i.e., an agent that kills bacteria.

Examples of bactericidal antibiotics that can be used in conjunction with the asparagine reducing agent include, but are not limited to, antibiotics that inhibit cell wall synthesis e.g., the beta-lactam antibiotics (e.g., penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems and vancomycin.

Also bactericidal are daptomycin, macrolides, lincosamide, fluoroquinolones, metronidazole, nitrofurantoin, co-trimoxazole, telithromycin. Aminoglycosidic antibiotics are usually considered bactericidal, although they may be bacteriostatic with some organisms.

Generally, the antibiotic is administered in a bactericidal/bactericidal amount, dependent on the mechanism of action.

Other antibiotics which can be used in conjunction with the asparagine reducing agent include, but are not limited to, quinolones (e.g., ciprofloxacin), and novobiocin.

According to a specific embodiment, the antibiotic is selected from the group consisting of as penicillin, clindamycin, erythromycin and ampicillin/sulbactam.

The asparagine reducing agent (e.g., with or without the additional antibiotic agent, e.g., as described above) of some embodiments of the invention can be administered to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the asparagine reducing agent (with or without additional active agents, e.g., antibiotics such as in a co -formulation) accountable for the biological effect.

Hereinafter, the phrases "physiologically acceptable carrier" and

"pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in

"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference. Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

According to a specific embodiment, the pharmaceutical composition is formulated for topical administration.

According to one embodiment, the pharmaceutical composition described herein (e.g., asparagine reducing agent, e.g., asparaginase) are used to treat a topical infection (i.e. infection of the skin) and are provided in a topical formulation.

According to another embodiment, the agents (e.g., asparagine reducing agent, e.g., asparaginase) are used to treat a local or systemic infection inside the body.

As mentioned the administration may be done in order to achieve a systemic effect or a local effect.

The term "tissue" refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.

Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For topical administration the compositions of some embodiments of the present invention also include a dermatologically acceptable carrier.

The phrase "dermatologically acceptable carrier", refers to a carrier which is suitable for topical application onto the skin, i.e., keratinous tissue, has good aesthetic properties, is compatible with the active agents of the present invention and any other components, and is safe and non-toxic for use in mammals.

In order to enhance the percutaneous absorption of the active ingredients, one or more of a number of agents can be added to the compositions including, but not limited to, dimethylsulfoxide, dimethylacetamide, dimethylformamide, surfactants, azone, alcohol, acetone, propylene glycol and polyethylene glycol.

The carrier utilized in the compositions of the invention can be in a wide variety of forms. These include emulsion carriers, including, but not limited to, oil-in-water, water-in-oil, water-in-oil-in-water, and oil-in-water-in-silicone emulsions, a cream, an ointment, an aqueous solution, a lotion, a soap, a paste, an emulsion, a gel, a spray or an aerosol. As will be understood by the skilled artisan, a given component will distribute primarily into either the water or oil/silicone phase, depending on the water solubility/dispersibility of the component in the composition.

Emulsions according to the present invention generally contain a pharmaceutically effective amount of an agent disclosed herein and a lipid or oil. Lipids and oils may be derived from animals, plants, or petroleum and may be natural or synthetic (i.e., man-made). Examples of suitable emulsifiers are described in, for example, U.S. Pat. No. 3,755,560, issued to Dickert, et al. Aug. 28, 1973; U.S. Pat. No. 4,421,769, issued to Dixon, et al., Dec. 20, 1983; and McCutcheon's Detergents and Emulsifiers, North American Edition, pages 317-324 (1986), each of which is fully incorporated by reference in its entirety.

The emulsion may also contain an anti-foaming agent to minimize foaming upon application to the keratinous tissue. Anti-foaming agents include high molecular weight silicones and other materials well known in the art for such use.

Suitable emulsions may have a wide range of viscosities, depending on the desired product form.

Examples of suitable carriers comprising oil-in-water emulsions are described in U.S. Pat. No. 5,073,371 to Turner, D. J. et al., issued Dec. 17, 1991, and U.S. Pat. No. 5,073,372, to Turner, D. J. et al., issued Dec. 17, 1991 each of which is fully incorporated by reference in its entirety. An especially preferred oil-in-water emulsion, containing a structuring agent, hydrophilic surfactant and water, is described in detail hereinafter.

A preferred oil-in-water emulsion comprises a structuring agent to assist in the formation of a liquid crystalline gel network structure. Without being limited by theory, it is believed that the structuring agent assists in providing rheological characteristics to the composition which contribute to the stability of the composition. The structuring agent may also function as an emulsifier or surfactant.

A wide variety of anionic surfactants are also useful herein. See, e.g., U.S. Pat. No. 3,929,678, to Laughlin et al., issued Dec. 30, 1975 which is fully incorporated by reference in its entirety. In addition, amphoteric and zwitterionic surfactants are also useful herein.

The topical compositions of the present invention can be formulated in any of a variety of forms utilized by the pharmaceutical industry for skin application including solutions, lotions, sprays, creams, ointments, salves, gels, oils, wash, etc., as described below.

For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (e.g., asparagine reducing agent) effective to prevent, alleviate or ameliorate symptoms of a bacterial infection (e.g., rheumatic fever, septic sore throat (pharyngitis), tonsillitis, toxic shock syndrome, bacteremia, pneumonia, meningitis, osteomyelitis, endocarditis, sinusitis, vaginitis, arthritis, urinary tract infection, acute glomerulonephritis, impetigo, acne, acne posacue, cellulitis, wound infection, born infection, fascitis, bronchitis, abscess, erysipelas, scarlet fever, PANDAS, post- streptococcal glomerulonephritis , nosocomial infection and opportunistic infection) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. l).

Dosage amount and interval may be adjusted individually to provide an asparagine tissue concentration which incurs a cytostatic effect to the invading bacteria (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations. Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Exemplary dose ranges for asparaginase (e.g., L- Asparaginase) include but are not limited to 400-40,000 IU/Kg, 500-30,000 IU/Kg, 1000-20,000 IU/Kg, 1000-10,000. For topical administration, of L-asparaginase, exemplary dose ranges include, but are not limited to, 4-400 IU/cm², 4-400 IU/cm², 50-400 IU/cm², 100-400 IU/cm².

Treatment can last from several days 1-6, 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, 4-5. 1- 4, 2-4, 3-4, 1-3 2-3 or 1-2, to several weeks, 1-4, 2-4, 3-4 or 1-2, 2-3.

Alternatively or additionally, treatment may be terminated and resumed at a later stage as needed.

Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient (e.g., asparagine reducing agent with or without an antibiotic).

Also provided is an article-of-manufacture or kit comprising in separate packaging an asparagine -reducing agent and an antibiotic agent, wherein the article-of- manufacture further comprise instructions for use in reducing infection of Group A Streptococcus bacteria or Group G Streptococcus bacteria.

The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

The agents of the invention may be further adsorbed, attached, immobilized or included in medical devices, such as patches, stents, catheters and the like.

As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to".

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference. MATERIALS AND EXPERIMENTAL PROCEDURES Materials

The reagents: cytochalasin D (Sigma), staurosporine (STS) (Sigma), chloroquine (Sigma), wortmannin (Sigma), benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z- VAD) (R&D systems), TNFa (Peprotech), necrostatin-1 (Nec-1) (Sigma), thapsigargin (TG) (Sigma) and dithiothreitol (DTT) (Sigma) per se did not exerted any effect on GAS growth or sil activation at the used concentrations. All other reagents were of the highest purity available.

Eukar otic cells

The human HeLa epithelial cell line (HeLa ATCC^® Catalog No. CCL-2™), the mouse embryonic fibroblasts (MEF), and Atg5 ⁷ MEF, the mouse subcutaneous fibroblast L cell line clone 929 (ATCC^® Catalog No. CCL-1™) and the mouse leukemic monocytes/macrophages Raw 264.7 cells (ATCC^® Catalog No. TIB-71™) were cultured in Dulbecco's Modified Eagles Medium (DMEM, Sigma) containing with 10% (v/v) fetal calf serum (FCS) (termed here DMEM medium) (Biological Industries). The Lung alveolar adenocarcinoma A549 cells (ATCC^® Catalog No. CCL-185™) were cultured in Ham's F-12 medium (Biological Industries) supplemented with 10 % FCS (v/v). All cell lines were grown at 37 °C in an atmosphere containing 5 % C0₂.

HeLa Lysates

HeLa cells were cultured in 24-well plate in DMEM medium. The cells were wash with cold PBS, scraped into a fresh cold DMEM medium and adjusted to 2.5 x 10⁵ cells/ml. Lysis was performed on ice by two cycles of sonication each of 15 seconds, using the exponential probe of Soniprep 150 Plus (MSE); complete lysis was verified by microscopic visualization.

LDH activity assay

MEF cells were grown in DMEM medium that lacks phenol red indicator. Cells were infected with

as described in the text. At desired time points 0.5ml supernatant were withdrawn and the LDH activity was determined using CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) according to manufacturer's instructions.

Bacterial strains and culturing (The bacterial strains used in this study are listed in Table 1) For cloning, Escherichia coli strains JM109 and SCSI 10 were used, which were cultured in Laria-Bertani broth (LB), Lennox (Becton, Dickinson, and Sparks) at 37 °C with agitation. GAS and GGS were cultured either in THY or in DMEM containing 10 % FCS and various combinations of the following amino acids (AA) (proline 35 mg/L, aspartic acid 13 mg/L, glutamic acid 15 mg/L, alanine 9 mg/L and ASN 15 mg/L), at 37 °C in sealed tubes, or 24-well plates without agitation at 37 °C in 5 % C0₂ incubator. To produce solid LB and THY, Bacto™ Agar (Becton, Dickinson) was added to a final concentration of 1.4 %. When necessary, antibiotics were added at the following concentrations: for GAS and GGS: 250 μg/ml kanamycin (Km), 50 μg/ml spectinomycin (Spec) and 1 μg/ml erythromycin (Erm); for E. coli: 100 μg/ml ampicillin (Amp), 50 μg/ml Spec, 750 μg/ml Erm and 50 μg/ml Km. All the antibiotics were purchased from Sigma-Aldrich.

Construction of bacterial mutants

The vectors and plasmid used for construction are presented in Table 2 and the primers used are listed in Table 3.

Construction of JS95_AT_G

To change the ATA start codon of silCR in JS95_ATA to ATG, a DNA segment containing silCR as well as sequences up and down-stream were PCR amplified with the primers KK-Cl and KK-Dl using genomic DNA of NS35, a GAS strain containing silCR with an ATG start codon, as a template. The 1562 bp PCR fragment was cloned by AT cloning into pGEM-T-Easy (Promega), yielding the plasmid PGKKC I-DI_ATG- The insert was released by a digestion with Ncol, followed by end blunting using DNA polymerase I, Large (Klenow) fragment (New England Biolabs), and a second digestion with Pstl. This insert was then cloned into a PstI/Eco ?V digested pJRS233, a temperature-sensitive shuttle plasmid [Perez-Casal, supra], to generate PJKKCI-DI_ATG- The resulting plasmid was transformed into JS95A5z7C [Hidalgo-Grass, C, et al. A locus of group A Streptococcus involved in invasive disease and DNA transfer. Mol Microbiol 46, 87-99 (2002)] and clones that lost antibiotic resistance to both Spec and Erm, were selected as previously described [Perez-Casal, J., Caparon, M.G. & Scott, J.R. Mry, a trans-acting positive regulator of the M protein gene of Streptococcus pyogenes with similarity to the receptor proteins of two-component regulatory systems. Bacteriol 173, 2617-2624 (1991)] . The presence of silCR with an ATG starting codon in the resulting mutant was confirmed by sequencing of a KK-C1- KK-D1 PCR fragment.

Construction of JS95 _{A G}A.silAB and JS95_ATG S E^'

The

mutant was constructed using pJsilAB-Ω,Κιη as previously described [Belotserkovsky, I., et al. Functional analysis of the quorum- sensing streptococcal invasion locus (sil). PLoS Pathog 5, el000651 (2009)] . The JS95_ATG*^E^~ mutant was constructed using pJsilE as previously described [Eran, Y., et al. Transcriptional regulation of the sil locus by the SilCR signalling peptide and its implications on group A streptococcus virulence. Mol Microbiol 63, 1209-1222 (2007)] .

Construction of ])P4-luc

The Luciferase encoding gene (luc) was PCR amplified with the primers LucRBS-F and Luc-R using pGL3 plasmid (Promega) as a template (see Table 4). The resulting 1672 bp fragment was AT cloned into pGEM-T-Easy, released by digestion with EcoRI and cloned into pP4-gfp¹ from which the gfp gene was removed by EcoRI digestion.

Construction of JS95_ATGtrjc/?^"

Insertion inactivation of trxR in JS95ATG was performed using p233- 10R as previously described [Leday, T.V., et al. TrxR, a new CovR-repressed response regulator that activates the Mga virulence regulon in group A Streptococcus. Infect Immun 76, 4659-4668 (2008)] .

Construction of JS95_ATGJS/O

A 1639 bp fragment containing most of the slo gene was PCR amplified with the primers SLO-F-Bam and SLO-R-Hindlll, using JS95ATG genomic DNA as a template (see Primer Table 4). The resulting fragment was AT cloned to pGEM-T-easy and transformed into the methylation deficient E. coli strain SCS I 10. The resulting plasmid, pGslo was then purified and a 528 bp portion from inside the slo was excised by digestion with SexAI, followed by end blunting, and digestion with EcoRV. Ω,Κιη cassette [Perez-Casal, J., Caparon, M.G. & Scott, J.R. Mry, a trans-acting positive regulator of the M protein gene of Streptococcus pyogenes with similarity to the receptor proteins of two-component regulatory systems. J Bacteriol 173, 2617-2624 (1991)] was ligated into the blunt ends resulting in the plasmid, pGslo, Km, that was subsequently digested with Notl and Ncol to release a fragment containing the Ω,Κιη flanked by sequences of 5' and 3' regions of slo. This 3.3 kb fragment was cloned into EcoRV digested pJRS233 generating pJslo, Km, which was transformed into JS95ATG- Mutants which underwent through double recombination events were selected as previously described [Perez-Casal supra] . The resulting Aslo mutants failed to produce detectable SLS activity as determined by Western bolting using anti-Streptolysin O antibody (Bio Academia).

Construction of JS95_ATGsag/~ and JS95 _{A G}A.slo,s gF

A 333 bp fragment of the sagl gene was PCR amplified using the primers Sagl- Fwd and Sagl-Rev (Nizet, V., et al. Genetic locus for streptolysin S production by group A streptococcus. Infect Immun 68, 4245-4254 (2000) and primer Table 4) and JS95 genomic DNA as a template. The fragment was AT cloned into the plasmid pJRS233-T prepared by EcoRV digestion and T-tailing by terminal transferase as described before [Zhou, M.Y. & Gomez-Sanchez, C.E. Universal TA cloning. Current issues in molecular biology 2, 1-7 (2000)] . The resulting plasmid, pJsagl, was transformed into JS95ATG and

and clones resistant to Erm were selected as previously described [Perez-Casal supra] , sagl mutants failed to produce detectable SLS activity as determined by loss of β-hemolysis on blood agar plates.

Construction of the complementation mutant

A 1735 bp fragment containing the complete sagl sequence and the sag operon terminator was PCR amplified with the primers sagHI-Fwd and sagDown-Rev (Nizet supra, and Primer Table 4) using JS95 genomic DNA as a template. The fragment was AT cloned to pGEM-T-easy yielding the plasmid pGsagI A 1229 bp fragment containing the sag promoter, sagA and the rho-independent terminator sequence downstream of sagA⁹ was PCR amplified with the primers sagup-Fwd-Apa and sagAB- Rev-SacII (Nizet supra and Primer Table 4) using M1T1 (Table 1) genomic DNA as a template (these primers did not yield a clear PCR product when a genomic DNA of JS95ATG was used). The PCR product was digested with Apal and SacII and cloned upstream to sagl in pGsagI digested with the same enzymes. The resulting plasmid, pGsagAsagI, was digested with Apal and PstI to release the entire cloned fragment which was subsequently cloned into Apal/Pstl digested PLZ12-Spec generating pLZsagl. pLZsagl was introduced into

by electroporation and transformants resistant to Spec were selected. Complementation was verified by the presence of β-hemolytic transformants on blood agar plates.

Construction of the complementation mutant JS95_Aj_GAslo,sagI-])LZslo

A 1740 bp fragment containing the complete slo sequence was PCR amplified with the primers SLO-F and SLO-R-Bam using JS95A_TG genomic DNA as a template. The fragment was AT cloned to pGEM-T-easy yielding the plasmid pGEMs/o. A 2203 bp fragment containing the upstream region of slo, which includes the gene encoding NAD-glycohydrolase (nga) and its promoter [Savic, D.J., McShan, W.M. & Ferretti, J.J. Autonomous expression of the slo gene of the bicistronic nga-slo operon of Streptococcus pyogenes. Infect Immun 70, 2730-2733 (2002)], was PCR amplified with the primers SLO-up-F-Ncol and SLO-up-R-SacII, using JS95A_TG genomic DNA as a template. The PCR product was digested with Ncol and SacII and cloned into pGEMs/o digested with the same enzymes. The resulting plasmid, pGsloup,slo was digested with Apal and BamHI to release a fragment containing the slo gene and its upstream region, and the latter fragment was cloned into ApaJVBamHI digested PLZ12-Spec generating phZslo. phZslo was transformed into

by electroporation and transformants resistant to Spec were selected. Complementation was verified by Western blotting.

Table 1- Strains

A "Jump Start" positive emmll type GAS wound isolate containing

NS144(pP4-gfp) the reporter plasmid pP4-gfp This study and Moses

A "Jump Start" positive emmll type GAS wound isolate containing

NS165(pP4-gjp) the reporter plasmid pP4-gfp This study and

JS95_ATGA«7A5(pP4-gfp) JS95_ATGAiM5 containing the reporter plasmid pP4-gfp This study

JS95_ATGsagr sagl insertion-inactivated derivative of JS95_ATG This study

JS95_ATGAslo slo deletion mutant of JS95_ATG This study

slo deletion and sagl insertion-inactivated mutant of JS95_ATG This study

JS95 _ATGAslo,sagI^'pLZsagI sagl complementation mutant of JS95_ATGAi o sagl^' This study

JS95_ATGAslo,sagI^'pLZslo slo complementation mutant of JS95_ATGAi o sagl^' This study lS95_ATGtrxR^' trxR insertion-inactivated derivative of JS95_ATG This study

JS95_ATG/f¾i?^~ which lost the p233-10R plasmid and reversed back to

JS95 _ATGtrxR^' cured JS95_ATG genotype This study

M1T1 M1T1 GAS clinical isolate 13

emm77 strain isolated from a cellulitis patient containing SilCR

NS35 with an ATG start codon Moses

Belotserkovsky, I., et al. Functional analysis of the quorum-sensing streptococcal invasion locus (sil). PLoS Pathog 5, el000651 (2009)

Hidalgo-Grass, C, et al. A locus of group A Streptococcus involved in invasive disease and DNA transfer. Mol Microbiol 46, 87-99 (2002)

5 Moses, A.E., et al. Invasive group a streptococcal infections, Israel. Emerg Infect

Dis 8, 421-426 (2002)

Table 2 - Plasmids

riiixinid Di'M i i plioii Kclt-mii't- pGEM-T-Easy A commercial T- vector for AT cloning Promega

pGEM-T A commercial T- vector for AT cloning Promega

pGEM-T-Easy vector containing a 1562bp fragment of silCR with pGKKCl-Dl_ATG an ATG start codon flanked by silB and silD partial sequences

pJRS233 Streptococcus-E. coli temperature sensitive shuttle vector Perez-Casal pJRS233-T pJRS233 T-vector for AT cloning This study

pJRS233 plasmid containing a 1562bp fragment of silCR with an

pJKKCl-Dl_ATG ATG start codon flanked by silB and silD partial sequences This study

pJRS233 plasmid containing an OKm resistance cassette flanked by philAB-QKm -500 bp fragments of silA and silB Belotserkovsky pKSM 410 Streptococcus-E. coli shuttle vector harboring a promoterless gfp Almengot

pKSM 410 plasmid containing the 154 bp sil promoter region

located upstream to blpM (contains only DR2) Belotserkovsky pP4-gfp plasmid containing silA and silB together with P_! and a

pP_fgJp silAB transcriptional terminator Belotserkovsky pJsilE^' pJRS233 plasmid containing a 635 bp internal fragment of silE Eran pGL3 A commercial Luciferase Reporter Vector Promega pP₄-luc pP_fgfp plasmid containing luc instead of gfp This study

p233-10R pJRS233 plasmid containing a fragment of trxR Leday

pGslo pGEM-T vector containing a 1639bp fragment of slo This study

pGEM-T vector containing an ΩΚπι resistance cassette flanked by pGsloQKm slo sequences This study

pJRS233 plasmid containing an OKm resistance cassette flanked by pJsloQKm slo sequences This study

pJsagl pJRS233-T plasmid containing a 333 bp fragment of sagl This study

pGEM-T-Easy vector containing the complete sagl sequence and

pGsagl the downstream sag operon terminator This study

pGEM-T-Easy vector containing the sag promoter, sagA, sagA

terminator, sagl and the downstream sag operon terminator

pGsagAsagl sequence This study

pLZ12-spec E. co¾^'-Gram-positive expression vector Human

Perez-Casal, J., Caparon, M.G. & Scott, J.R. Mry, a trans-acting positive regulator of the M protein gene of Streptococcus pyogenes with similarity to the receptor proteins of two-component regulatory systems. J Bacteriol 173, 2617-2624 (1991)

Leday, T.V., et al. TrxR, a new CovR-repressed response regulator that activates the Mga virulence regulon in group A Streptococcus. Infect Immun 76, 4659-4668 (2008)

Husmann, L.K., Scott, J.R., Lindahl, G. & Stenberg, L. Expression of the Arp protein, a member of the M protein family, is not sufficient to inhibit phagocytosis of Streptococcus pyogenes. Infect Immun 63, 345-348 (1995)

Eran, Y., et al. Transcriptional regulation of the sil locus by the SilCR signalling peptide and its implications on group A streptococcus virulence. Mol Microbiol 63, 1209-1222 (2007)

Almengor, A.C. & Mclver, K.S. Transcriptional activation of sclA by Mga requires a distal binding site in Streptococcus pyogenes. Bacteriol 186, 7847-7857 (2004) Table 3- Primers/SEQ ID NO:

Nizet, V., et al. Genetic locus for streptolysin S production by group A streptococcus. Infect Immun 68, 4245-4254 (2000)

Leday, T.V., et al. TrxR, a new CovR-repressed response regulator that activates the Mga virulence regulon in group A Streptococcus. Infect Immun 76, 4659-4668 (2008) Sage, A. P., Lu, J., Tintut, Y. & Demer, L.L. Hyperphosphatemia-induced nanocrystals upregulate the expression of bone morphogenetic protein-2 and osteopontin genes in mouse smooth muscle cells in vitro. Kidney international 79, 414-422 (2011)

Table 4 - Statistical Analysis

Fig. 3a + STS No addition 2 6 One-tail 0.0023

Fig. 3D MEF Atg5^+/+ MEF Atg5^' 2 5 One-tail 0.8930

Fig. 3E No addition + Wortmannin 2 7 One-tail 1.0000

Fig. 3E No addition + Chloroquine 2 7 One-tail 1.0000

Fig. 3F No addition + Z-VAD 2 5 One-tail 0.352

Fig. 3G +Z-VAD + TNF-a No addition 2 6 One-tail 0.838

Fig. 3G + Z-VAD + TNFa + No addition 2 6 Two-tail 0.0155

Nec-1

Fig. 3b +TG No addition 2 6 One-tail 0.0046

Fig. 3b +DTT No addition 2 6 One-tail 0.0046

Fig. 3c left panel JS95_ATG + STS JS95_ATG 4 5 One-tail 0.0002

Fig. 3c left panel JS95 _ATG slo,sagr + STS JS95_ATa/lslo, sagl^' 2 5 One-tail 0.0155

Fig. 3c right JS95_ATG + TG JS95_ATG 2 5 One-tail 0.0282 panel

Fig. 3c right lS95_ATGAslo,sagr + TG

sagl 2 5 One-tail 0.0282 panel

Fig. 4c left panel + 4AA No addition 2 5 One-tail 0.4810

Fig. 4c left panel + ASN No addition 2 5 One-tail 0.0232

Fig. 4c left panel + 4AA+ASN No addition 2 5 One-tail 0.0232

Fig. 4D 0.60mg/L ASN 0.45mg/L ASN 2 5 One-tail 0.0155

Fig. 4a 0.45mg/L ASN O.OOmg/L ASN 2 5 One-tail 0.1850

Fig. 4E JS95_ATG trxR - ASN JS95_ATG - ASN 2 5 Two-tail 0.0255

Streptococcal infection of mice, human blood and eukaryotic cells.

Streptococcal strains and variant, construction of mutants and DNA manipulations as well as some of the methods used for generating the data presented herein is provided in this section. For the murine models of human GAS soft-tissue and bacteremia infections strains were cultured overnight in Todd-Hewitt medium supplemented with 0.2 % yeast extract (THY). Then, bacteria were grown in THY to mid-log phase (OD₆oo of 0.3-0.5), washed and resuspended in PBS (Biological Industries). For soft-tissue infection, 10 g BALB/c female mice (Harlan Laboratories) were injected under the skin with 1 x 10 CFU of GAS in 100 ul, as previously described 25 ' 29. Eight mm punch biopsies (Acuderm), taken from the injection site from euthanized mice, were immersed in 4 % paraformaldehyde solution for 4 hours at room temperature (RT). Then, the samples were transferred to 30 % sucrose solution for 16 hours at 4 °C, embedded in Tissue-Tek® OCT™ Compound (Sakura) and refrigerated at -20 °C. Transverse sections 20 μιη thick were cut using a Leica CM3000 cryostat and thaw-mounted on Superfrost* Plus and ColorFrost* Plus Microscope slides (Thermo). The preparations were stained with 2 μg/ml DAPI (Invitrogen) and 5 μg/ml Phalloidin (Invitrogen) for 0.5 hour, mounted with Permafluor solution (Thermo) and analyzed with fluorescent confocal microscope (Zeiss LSM 710). The images were analyzed using ZEN 2009 Light Edition software, while keeping the intensity of GFP (green) at equal level for all examined samples. Concurrently, biopsies were homogenized in 0.5 ml PBS for 30 seconds on ice, using Polytron® PT2100 (Kinematica). Two samples from each homogenate of 100 μΐ were transferred to 96-wells plate and the luminescence of firefly luciferase (Luc) (Promega) was determined with Mithras Multimode Microplate Reader LB 940 (Berthold Technologies) after injection of 50 μΐ of luciferin (Promega). To normalize the luminescence readings according to the amount of bacteria present in the corresponding biopsies, an additional 100 μΐ sample from each homogenate were subjected to serial dilutions on blood-agar plates and β-hemolytic colonies (CFU) were enumerated. For the bacteremia model, a 100 μΐ of GAS containing 3 x 10' CFU, were injected via to the lateral tail vein of 7 -week-old BALB/c male mice (Harlan Laboratories). Kidrolase^® (EUSA Pharma S.A.) 200 I.U. in 50 ul PBS were injected either once, together with the bacteria at time 0, or once more 24 hours after the first injection. The control group of mice received 2 injections of Kidrolase^® (200 I.U. in 150 μΐ PBS) only, as described above. Mice were monitored daily, and Kaplan-Meier survival curves were generated and analyzed for statistical significance using log rank (Mantel-Cox) test. The Institutional Ethics Committee for animal care approved all animal procedures (approval No. MD-10-12267-4). The ability of GAS to survive in human blood was tested by the direct bactericidal test of Lancefield as described previously⁶¹. In brief, GAS was grown to OD₆₀₀ of 0.16 in DMEM medium + 5AA and then diluted 1:40,000 into a fresh DMEM medium + 5AA. 0.2 ml containing 300-700 CFU was mixed with 0.6 ml of freshly drawn heparinized human blood. 4.0 units of Kidrolase® were added to the assay at various time points and the number of CFU after 3 hours was enumerated. Multiplication factor was calculated by dividing the CFU at the end of the assay by the CFU at time zero.

Eukaryotic cells were cultured in a DMEM (Sigma) containing 10 % FCS (Biological Industries) (DMEM medium) in 24-well plates (Nunc™) at 37 °C in a 5 % CO2 incubator. Bacteria were grown in THY until an early log phase (OD₆oo=0.3-0.4), washed in PBS by centrifugation and concentrated to an OD₆oo of 8 (10⁹ CFU/ml), and finally diluted to form a bacterial suspension containing 250,000 CFU in 25 μΐ of PBS. Prior to infection, the eukaryotic cells were washed with PBS and then incubated in a fresh DMEM medium into which the bacterial suspension was added, generating MOI of ~ 1. Plates were then centrifuged at 300 x g for 5 minutes and further incubated at 37 °C in the C0₂ incubator.

Determination of sil activation.

For determination of SLS activity, tested bacteria were grown in THY to an early log phase (OD₆₀₀=0.3), washed with PBS, concentrated to OD₆₀₀ of 8 (10⁹ CFU/ml), diluted 4,000-folds into 24-well plates containing DMEM medium supplemented with 5AA and grown at 37°C in an atmosphere containing 5% C0₂. At different time points, plates were centrifuged and supernatants were removed and mixed with equal volumes of 2.5% (v/v) defibrinated sheep erythrocytes in PBS. The mixture was incubated for 1 hour at 37°C, centrifuged (3000 x g for 5 min) and readings of the supernatants at ODs₄o were determined. Values of complete hemolysis representing 100% were determined by lysing the equivalent erythrocytes suspension with 1% Triton X-100. Trypan blue at 13 μg/ml completely inhibited hemolysis, indicating that under the aerobic assay conditions the contribution of SLO to hemolytic activity was negligible. For determination of SLO- mediated hemolytic activity, tested bacteria were grown overnight at 37 °C anaerobically, in 24-well plate containing DMEM supplemented with 5AA, 10 μΜ of the protease inhibitor E-64, and when necessary appropriate antibiotics. The overnight cultures were centrifuged and the supernatants of 0.5 ml were reduced by treatment with 20 mM L-cysteine for 10 min at RT. An equal volume of 4% (v/v) defibrinated sheep erythrocytes in PBS was added, and the samples were incubated at 37 °C for 1 hour. After centrifugation, ODs₄o readings were recorded, and 100% hemolysis was determined as described above.

The indirect assay assesses sil self-activation by quantifying SilCR production using the reporter strains iS95 p_i p_ipP4-gfp or iS95 p_i p_isilE^~pP4-gfp (erm -resistant). This assay was mainly used when the tested strains contained 2 antibiotic resistance genes or an erythromycin resistant cassette that hampered the transformation with pP4-gfp or affected the rate of bacterial growth. Tested strains were grown and resuspended in PBS (250,000 CFU in 25 μΐ) as described for the direct assay, and used to infect eukaryotic cells or seeded in DMEM media containing AA. Then, 0.2 ml from the respective culture media (free of eukaryotic cells and of bacteria) were mixed with 0.8 ml of the reporter strains, (grown overnight in THY and diluted 1:25 into a fresh THY medium). The mixtures were incubated for 2 hours at 37°C without agitation in sealed Eppendorf tubes. Reporter strains were washed by centrifugation and resuspended in 0.6 ml PBS, and samples of 0.2 ml were transferred into 96-well flat bottom transparent plates (FluoroNunc™). The fluorescence intensity of GFP was measured with Infinite® F200 (Tecan, Austria GmbH), using the filter sets 485/20 nm for excitation and 535/25 nm for emission. The fluorescence data were normalized according to the density of the cultures, which was determined by measuring OD₅9₅. The relative fluorescence values shown reflect the amount of SilCR present in the culture medium of the tested strains 27.

Immunofluorescence of MEF cells infected with GAS.

Infected MEF cells were fixed with 4 % paraformaldehyde in PBS at RT, and then permeabilized with 0.1 % Saponin (Sigma). Cells were washed with PBS and blocked for 0.5 hour at RT with Image-iT FX Signal Enhancer (Invitrogen). Anti- Streptococcus pyogenes group A carbohydrate antibody (Abeam) was incubated with the permeabilized cell preparation (1: 250) at 4 °C overnight and washed away entirely. A secondary donkey anti-goat IgG Alexa Fluor 647 (Invitrogen) antibody was added (1 μg/ml) for 1 hour at RT, washed away and cells were stained with Alexa Fluor 568 Phalloidin and NucBlue (Invitrogen), according to manufacturer's instructions. Immunofluorescently stained cells were analyzed using the laser scanner of the Nikon Al confocal microscope. The series of z stack images were generated with a 0.8 μιη step size, using a Plan Apo VC 60X/1.4 oil objective.

Depletion of DMEM medium of ASN.

DMEM containing 10% FCS was incubated with E. coli L-ASNase 0.15 units/ml (Sigma) at 37°C for 2 hours and inactivated by heating the medium at 80°C for 1 hour. ASN depletion was confirmed by chromatography-tandem mass spectrometry (see hereinbelow).

RNA isolation and Real time RT-PCR.

MEF cell were infected with GAS strains or treated with TG as described in herein. After 0, 2, 4, 7 or 9 hours of incubation, cells were washed, scrapped into 1 ml cold PBS and centrifuged. RNA was purified from the cell pellet by SV Total RNA isolation system (Promga) according to manufacturer's instructions. RNA was subjected to cDNA synthesis using MMLV reverse transcriptase (Promega), according to the manufacturer's protocols. Standard real time RT-PCR reactions were conducted using SYBR-green mix (Absolute SYBR GREEN ROX MIX, ABgene) and fluorescence detection was performed using Rotor-Gene 3000 A (Corbett life Science, Qiagen) according to manufacturer's instructions. The RT-PCR primers (Table 3) were designed using Primer Express™ software v2.0 (Applied Biosystems) for the mouse asns gene. The cDNA amount of β-actin was used to normalization. The data were analyzed according to the standard curve method (Rotor-gene analysis software 6.0) and are presented as abundance of transcript amount relative to that of β-actin.

Statistics.

All values are expressed as means + S.D. Where indicated, the non-parametric one or two-tailed exact Wilcoxon matched-pairs signed-ranks test were applied. The test was conducted on experiments of n>5 and for each n median of three determinations was used. Since sil activation could be detected only after 2 hours in the eukaryotic cell- infection experiments and in experiments of bacterial culturing in DMEM medium supplemented with AA, prior measurements were not included in these statistical analyses. In the experiments using conditioned media, sil activation was detected after one hour, thus one hour and further time points were included in the test. P-values from independent experiment were combined using Fisher's exact test and multiple comparisons were adjusted using the Bonferroni correction procedure. The Mantel-Cox method was used to assess mice survival curves and Mann-Whitney U test (two tails) was used to evaluate significant differences in luciferase activity determinations. P < 0.05 was considered as significant.

Determination of SLS and SLO hemolytic activities

For determination of SLS activity, tested bacteria were grown in THY to an early log phase (OD₆oo=0.3), washed with PBS, concentrated to OD₆oo of 8 (10⁹ CFU/ml), diluted 4,000-folds into 24-well plates containing DMEM medium supplemented with 5AA and grown at 37 °C in an atmosphere containing 5 % C0₂. At different time points, plates were centrifuged and supernatants were removed and mixed with equal volumes of 2.5% (v/v) defibrinated sheep erythrocytes in PBS. The mixture was incubated for 1 hour at 37 °C, centrifuged (3000 x g for 5 min) and readings of the supernatants at ODs₄o were determined. Values of complete hemolysis representing 100 % were determined by lysing the equivalent erythrocytes suspension with 1 % Triton X- 100. Trypan blue at 13 μg/ml completely inhibited hemolysis, indicating that under the aerobic assay conditions the contribution of SLO to hemolytic activity was negligible. For determination of SLO-mediated hemolytic activity, tested bacteria were grown overnight at 37 °C anaerobically, in 24-well plate containing DMEM supplemented with 5AA, 10 μΜ of the protease inhibitor E-64, and when necessary appropriate antibiotics. The overnight cultures were centrifuged and the supernatants of 0.5 ml were reduced by treatment with 20 mM L-cysteine for 10 min at room temperature (RT). An equal volume of 4 % (v/v) defibrinated sheep erythrocytes in PBS was added, and the samples were incubated at 37 °C for 1 hour. After centrifugation, ODs₄o readings were recorded, and 100 % hemolysis was determined as described above.

Chromatography-tandem mass spectrometry (HPLC-MS/MS)

Asparagine (ASN) was depleted from the DMEM medium by treatment with

Aspariginase (ASNase) and filtered through 3000 NMWL membrane (Amicon, MILLIPORE). The filtered solution was subjected to HPLC-MS/MS on an Accela HPLC linked to a TSQ Quantum Access Max mass spectrometer (Thermo Scientific) via a heated electrospray ionization (H-ESI) interface.

HPLC conditions: Separations were performed on a Hypersil GOLD C8

(Thermo Scientifics), 5 μιη, 150 x 4.6 mm column. For this test LC/MS-grade acetonitrile, methanol and water were purchased from Biolab Ltd., whereas the ion- pairing agent HFBA (Heptafluorobutyric acid) was purchased from Sigma- Aldrich. The chromatographic separation was performed using a gradient program at a flow rate of 1 ml/min over a total run time of 8 min. An outline of the mobile phase gradient program is summarized in Table 5 below. Solvent A is composed of a water solution containing 0.1 % formic acid and 0.3 % HFBA. Solvent B is composed of 0.1 % formic acid in methanol. The column temperature was set to 35 °C, the autosampler tray temperature was maintained at 5°C and the injection volume was 5 ul.

- Outline of mobile phase gradient program

MS/MS conditions: The mass spectrometer was operated in positive ionization mode and detection and quantification were performed using multiple reactions monitoring (MRM). The pressure of the nitrogen sheath gas and auxiliary gas were set at 25 and 1 (arbitrary units). The ionization spray voltage, capillary transfer tube temperature, tube lens and skimmer offset were set at 4.5 kV 300°C, 87V and 10V, respectively. The vaporizing temperature within the H-ESI source was maintained at 300°C. The scan time was 0.05 seconds, with a scan width of 0.1 m/z. TSQ Tune Software (Thermo Scientific) was used for the optimization of tuning parameters. Data acquisition and processing were carried out using the Xcalibur program (Thermo Scientific). F or ASN (133.08 m/z), the retention time was 3.15 min and 3 transitions were monitored: 74.25 m/z (collision energy (CE) 16V), 87.22 m/z (CE 16V) and 116.12 m/z (CE 10V).

EXAMPLE 1

The Quorum Sensing Locus sil is Activated In Vivo

The QS streptococcal invasion locus (sil) was previously identified in the NF

GAS strain JS95 (Hidalgo-Grass et al., 2004; Hidalgo-Grass et al., 2002) (termed here JS95ATA)- In JS95ATA the start codon of the autoinducer peptide SilCR is ATA. Thus, this strain is unable to produce SilCR but is capable to sense minute concentrations of the peptide through its two-component system (TCS) SilA/B and turn on the autoinduction cycle as depicted in Figure 1A. By characterizing the promoters that are highly stimulated by SilCR through SilA/B (Figure 1A), the present inventors have developed the ability to quantify SilCR-mediated signaling by measuring GFP or luciferase (Luc) accumulation, and identified GAS and group G streptococcal (GGS) strains that preserved the ability to both sense and produce SilCR (Belotserkovsky et al., 2009). The start codon of silCR was changed from ATA to ATG. Indeed it was demonstrated that the resulting strain, JS95A_TG, acquired the ability to produce SilCR when minute quantities of synthetic SilCR were added to the culture medium and initiated the autoinduction cycle (Figure IE, Figure 1A). To test if sil would be self- activated in vivo, JS95A_TA and JS95A_TG were transformed with pP4-gfp or pP4-luc (Figure 1A). The corresponding strains were injected subcutaneously into mice and punch biopsies of soft-tissue were taken (Hidalgo-Grass et al., 2006). GFP-labeled bacteria were detected in mice injected with

gfp (Figures 1B,C). Furthermore, GFP expression was apparent as early as 6 hours after mice injection (Figures 1B,C). Only a portion of the bacteria present in the examined fields was expressing GFP, as evident by comparing GAS staining by DAPI and GFP (Figures 1B,C). To provide a quantitative measure of sil activation in vivo, infected tissue samples were collected from mice challenged either with

or

Samples were homogenized, and Luc activity was determined and normalized to the number of colony forming units (CFU). Luc activity in mice infected with

luc (Figure ID). The activation was transient and was detected at 6 and 12 hours after inoculation, but not at 3 and 24 hours (Figure ID). Taken together, these results show that the host microenvironment that exists during the initial stages of GAS infection is suitable for turning on sil naturally.

EXAMPLE 2

sil Activation Occurs Ex Vivo During GAS Adherence to Mammalian Cells

To test that sil activation occurs ex vivo, HeLa cells were infected with either

The infection was conducted at MOI (multiplicity of infection) of ~ 1.0. At various time points after infection, samples from the culture media containing bacteria were withdrawn and subjected to cell sorting (FACS) analysis (Figures IF, G) or to determination of SilCR production using reporter strains (Figure IF). A time-dependent increase in GFP expression was detected in the medium of HeLa cells infected with

(Figure 2A,C) that peaked at 7 hours after infection and was detectable even after 22 hours (Figure 2C). In sharp contrast, no significant activation was detected in the medium of HeLa cells infected with JS95 AT _ApP4-gfp (Figure 2B). Subsequent studies showed that the presence of HeLa cells is absolutely necessary for activation (Figure 2C), and intact but not lysed cells support this process (Figure 2G). As expected, activation required intact SilA/B (Figure 2C), and was specifically blocked by SilCR antiserum (Figure S2H). The process of self-activation was not restricted to HeLa cells but occurred also when mouse embryonic fibroblasts (MEFs) (Figures 2E; 3A-C; 4B,C; 2L, 3D-3F), L929 cells (Figure 3G), and Raw 264.7 cells (not shown), were infected with JS95_ATGpP4-gfp. TO demonstrate that self- activation also occurs in strains possessing naturally active sil [(Belotserkovsky et al., 2009)], GAS and GGS sz7-active isolates were transformed with pP4-gfp and then infected MEF cells with the corresponding strains. Activation occurred in the presence but not in the absence of MEF cells (not shown) and was inhibited by the addition of SilCR antiserum but not by a control serum (Figure 21). To demonstrate that self- activation occurs also in vivo for a strain possessing naturally active sil, GAS strain NS 144 of emm77-type was transformed with pP4-luc. It was demonstrated that Luc is produced under the settings that are described in Figure ID (Figure 2J).

Although GAS is considered an extracellular pathogen, GAS adherence to eukaryotic cells may lead to its internalization (Courtney et al., 2002). To test whether GAS internalization contributes to sil activation, HeLa cells were treated with cytochalasin D (Ozeri et al., 2001) and it was found that while this treatment reduced the level of intracellular GAS by ~ 200-fold it did not affect sil activation (Figure 2K). Next, it was tested whether activation occurs when bacteria and eukaryotic cells are separated by a membrane preventing physical contact, but allowing diffusion of small molecules. HeLa cells were grown in a Transwell™ device and infected with JS95_ATGpP4-gfp added either to the upper Transwell™ chamber (separated from HeLa cells) or to the lower Transwell™ chamber (together with HeLa cells). As expected, addition of GAS to the lower chamber resulted in sz7-activation that was apparent after 4 hours and peaked at 7 hours post infection, and the presence of HeLa cells was necessary for the activation. However, addition of the bacteria to the upper chamber significantly delayed sil- activation which was apparent between 8 to 10 hours after infection (Figure 2D). These results show that a physical contact between GAS and HeLa cells facilitates sil- activation but activation can also occur independently of direct contact between the cells, probably due to GAS soluble products.

Contact-dependent followed by contact-independent activation of sil alluded to the possible involvement of GAS streptolysins, since both toxins are more efficiently delivered into eukaryotic cells during GAS adherence and less efficiently as soluble products (Ofek et al., 1990; Ruiz et al., 1998). To test for the involvement of SLS and SLO, mutants of JS95A_TG lacking either SLS or SLO, and a double mutant lacking both toxins were constructed. While the double mutant lost completely the ability to activate sil upon contact with MEFs, isogenic mutants of either SLS or SLO were able to activate sil (Figure 2E). The fact that each toxin can mediate sil activation independently of the other was exemplified by the complementation experiments showing that expression of either SLO or SLS in the double mutant restored sil activation (Figure 2L). In the case of the complementing mutant that expressed SLS from a plasmid, sil activation was significantly higher than that of JS95A_TG (Figure 2L), due to an increased amount of SLS expression as reflected by measuring of hemolytic activity (Figure 2M). To verify that the ex vivo activation of sil, shares the same properties as the in vivo one, the requirement for SLO and SLS was tested in the experimental settings described in Figure ID. The results obtained clearly demonstrated that SLO and SLS significantly contribute to sil activation under both conditions (Figures 2E,F).

EXAMPLE 3

Triggering ofER Stress Leads to sil Activation

It was reported that SLO or SLS can participate in GAS -mediated induction of major cellular responses such as: autophagy (Nakagawa et al., 2004), necrosis (Miyoshi- Akiyama et al., 2005), apoptosis (Timmer et al., 2009), oncosis (Goldmann et al., 2009) and endoplasmic reticulum (ER) stress (Cywes Bentley et al., 2005). Since staurosporine (STS) induces these responses (Christensen et al., 1998; Dunai et al., 2012; Short et al., 2007), next thing tested was if pretreatment of MEF cells with STS would generate a conditioned medium capable of activating sil. Indeed, it was found that sil was rapidly and strongly activated by a conditioned medium generated by treating MEFs with STS (Figure 3 A).

To delineate which of the cellular processes mentioned above is involved in sil activation, it was tested if MEF cells deficient of Atg5, [which is essential for GAS- mediated autophagy (Nakagawa et al., 2004)], would support sil activation. MEF cells deficient of Atg5 supported sil activation similarly to Atg5 proficient cells (Figure 3D). Furthermore, the inhibitors of autophagy, chloroquine and wortmannin, did not inhibit sil activation (Figure 3E), thus collectively suggesting that autophagy is not involved in this process. For testing the involvement of apoptosis the cell-permeable pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD) was used. It was found that it did not exert any effect on activation (Figure 3F). Necroptosis is a specialized pathway of programmed necrosis that is initiated by ligating death receptors (such as tumor necrosis factor (TNF) receptor 1) and involves activation of kinase receptor-interacting proteins, RIP1 and RIP3, under caspase-compromised conditions (Vandenabeele et al., 2010). To test if necroptosis is linked to sil activation, L929 cells were used that undergo necroptotic cell-death when exposed to TNFa and Z-VAD (Wu et al., 2011). Treatment of L929 cells with these compounds led to cell necrosis that was apparent by microscopic visualization after 2 hours (Figure 3G), yet the resulting conditioned medium was unable to activate sil (Figure 3G). Conversely, when the inhibitor of RIP 1 kinase, necrostatin-1, (Nec-1) (Degterev et al., 2008) was included, the conditioned medium activated sil effectively (Figure 3G). Because Nec-1 protected L929 cells from necrotic death (Figure 3G), these results suggested that TNFa causes sil activation through another route that is hindered by cell necrosis.

It was reported that treatment of L929 cells with TNFa causes ER stress (Xue et al., 2005). Therefore, it was tested if the chemical inducers of ER stress thapsigargin (TG), and dithiothreitol (DTT) would produce conditioned media capable of activating sil. MEFs treated by these compounds produced conditioned media that rapidly activated sil (Figure 3B). Furthermore, STS and TG considerably enhanced sil activation when added to MEFs infected with JS95A_TG, or even when MEF cells were infected with the SLO, SLS double mutant (Figure 3C). This suggested that both STS and TG bypass the requirement for SLO and SLS in sil activation. EXAMPLE 4

Host asns Transcription is upregulated during GAS Infection of Eukaryotic Cells

Studies of Kilberg et al. demonstrated that ER-stress triggered by TG in HepG2 cells, up-regulates the expression of asparagine synthetase (ASNS) through the ATF4 pathway (Barbosa-Tessmann et al., 1999; Gjymishka et al., 2009). Since treatment of MEFs with TG generated a conditioned medium capable of activating sil (Figure 3B), the present inventors examined if infection of MEF cells with GAS would also increase the asns transcript level. In an attempt to delay cell necrosis that may obscure asns transcript determinations, the concentration of FCS was reduced in the DMEM medium to 5%. This decreased the rate of GAS proliferation and delayed MEF cell-necrosis (Figure 4D). Infection of MEFs with JS95A_TG under these conditions resulted in increased asns transcript level compared to uninfected cells or cells infected with the double mutant lacking both SLO and SLS (Figure 4A inset). The increase in transcription started to be apparent after 4 hours and within 7 to 9 hours reached 15 to 20 % of that caused by TG (Figure 4A). Nevertheless, asns transcription triggered by the double mutant complemented with a plasmid overexpressing SLS (Figures 2L-M) reached a level of about 50 % of that induced by TG (Figure 4A).

To visualize sil activation under the above-described conditions (5% FCS), the present inventors analyzed MEF cells infected with

by fluorescence microscopy. At 4 hours after infection few adhering GAS were observed but no sil activation was detected (Figure 4B). At seven hours post infection, many adhering bacteria were seen, yet only a small fraction of bacteria which resided in clumps on some of the infected cells, expressed GFP (Figure 4B). In contrast, TG that strongly upregulates asns transcription (Figure 4A) significantly augmented sil activation when added to MEFs infected with GAS. Already 4 hours after infection almost all adhering bacteria produced GFP (Figure 4C). In the absence of TG, a similar level of GFP- producing bacteria was apparent only after 9 hours of infection (Figure 4B). Since asns transcription did not change significantly between 7 to 9 hours of GAS infection (Figure 4A inset) it is conceivable that the substantial activation of sil occurring between 7 to 9 hours (Figure 4B) is caused by sil autoinduction. EXAMPLE 5

Asparagine is Essential for sil Activation and Promotes Bacterial Proliferation

While testing lung alveolar adenocarcinoma A549 cells for their ability to facilitate sil activation, it was discovered that the nutrient mixture F-12 (HAM) per se activates sil (Figure 5A). To delineate the component of F-12 (HAM) that is responsible for sil activation, DMEM was supplemented with the components that are present in F- 12 (HAM), but are absent in DMEM. Both media were supplemented with 10 % fetal calf serum (FCS) that is essential for sil activation as well as for GAS growth. It was found that a group of 5 amino acids (5AA) containing proline, aspartic acid, glutamic acid, alanine, and asparagine (ASN), was accountable for sil activation (Figure 5B). While the rate of JS95A_TG growth in DMEM medium fortified with the 4AA (proline, aspartic acid, glutamic acid and alanine) was slightly higher than the rate of growth in DMEM supplemented with ASN alone (Figure 5G), only the latter supported sil activation (Figure 5C). Nevertheless, when ASN was added to the medium containing the 4AA, it increased the rate of JS95A_TG growth (Figure 5G) and triggered sil activation as well (Figure 5C). In attempt to distinguish between these two ASN-mediated effects, the concentration of ASN was progressively increased in DMEM medium supplemented with the 4AA and sil activation and bacterial growth were monitored. When the concentration of added ASN was increased from 0.45 to 0.60 mg/L, there was an abrupt increase in sil activity that peaked at 6 hours after incubation start (Figure 5H). The CFUs of JS95A_TG grown for 6 hours in media containing 0.45 or 0.60 mg/L ASN were almost identical (Figure 5H), suggesting that sil activation is triggered by sensing of ASN.

It was reported that in the chemotaxis receptor McpB of Bacillus subtilis the upper PAS domain binds ASN (Glekas et al., 2010). Protein-BLAST search with the McpB binding domain for ASN (AA 35 to 279) against all GAS genomes identified 9 TCS with significant but low expected values. All of these, exhibited homology with the sensor kinase YesM of the Bacillus subtilis TCS YesMN. Subsequent Protein-Blast of the 12 TCS of M1T1 MGAS5005 with YesM identified 3 TCS sensors including TrxS (Leday et al., 2008). The protein sequence homology between the surface exposed TrxS domain, (AA 50 to 283) and the McpB binding domain for ASN (AA 35 to 279) exhibited an insignificant level of global homology [(14.2% identity 27.8% similarity, EMBOSS (Rice et al., 2000)]. Nonetheless, both domains showed a significant level of structural homology (Figure 51), as identified by analysis using 3 different programs for protein structure prediction [(Phyre², I-TASSER, or LOMETS (Soding, 2005; Wu and Zhang, 2007; Zhang, 2008)].

To test the possible involvement of TrxSR in sil regulation, the response regulator trxR was inactivated by insertion mutagenesis. The resulting mutant gained the ability to produce SilCR even in the absence of ASN in the medium (Figure 5D). Furthermore, clones that were cured of the insertional plasmid reverted to the original phenotype, thus demonstrating absolute dependence of sil activation on the presence of ASN (Figure 5E). These results taken together suggest that TrxRS activation via ASN releases sil repression exerted either directly or indirectly. To demonstrate that ASN- mediated signaling occurs also in vivo the experimental settings described in Figure ID was used to examine the ability of Kidrolase^®, a trade name of L-asparginase (ASNase), (an FDA approved E. coli enzyme for the treatment of acute lymphocytic leukemia), to prevent Luc production when injected together with the bacteria. The results shown in Figure 5J demonstrate clearly that Kidrolase^® specifically blocked Luc formation in vivo.

The 10 % FCS present in the DMEM medium contains a residual amount of ASN. To remove ASN completely, the medium was treated with ASNase and after that ASNase was heat-inactivated. Complete removal of ASN was verified by chromatography-tandem mass spectrometry. JS95A_TG growth was significantly slowed in the absence of ASN even compared to a medium that was supplemented with ASN concentration as low as 0.015 mg/L ASN. The growth reached its maximal rate at 1.5 mg/L of ASN (Figure 5F). A strong dependence of growth on ASN was also observed for the pandemic M1T1 GAS clone (Maamary et al., 2012), albeit the latter grew more rapidly both in the presence and absence of ASN (Figure 5K). In the absence of ASN, both GAS strains reached maximal level of growth after overnight incubation, suggesting that GAS is not auxotrophic for ASN, but produces this AA in DMEM medium at a low rate. Indeed the results of transcriptome profiling described below demonstrate that the transcription of ASNS in GAS is upregulated in the absence of ASN (Table S5 item number 50). Most fascinating was the finding that in the absence of added ASN, growth of both GAS strains was arrested by the addition of increasing concentration of ASNase (Figure 5L), suggesting that even the self -produced ASN could be accessible to the extracellularly added enzyme.

EXAMPLE 6

ASN Sensing Affects the Expression of nearly 17% of GAS Genes

In attempt to identify genes whose response to ASN is independent of sil, a transcriptome profiling of wild type (WT) MGAS5005 and of its trxR- utant (which do not possess sil), was performed using RNA sequencing (RNA-seq). For this purpose, these strains were grown in DMEM + 10 % FCS that was fortified with the 5AA to early log phase, then Kidrolase^® was either added or not, RNA was harvested at 0.5 and 1.0 hours later and subjected to RNA-seq analysis. Comparison of transcriptome profiles in the presence and absence of kidrolase^® in WT MGAS5005 strain showed that 311 genes (16.7 %) were significantly altered. Out of the 311 genes, 194 genes (10.4%) were significantly altered also in the trxK mutant in the presence and absence of Kidrolase^®. 117 genes were significantly altered only in the WT and not in the trxK mutant, thus TrxSR is involved in regulating the transcription of about 1/3 of the genes that respond to ASN depletion. Cluster analysis of differential expression of MGAS5005 and its isogenic TrxR mutant in the presence and absence of ASN is shown in Figure 6F. The list of the 311 genes (Table 5S of Baruch et al. Cell 2014 Jan 16;156(l-2):97-108) on which the analysis was performed contains gene products involved in metabolism, virulence, gene regulation, replication and other functions. To examine if similar genes would be affected upon ASN depletion in the strain JS95A_TG, the bacteria were grown and RNA harvested under the same conditions described for the strain MGAS5005. The transcription of 12 genes was analyzed using RT-RT-PCR. As shown in Table 5S Baruch et al. Cell 2014 Jan 16; 156( l-2):97- 108, expression of all 12 JS95_ATG genes were comparable to the corresponding genes in MGAS5005 with respect to ASN depletion, albeit the influence of TrxR varied for some genes (Table S5 Baruch et al. supra). As expected from the absolute dependence of sil activation on ASN and its repression by TrxR (Figure 5E), the transcription of silE and blpM (Belotserkovsky et al., 2009) was strongly upregulated in the trxK mutant both in the presence and absence of ASN (Figure 6G-H). Most intriguing was the finding that the transcription of the genes encoding SLO (slo) and SLS (sagA-I operon) was strongly upregulated in the absence of ASN both in MGAS5005 and in JS95_ATG, yet in MGAS5005 their dependency on TrxR was less pronounced compared to that in JS95A_TG (Figure 6A-D). This finding suggests that in the absence of ASN the bacterium increases the production of SLO and SLS to gain more ASN from the host. Finally, the transcription of genes involved in GAS replication such as polA, lig and others (see Table S5 Baruch et al. supra) were significantly down-regulated in the absence of ASN, suggesting that there is a tight linkage between ASN sensing and GAS proliferation. Taken together, the results shown are consistent with a model whereby the delivery of SLO and SLS through adhering GAS induces ER stress. This in turn results in ASN production. The released ASN is sensed by GAS via TrxSR and other yet unidentified system(s) to both alter the expression of nearly 17% of GAS genes and to accelerate GAS rate of multiplication (Figure 6E).

EXAMPLE 7

The Therapeutic Effect of ASN as e against GAS Bacteremia

The fact that ASN is required for optimal growth of GAS (Figures 5F and 5K) and that ASNase blocks GAS growth in DMEM medium (Figure 5L) suggested that the latter may prevent the ability of GAS to proliferate in human blood, a property that is the hallmark for GAS virulence (Cunningham, 2000). In a classical GAS blood survival assay, addition of Kidrolase^® at time 0, 0.5, or even 1.0 hour after infection prevented JS95A_TG proliferation (Figure 7A). Similar results were obtained for the pandemic M1T1 5448 strain (Figure 7C). To rule out that the Kidrolase^®-mediated arrest of GAS proliferation results from adverse reaction of Kidrolase^® on phagocytic killing which is independent of breakdown of ASN to aspartic acid and ammonia, the ability of Staphylococcus epidermidis ATCC 12228 to survive in human blood in the presence and absence of Kidrolase^® was tested. It was found that S. epidermidis grew at the same rate in DMEM medium in the presence and absence of Kidrolase^® and its survival rate in whole human blood was unaffected by the drug, thus ruling out that possibility that the latter adversely affects bacterial phagocytic killing (Figure 7D).

To test the ability of Kidrolase^® to control GAS bacteremia, the bacteremia mouse model developed by Medina and colleagues (Medina et al., 2001) was employed. Mice were injected i.v. with 10⁷ CFU of JS95A_TG with or without Kidrolase^®. All mice that received 2 consecutive (24 hours apart) injections of Kidrolase^® but no GAS, survived (Figure 7B). Six out of 7 mice that received GAS only, died within 10 days (Figure 7B). In the group of mice that received GAS and a single injection of Kidrolase^®, 3 out of 7 mice died at days 4 to 5 after GAS injection. No significant difference in the death rates of this group and the group receiving GAS only was observed (P= 0.168, Log rank (Mental-Cox). However, in the group of mice that received GAS and 2 consecutive injections of Kidrolase^®, 3 mice died between day 8 and 9 (Figure 7B). The Kaplan-Meier analysis showed that there is a significant difference in death rates between this group and the group of mice receiving GAS only [(P= 0.0283, Log rank (Mental-Cox)].

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

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Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting pathogenicity of Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteriain a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an asparagine-reducing agent, thereby inhibiting pathogenicity of the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria.

2. A method of reducing infection of Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an asparagine-reducing agent, thereby reducing infection of the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria in the subject.

3. A method of arresting growth of Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria, the method comprising reducing availability of asparagine to the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria, thereby arresting growth of the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria.

4. The method of claim 3, wherein said reducing availability of asparagine to the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria is performed by contacting the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria or a host infected therewith with an effective amount of an asparagine-reducing agent.

5. An asparagine-reducing agent for treating infection of a Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria in a subject in need thereof.

6. The method of claim 1, wherein said administering comprises topical administering.

7. The method of claim 3, being an ex vivo or in vitro method.

8. The method of claim 4, wherein the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria are in a biological sample.

9. An article-of-manufacture comprising in separate packaging an asparagine-reducing agent and an antibiotic agent, wherein the article-of-manufacture further comprises instructions for use in reducing infection of Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria.

10. A pharmaceutical composition formulated for local administration comprising as an active ingredient an asparagine-reducing agent.

11. The pharmaceutical composition of claim 10, being a topical formulation.

12. The pharmaceutical composition of claim 10 or 11, being formulated as lotion, cream, gel, ointment or spray.

13. The method, asparagine-reducing agent, article-of-manufacture or pharmaceutical composition of any one of claims 1-2, 5 and 9-12, wherein said asparagine-reducing agent is selected from the group consisting an agent which increases asparagine degradation, an agent which reduces asparagine synthesis, an agent which reduces uptake by the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria, an agent which reduces asparagine excretion, an agent which sequesters free asparagine.

14. The method, asparagine-reducing agent, article-of-manufacture or pharmaceutical composition of claim 13, wherein said agent which increases asparagine degradation comprises an asparaginase (EC 3.5.1.1).

15. The method or asparagine-reducing agent of any one of claims 2, 5 and 13-14, wherein said subject is inflicted with- or is at risk of septic sore throat (pharyngitis), tonsillitis, impetigo, cellulitis, erysipelas, necrotizing fasciitis, sinusitis, otitis, pneumonia, meningitis, septic arthritis, osteomyelitis, vaginitis, endocarditis, myositis, bacteremia, toxic shock syndrome, scarlet fever, rheumatic fever, post streptococcal glomerulonephritis and PANDAS (Pediatric Autoimmune Neuropsychiatry Disorders).

16. The method or asparagine-reducing agent of any one of claims 2, 5 and 13-15, wherein the subject is not inflicted with cancer.

17. The method of any one of claims 1, 2, and 13-15 or asparagine-reducing agent of claim 5, further comprising administering to the subject a therapeutically effective amount of an antibiotic agent or an anti-fungal agent.

18. The article of manufacture of claim 9 or asparagine-reducing agent or the method of claim 17, wherein said antibiotic agent is a cytotoxic antibiotic.

19. The method of claim 3 or 4, wherein said contacting is performed in vivo.