WO2015033344A1 - Procédés et nécessaires permettant d'inhiber la pathogénicité d'un streptocoque du groupe a (sga) ou d'un streptocoque du groupe g (sgg) - Google Patents

Procédés et nécessaires permettant d'inhiber la pathogénicité d'un streptocoque du groupe a (sga) ou d'un streptocoque du groupe g (sgg) Download PDF

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Publication number
WO2015033344A1
WO2015033344A1 PCT/IL2014/050795 IL2014050795W WO2015033344A1 WO 2015033344 A1 WO2015033344 A1 WO 2015033344A1 IL 2014050795 W IL2014050795 W IL 2014050795W WO 2015033344 A1 WO2015033344 A1 WO 2015033344A1
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streptococcus
group
bacteria
asparagine
gas
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PCT/IL2014/050795
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English (en)
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Emanuel Hanski
Miriam RAVINS
Baruch HERTZOG
Moshe Baruch
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Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
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Publication of WO2015033344A1 publication Critical patent/WO2015033344A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions

Definitions

  • 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).
  • GAS group A Streptococcus
  • GGS group G Streptococcus
  • GAS The group A Streptococcus
  • NF necrotizing fasciitis
  • streptococcal toxic shock syndrome 1 ' 2 The group A Streptococcus
  • 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 intervention 4 .
  • TrxR a new CovR-repressed response regulator that activates the Mga virulence regulon in group A Streptococcus. Infection and immunity 76, 4659-4668 (2008).
  • a method of inhibiting pathogenicity of Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteriain a subject in need thereof 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.
  • a method of reducing infection of Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria in a subject in need thereof 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.
  • GAS Group A Streptococcus
  • GGS Group G Streptococcus
  • a method of arresting growth of Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria 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.
  • 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.
  • the administering comprises topical administering.
  • the method is an ex vivo or in vitro method.
  • the Group A Streptococcus (GAS) bacteria or Group G Streptococcus (GGS) bacteria are in a biological sample.
  • 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.
  • GAS Group A Streptococcus
  • GGS Group G Streptococcus
  • a pharmaceutical composition formulated for local administration comprising as an active ingredient an asparagine-reducing agent.
  • the pharmaceutical composition is a topical formulation.
  • the pharmaceutical composition is formulated as lotion, cream, gel, ointment or spray.
  • 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.
  • GAS Group A Streptococcus
  • GGS Group G Streptococcus
  • the agent which increases asparagine degradation comprises an asparaginase (EC 3.5.1.1).
  • 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).
  • 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 rhe
  • the subject is not inflicted with cancer.
  • the method further comprises administering to the subject a therapeutically effective amount of an antibiotic agent or an anti-fungal agent.
  • the antibiotic agent is a cytotoxic antibiotic.
  • the contacting is performed in vivo.
  • FIGs. 1A-G show that sil is activated in vivo.
  • 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.
  • 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.
  • RLU relative luminescence units
  • FIG. 1 Schematic representation of the two assays performed to assess sil activation during mammalian cells infection with GAS and GGS.
  • 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 i silE ⁇ 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.
  • 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 ATG pP4-gfp (A) or JS95 AT AP ⁇ - ⁇ (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.
  • 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.
  • 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 ⁇ STS.
  • 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 ⁇ ), or wortmannin (1 ⁇ ) then infected with JS95A TG P ⁇ 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.
  • L929 cells were treated with TNF-a (10 ng/ml) and Z-VAD (100 ⁇ ) or with TNF-a plus Z-VAD (100 ⁇ ) and necrostatin-1 (Nec-1, 100 ⁇ ).
  • the culture media of L929 cells, (treated as indicated in the upper panel) were collected in the indicated times and added to JS95A TG PP4-,? 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 ⁇ ) was added. At the indicated time points asns transcript level was determined by RT-RT-PCR and normalized to the transcript level of ⁇ -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).
  • FIGs. 5A-L show that ASN is essential for sil activation and promotes bacterial proliferation.
  • 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.
  • 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.
  • DMEM medium containing 4AA 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 A1G pP4-luc + PBS, JS95 ATA pP4-luc + PBS or JS95 ATG pP4-luc + 200 I.U Kidrolase®.
  • Punch biopsies were taken 6 hours after injection, homogenized, and relative luminescence units (RLU) were normalized to the CFUs.
  • RLU relative luminescence units
  • 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.
  • 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 OD 6 oo- 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.
  • JS95 ATG the transcription level of sagA (A), sagB (B), sagD (C), and slo (D) were determined by RT-RT-PCR.
  • the amount of cDNA was normalized to that of gyrA in each RNA sample.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • GAS group A Streptococcus
  • GGS group G Streptococcus
  • GAS quorum sensing
  • SLO streptolysin O
  • SLS streptolysin S
  • 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.
  • asparagine significantly increases the growth rate of GAS.
  • a method of arresting growth of Group A Streptococcus bacteria or Group G Streptococcus bacteria 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)
  • GAS Group A streptococcus
  • 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.
  • GAS strains contemplated according to the present teachings can be M- type of non-M-type.
  • Group G Streptococcus 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.
  • the Gram-positive bacteria are Group A Streptococcus (GAS) type bacteria, also referred to as Streptococcus pyogenes.
  • GAS Group A Streptococcus
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • contacting can occur ex vivo such as in a biological sample which may comprise eukaryotic cells.
  • 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.
  • the biological sample may comprise tissues (biopsies) or organs.
  • 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.
  • 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.
  • a method of inhibiting pathogenicity of Group A Streptococcus bacteria or Group G Streptococcus bacteria in a subject in need thereof 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.
  • a method of reducing infection of Group A Streptococcus bacteria or Group G Streptococcus bacteria in a subject in need thereof 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.
  • a method of treating a disease e.g., medical condition, syndrome
  • a disease e.g., medical condition, syndrome
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 rhe
  • 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.
  • the subject has a life threatening condition.
  • 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.
  • 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.
  • ALL acute lymphyblastic leukemia
  • B-cell and T-cell leukemias chronic leukemias
  • acute undifferentiated leukemia e.g., acute lymphyblastic leukemia (ALL), acute non-lymphocytic leukemias, B-cell and
  • 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.
  • infectious diseases such as those caused by HIV infection (i.e., AIDS), rheumatoid arthritis, SLE, autoimmune, collagen vascular diseases, AIDS, osteoarthritis, I
  • terapéuticaally effective amount refers to the amount of the agent that is sufficient to cause, for example, a bacteristatic effect.
  • 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.
  • 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
  • the agent may target a bacterial gene or a host cell gene.
  • 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.
  • 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.
  • 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.
  • the bacterial L-ASP is E. coli L-ASP, e.g., Merck's Elspar R TM, Kidrolase R TM.
  • Other suitable L-ASPs include those obtained from Erwinia chrysanthemi, e.g., Erwinase, Serratia marcescens, guinea pig, and Caviodea.
  • the L-ASP contains alternative or additional groups.
  • 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 TM 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.
  • 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.
  • ATP is used to activate aspartate, forming ⁇ -aspartyl-AMP.
  • Glutamine donates an ammonium group, which reacts with ⁇ -aspartyl-AMP to form asparagine and free AMP.
  • 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.
  • such an agent reduces expression or activity of an asparagine binding molecule such as TrxS (having an asparagine binding domain) expressed on the bacterial cell.
  • an asparagine binding molecule such as TrxS (having an asparagine binding domain) expressed on the bacterial cell.
  • TrxS asparagine binding domain
  • the agent targets a bacterial target such as TrxS, SUA, SilB, SilCR, M5005_s/ry_1359, M5005_s/ry_0743 or M5005_3 ⁇ 4ry_0745.
  • 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).
  • eukaryotic target i.e., of the host
  • NRE1/2 nutrient sensing-response elements
  • an agent capable of downregulating a target is an antibody or antibody fragment capable of specifically binding thereto.
  • the antibody specifically binds at least one epitope of the target.
  • epitope refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • 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
  • 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.
  • RNA silencing agent refers to an RNA which is capable of specifically inhibiting or “silencing" the expression of a target gene.
  • 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.
  • RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.
  • the RNA silencing agent is capable of inducing RNA interference.
  • the RNA silencing agent is capable of mediating translational repression.
  • 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.
  • target RNA e.g., a gene product of the asparagine synthesis pathway
  • RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs).
  • siRNAs short interfering RNAs
  • 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.
  • dsRNAs double-stranded RNAs
  • RNA-induced silencing complex RISC
  • some embodiments of the invention contemplates use of dsRNA to downregulate protein expression from mRNA.
  • the dsRNA is greater than 30 bp.
  • the use of long dsRNAs i.e. dsRNA greater than 30 bp
  • 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.
  • the invention 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.
  • long dsRNA over 30 base transcripts
  • the invention also contemplates introduction of long dsRNA specifically designed not to induce the interferon and PKR pathways for down-regulating gene expression.
  • 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.
  • siRNAs small inhibitory RNAs
  • RNA refers to small inhibitory RNA duplexes (generally between
  • RNA interference RNA interference
  • 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.
  • RNA silencing agent of some embodiments of the invention may also be a short hairpin RNA (shRNA).
  • shRNA 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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].
  • UTRs untranslated regions
  • 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).
  • 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
  • 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.
  • 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.
  • a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
  • 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.
  • the RNA silencing agent provided herein can be functionally associated with a cell-penetrating peptide.
  • 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 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 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 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).
  • a zinc-finger protein 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.
  • 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.
  • ZFN zinc-finger nuclease
  • Cleavage domains and cleavage half domains can be obtained, for example, from various restriction endonucleases and/or homing endo nucleases.
  • the cleavage half-domains are derived from a Type IIS restriction endonuclease (e.g., Fok I).
  • the zinc finger domain recognizes a target site e.g., of TrxS, TrxR.
  • the zinc finger domain comprises 5 or 6 zinc finger domains and recognizes a target site in a bacterial target.
  • the TALE Transcription activator like
  • a region of interest e.g., e.g., of TrxS, TrxR
  • the TALE comprises one or more engineered TALE binding domains.
  • 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.
  • the cleavage half-domains are derived from a Type IIS restriction endonuclease (e.g., Fok I).
  • the TALE DNA binding domain recognizes a target site in a target e.g., of TrxS, TrxR.
  • 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).
  • a region of interest e.g., a highly expressed gene, a disease associated gene or a safe harbor gene
  • 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.
  • 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.
  • 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.
  • nucleic acid agents or proteins can be provided per se.
  • a nucleic acid sequence encoding same is ligated into a nucleic acid construct suitable for mammalian/bacterial cell expression.
  • 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.
  • the asparagine-reducing agent is administered prior to the antibiotic agent.
  • the antibiotic agent is a bactericidal agent i.e., an agent that kills bacteria.
  • 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.
  • the beta-lactam antibiotics e.g., penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems and vancomycin.
  • 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.
  • the antibiotic is administered in a bactericidal/bactericidal amount, dependent on the mechanism of action.
  • antibiotics which can be used in conjunction with the asparagine reducing agent include, but are not limited to, quinolones (e.g., ciprofloxacin), and novobiocin.
  • 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
  • the asparagine reducing agent can be administered to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • 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.
  • 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.
  • pharmaceutically acceptable carrier refers 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.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • 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.
  • 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
  • pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers)
  • 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).
  • the pharmaceutical composition is formulated for topical administration.
  • the pharmaceutical composition described herein e.g., asparagine reducing agent, e.g., asparaginase
  • a topical infection i.e. infection of the skin
  • a topical formulation i.e., asparaginase
  • the agents e.g., asparagine reducing agent, e.g., asparaginase
  • asparagine reducing agent e.g., asparaginase
  • the agents are used to treat a local or systemic infection inside the body.
  • the administration may be done in order to achieve a systemic effect or a local effect.
  • 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.
  • 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.
  • 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.
  • 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.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • 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).
  • 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.
  • 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.
  • 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.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • compositions of some embodiments of the present invention also include a dermatologically acceptable carrier.
  • keratinous tissue 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • amphoteric and zwitterionic surfactants are also useful herein.
  • 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.
  • 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.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • 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.
  • compositions 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.
  • 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.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • 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.
  • 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.
  • active ingredients e.g., asparagine reducing agent
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • 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).
  • MEC minimum effective concentration
  • 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.
  • compositions 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 include but are not limited to 400-40,000 IU/Kg, 500-30,000 IU/Kg, 1000-20,000 IU/Kg, 1000-10,000.
  • exemplary dose ranges include, but are not limited to, 4-400 IU/cm 2 , 4-400 IU/cm 2 , 50-400 IU/cm 2 , 100-400 IU/cm 2 .
  • 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.
  • 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).
  • a pack or dispenser device such as an FDA approved kit
  • the active ingredient e.g., asparagine reducing agent with or without an antibiotic.
  • 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.
  • compositions, 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.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the human HeLa epithelial cell line (HeLa ATCC ® Catalog No. CCL-2TM), the mouse embryonic fibroblasts (MEF), and Atg5 7 MEF, the mouse subcutaneous fibroblast L cell line clone 929 (ATCC ® Catalog No. CCL-1TM) and the mouse leukemic monocytes/macrophages Raw 264.7 cells (ATCC ® Catalog No. TIB-71TM) 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).
  • DMEM Dulbecco's Modified Eagles Medium
  • FCS fetal calf serum
  • the Lung alveolar adenocarcinoma A549 cells (ATCC ® Catalog No. CCL-185TM) were cultured in Ham's F-12 medium (Biological Industries) supplemented with 10 % FCS (v/v). All
  • 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 10 5 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.
  • MSE Soniprep 150 Plus
  • 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)
  • 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 % C0 2 incubator.
  • AA amino acids
  • antibiotics were added at the following concentrations: for GAS and GGS: 250 ⁇ g/ml kanamycin (Km), 50 ⁇ g/ml spectinomycin (Spec) and 1 ⁇ g/ml erythromycin (Erm); for E. coli: 100 ⁇ g/ml ampicillin (Amp), 50 ⁇ g/ml Spec, 750 ⁇ g/ml Erm and 50 ⁇ g/ml Km. All the antibiotics were purchased from Sigma-Aldrich.
  • 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.
  • 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.
  • the mutant was constructed using pJsilAB- ⁇ , ⁇ 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)] .
  • 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-gfp 1 from which the gfp gene was removed by EcoRI digestion.
  • 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)] .
  • 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.
  • ⁇ , ⁇ 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 ⁇ , ⁇ flanked by sequences of 5' and 3' regions of slo.
  • 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)] .
  • pJsagl 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 ⁇ -hemolysis on blood agar plates.
  • 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 sagA 9 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 ⁇ -hemolytic transformants on blood agar plates.
  • 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.
  • 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.
  • 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
  • TrxR a new CovR-repressed response regulator that activates the Mga virulence regulon in group A Streptococcus. Infect Immun 76, 4659-4668 (2008)
  • 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 AT a/lslo, sagl ' 2 5 One-tail 0.0155
  • mice 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.
  • PBS Biological Industries
  • the preparations were stained with 2 ⁇ g/ml DAPI (Invitrogen) and 5 ⁇ g/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).
  • 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 ⁇ 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.
  • 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 ODs 4 o were determined.
  • the indirect assay assesses sil self-activation by quantifying SilCR production using the reporter strains iS95 p i p i pP4-gfp or iS95 p i p i silE ⁇ 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 ⁇ ) as described for the direct assay, and used to infect eukaryotic cells or seeded in DMEM media containing AA.
  • 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 (FluoroNuncTM).
  • 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 OD 5 9 5 .
  • the relative fluorescence values shown reflect the amount of SilCR present in the culture medium of the tested strains 27.
  • 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 ⁇ g/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 ⁇ step size, using a Plan Apo VC 60X/1.4 oil objective.
  • 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).
  • 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 ExpressTM software v2.0 (Applied Biosystems) for the mouse asns gene.
  • the cDNA amount of ⁇ -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 ⁇ -actin.
  • 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 ODs 4 o were determined.
  • Asparagine (ASN) was depleted from the DMEM medium by treatment with
  • Aspariginase 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.
  • 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).
  • the Quorum Sensing Locus sil is Activated In Vivo
  • 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.
  • 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.
  • TCS two-component system
  • JS95A T A 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
  • HeLa cells were grown in a TranswellTM device and infected with JS95 ATG pP4-gfp added either to the upper TranswellTM chamber (separated from HeLa cells) or to the lower TranswellTM chamber (together with HeLa cells).
  • GAS GAS
  • 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.
  • addition of the bacteria to the upper chamber significantly delayed sil- activation which was apparent between 8 to 10 hours after infection (Figure 2D).
  • 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).
  • 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).
  • TNF tumor necrosis factor
  • Asparagine is Essential for sil Activation and Promotes Bacterial Proliferation
  • 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.
  • ASNase an FDA approved E. coli enzyme for the treatment of acute lymphocytic leukemia
  • the 10 % FCS present in the DMEM medium contains a residual amount of ASN.
  • 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).
  • RNA sequencing RNA sequencing
  • 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
  • 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).
  • Kidrolase ® 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 10 7 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.
  • Nizet V. Streptococcal beta-hemolysins: genetics and role in disease pathogenesis. Trends in microbiology 10, 575-580 (2002).
  • TrxR a new CovR-repressed response regulator that activates the Mga virulence regulon in group A Streptococcus. Infection and immunity 76, 4659-4668 (2008).
  • Streptolysin S contributes to group A streptococcal translocation across an epithelial barrier. The Journal of biological chemistry 286, 2750-2761 (2011).
  • Tumor necrosis factor alpha induces the unfolded protein response (UPR) in a reactive oxygen species (ROS)-dependent fashion, and the UPR counteracts ROS accumulation by TNFalpha.
  • UPR unfolded protein response
  • ROS reactive oxygen species

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Abstract

La présente invention concerne un procédé d'inhibition de la pathogénicité d'une bactérie de type streptocoque du groupe A (SGA) ou d'une bactérie de type streptocoque du groupe G (SGG) chez un sujet en ayant besoin. Ledit procédé implique d'administrer au sujet une quantité thérapeutiquement efficace d'un agent capable de réduire l'asparagine, de façon à inhiber la pathogénicité d'une bactérie de type streptocoque du groupe A (SGA) ou d'une bactérie de type streptocoque du groupe G (SGG).
PCT/IL2014/050795 2013-09-05 2014-09-04 Procédés et nécessaires permettant d'inhiber la pathogénicité d'un streptocoque du groupe a (sga) ou d'un streptocoque du groupe g (sgg) WO2015033344A1 (fr)

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