The invention relates to materials and methods for treating infections, and more particularly to materials and methods for reducing endogenous microbial H2S levels.
Despite the phenomenal success of antibiotics, infectious diseases remain the second leading cause of death worldwide. About two million Americans are infected in hospitals each year (with 90,000 deaths as a result), and more than half of these infections resist at least one antibiotic. For example, pathogens can alarmingly become fully resistant to last resort antibiotics, such as vancomycin. The emergence of multidrug-resistant bacteria has created a situation in which there are few or no options for treating certain infections. Natural antibiotics and their derivatives are intrinsically prone to become obsolete because of preexisting genes that render pathogens resistant to them. Bacterial species share these genes, thus rapidly spreading resistance from hospitals and farms to surrounding communities.
H2S is a toxic gas that has been associated with beneficial functions in mammals, including vasorelaxation, cardioprotection, neurotransmission, and anti-inflammatory action in the gastrointestinal (GI) tract (1-5). The ability of H2S to function as a signaling molecule parallels the action of another established gasotransmitter, nitric oxide (NO) (6-8). Endogenous NO was demonstrated to protect certain Gram(+) bacteria against antibiotics and oxidative stress (10-12).
Like NO, H2S is produced enzymatically in various tissues (1, 4). Three H2S-generating enzymes have been characterized in mammals: cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). CBS and CSE produce H2S predominantly from L-cysteine (Cys). 3MST does so via the intermediate synthesis of 3-mercaptopyruvate by cysteine aminotranferase (CAT), which is inhibited by aspartate (Asp) competition for Cys on CAT (9) (
As indicated in the Background section, above, there is a great need in the art to develop new effective treatments for microbial infections and to fight antibiotic resistance.
The present invention addresses these and other needs by providing methods and compositions for treating infections and enhancing effectiveness of antibiotics by reducing endogenous microbial H2S levels.
In one aspect, the invention provides a method for treating a subject having a microbial infection, said method comprising administering to said subject a therapeutically effective amount of at least one inhibitor of endogenous H2S production by an organism causing the microbial infection. In one embodiment, the method further comprises administering a second antimicrobial compound (e.g., a quinolone, an acridine, a phenothiazine, an aminoglycoside, a macrolide, an amphenicol, a steroid, an ansamycin, an antifolate, a polymyxin, a glycopeptide, a cephalosporin, a lactam, or any combination thereof). In one specific embodiment, the second antimicrobial compound is selected from the group consisting of novobiocin, norfloxacin, nalidixic acid, oxolinic acid, acriflavine, 9-aminoacridine, proflavine, trifluoperazine, promethazine, chlorpromazine, streptomycin, apramycin, tylosin, oleandomycin, erythromycin, josamycin, spiramycin, troleandomycin, chloramphenicol, fusidic acid, rifamycin SV, trimethoprim, 2,4-diamino-6,7-diisopropylpteridine, polymyxin B, colistin, vancomycin, cefsulodin, cephalothin, cefoperazone, oxacillin, nafcillin, phenethicillin, penicillin G, cloxacillin, moxalactam, carbenicillin, and any combination thereof. In another specific embodiment, the second antimicrobial compound is selected from the compounds disclosed in Tables 1 and 2 (below) and any combination thereof. The inhibitor of endogenous H2S production and the second antimicrobial compound can be administered simultaneously (within the same composition or in different compositions) or sequentially (with either the inhibitor of endogenous H2S production or the second antimicrobial compound administered first).
In another aspect, the invention provides a method for enhancing efficacy of an antimicrobial treatment in a subject having a microbial infection, wherein said antimicrobial treatment comprises administering to the subject a first antimicrobial compound that becomes compromised by H2S or natural products of H2S metabolism in vivo, said method comprising co-administering said first compound with a therapeutically effective amount of a second compound which second compound is an inhibitor of endogenous H2S production by an organism causing the microbial infection. The first antimicrobial compound that becomes compromised by H2S or natural products of H2S metabolism in vivo can be without limitation, e.g., a quinolone, an acridine, a phenothiazine, an aminoglycoside, a macrolide, an amphenicol, a steroid, an ansamycin, an antifolate, a polymyxin, a glycopeptide, a cephalosporin, a lactam, or any combination thereof. In one specific embodiment, the first antimicrobial compound that becomes compromised by H2S or natural products of H2S metabolism in vivo is selected from the group consisting of novobiocin, norfloxacin, nalidixic acid, oxolinic acid, acriflavine, 9-aminoacridine, proflavine, trifluoperazine, promethazine, chlorpromazine, streptomycin, apramycin, tylosin, oleandomycin, erythromycin, josamycin, spiramycin, troleandomycin, chloramphenicol, fusidic acid, rifamycin SV, trimethoprim, 2,4-diamino-6,7-diisopropylpteridine, polymyxin B, colistin, vancomycin, cefsulodin, cephalothin, cefoperazone, oxacillin, nafcillin, phenethicillin, penicillin G, cloxacillin, moxalactam, carbenicillin, and any combination thereof. In another specific embodiment, the first antimicrobial compound that becomes compromised by H2S or natural products of H2S metabolism in vivo is selected from the compounds disclosed in Tables 1 and 2 (below) and any combination thereof. The first antimicrobial compound that becomes compromised by H2S or natural products of H2S metabolism in vivo and the inhibitor of endogenous H2S production can be administered simultaneously (within the same composition or in different compositions) or sequentially.
In yet another aspect, the invention provides a method for sensitizing a microbial pathogen to oxidative damage comprising administering to said pathogen an effective amount of at least one inhibitor of endogenous H2S production by said pathogen. In one embodiment, the pathogen is in a subject and the inhibitor of endogenous H2S production is administered to the subject.
In one embodiment, the inhibitor of endogenous H2S production useful in the methods of the present invention inhibits an H2S-generating enzyme within the organism causing the microbial infection. In one embodiment, the inhibitor of endogenous H2S production inhibits microbial cystathionine β-synthase (CBS) or paralogs thereof. In a specific embodiment, such CBS inhibitor can be co-administered (simultaneously in the same or separate compositions or sequentially) with an inhibitor of microbial cystathionine γ-lyase (CSE) or paralogs thereof. In a separate embodiment, the inhibitor of endogenous H2S production inhibits microbial 3-mercaptopyruvate sulfurtransferase (3MST) or paralogs thereof. In another embodiment, the inhibitor of endogenous H2S production inhibits microbial cystathionine γ-lyase (CSE) or paralogs thereof.
In one embodiment, the inhibitor of endogenous H2S production is amino-oxyacetate (AOAA). In another embodiment, the inhibitor of endogenous H2S production is hydroxylamine. In yet another embodiment, the inhibitor of endogenous H2S production is D,L-propargylglycine (PAG). In a further embodiment, the inhibitor of endogenous H2S production is β-cyano-L-alanine. In another embodiment, the inhibitor of endogenous H2S production is aspartate or a derivative thereof. In an additional embodiment, the inhibitor of endogenous H2S production is homocysteine.
In one embodiment, the inhibitors of endogenous H2S production useful in the methods of the present invention selectively inhibit H2S-generating enzyme(s) within the organism causing the microbial infection, but not H2S-generating enzyme(s) in the cells of the subject being treated. Such selective inhibitors can be identified using various screening methods known in the art, e.g., as described in International Application Publication No. WO 2011/130181.
The methods of the present invention are useful for treating infections caused by various microorganisms, including, e.g., bacteria, fungi and protozoa. Non-limiting examples of encompassed bacterial genera include, e.g., Bacillus, Brucella, Clostridium, Enterococcus, Escherichia, Francisella, Helicobacter, Klebsiella, Legionella, Listeria, Mycobacterium, Pseudomonas, Salmonella, Shigella, Staphylococcus, Streptococcus, Trypanasoma, Vibrio, and Yersinia. In one specific embodiment, bacteria are from a species selected from the group consisting of E. coli, S. aureus, B. anthracis, and P. aeruginosa.
Non-limiting examples of microbial infectious diseases which can be treated by the methods of the present invention include, e.g., pneumonia, bronchitis, diphtheria, pertussis (whooping cough), tetanus, endocarditis, sepsis, bacterial gastroenteritis, cholera, tuberculosis, gonorrhea, chlamydia, syphilis, bacterial meningitis, malaria; trachoma, leishmaniasis, chagas disease, trichomoniasis, lyme disease, and leprosy.
The methods of the present invention can be used to treat infections in various animals, including, e.g., mammals, birds and fish. In one specific embodiment, the methods of the present invention are used to treat infections in humans.
The methods of the present invention can further comprise administering an inhibitor of microbial endogenous nitric oxide (NO) production or a NO scavenger. Such inhibitor of microbial endogenous nitric oxide (NO) production or a NO scavenger can be administered simultaneously (within the same composition or in different compositions) or sequentially (in any order) with an inhibitor of endogenous H2S production and/or the second antimicrobial compound. Non-limiting examples of useful inhibitors of endogenous NO production include, e.g., L-arginine, NG-monomethyl-L-arginine, NG-nitro-L-arginine methyl ester, NG-nitro-L-arginine, NG-amino-L-arginine, NG,NG-dimethylarginine (asymmetric dimethylarginine), L-thiocitrulline, S-methyl-L-thiocitrulline, diphenyleneiodonium chloride, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy 3-oxide, 7-nitroindazole, N(5)-(1-iminoethyl)-L-ornithine, aminoguanidine, canavanine, ebselen, S-methyl-L-citrulline, S-methylisourea, and 2-mercaptoethylguanidine. In one specific embodiment (wherein the microbial infection is caused by Gram (+) bacteria), the inhibitor of endogenous NO production is an iNOS-specific inhibitor. Non-limiting examples of useful NO scavengers include, e.g., non-heme iron-containing peptides, non-heme iron-containing proteins, porphyrins, metalloporphyrins, dithiocarbamates, dimercaptosuccinic acid, phenanthroline, desferrioxamine, pyridoxal isonicotinoyl hydrazone, 1,2-dimethyl-3hydroxypyrid-4-one, [+] 1,2-bis(3,5-dioxopiperazinelyl)propane, and 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide. In one specific embodiment, the NO scavenger is a perfluorocarbon emulsion.
In conjunction with the methods of the present invention, provided herein are various combination pharmaceutical compositions. In one embodiment, the invention provides a pharmaceutical composition comprising (i) an antimicrobial compound that becomes compromised by H2S or natural products of H2S metabolism in vivo and (n) an inhibitor of microbial endogenous H2S production. In a specific embodiment, this composition further comprises (iii) an inhibitor of microbial endogenous nitric oxide (NO) production or a NO scavenger. In a separate embodiment, the invention provides a pharmaceutical composition comprising (i) an inhibitor of microbial endogenous H2S production and (ii) an inhibitor of microbial endogenous nitric oxide (NO) production or a NO scavenger.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
The present invention is based in part on the discovery that microbial H2S is cytoprotective. As described in the Examples below, the present inventors have discovered that (1) bacterial cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST) generate H2S under normal growth conditions and protect bacteria against a broad range of antibiotics, and (2) that the mechanism of H2S-mediated defense against antibiotics relies on protection against oxidative stress and the DNA-damaging Fenton reaction. As described herein, endogenous microbial H2S can diminish the effectiveness of clinically used antibiotics. Thus, inhibition of this “gasoprotector” may be useful as an augmentation therapy against a broad range of microbial pathogens, including bacteria, fungi and protozoa. As microbial CBS, CSE, and 3MST diverge significantly from their mammalian counterparts (see, e.g.,
In one aspect, the invention provides a method for treating a subject having a microbial infection, said method comprising administering to said subject a therapeutically effective amount of at least one inhibitor of endogenous H2S production by an organism (e.g., pathogenic bacteria, fungi, protozoa) causing the microbial infection. In one embodiment, the method further comprises administering a second antimicrobial compound.
In another aspect, the invention provides a method for enhancing efficacy of an antimicrobial treatment in a subject having a microbial infection, wherein said antimicrobial treatment comprises administering to the subject a first antimicrobial compound that becomes compromised by H2S or natural products of H2S metabolism in vivo, said method comprising co-administering said first compound with a therapeutically effective amount of a second compound which second compound is an inhibitor of endogenous H2S production by an organism (e.g., pathogenic bacteria, fungi, protozoa) causing the microbial infection.
In a separate aspect, the invention provides a method for sensitizing a microbial pathogen (e.g., pathogenic bacteria, fungi, protozoa) to oxidative damage comprising administering to said pathogen an effective amount of at least one inhibitor of endogenous H2S production by said pathogen. In one embodiment, the pathogen is in a subject and the inhibitor of endogenous H2S production is administered to the subject. Such sensitization can be important, for example, for facilitating mammal's own defense against infections. When mammalian immune system attempts to kill pathogens via oxidative stress, pathogens′ H2S provides protection against the immune response.
In one embodiment, the inhibitor of endogenous H2S production useful in the methods of the present invention inhibits an H2S-generating enzyme within the organism causing the microbial infection. For example, AOAA and hydroxylamine can be used to inhibit CBS; PAG and β-cyano-L-alanine can be used to inhibit CSE; and aspartate and derivatives thereof can be used to inhibit 3MST.
In one embodiment, the inhibitor of endogenous H2S production inhibits microbial cystathionine β-synthase (CBS) or paralogs thereof. In a specific embodiment, such CBS inhibitor can be co-administered (simultaneously in the same or separate compositions or sequentially) with an inhibitor of microbial cystathionine γ-lyase (CSE) or paralogs thereof. In a separate embodiment, the inhibitor of endogenous H2S production inhibits microbial 3-mercaptopyruvate sulfurtransferase (3MST) or paralogs thereof. In another embodiment, the inhibitor of endogenous H2S production inhibits microbial cystathionine γ-lyase (CSE) or paralogs thereof.
In one embodiment, the inhibitor of endogenous H2S production is amino-oxyacetate (AOAA). In another embodiment, the inhibitor of endogenous H2S production is hydroxylamine. In yet another embodiment, the inhibitor of endogenous H2S production is D,L-propargylglycine (PAG). In a further embodiment, the inhibitor of endogenous H2S production is β-cyano-L-alanine. In another embodiment, the inhibitor of endogenous H2S production is aspartate or a derivative thereof. In an additional embodiment, the inhibitor of endogenous H2S production is homocysteine. In one embodiment, the inhibitors of endogenous H2S production useful in the methods of the present invention selectively inhibit H2S-generating enzyme(s) within the organism causing the microbial infection, but not H2S-generating enzyme(s) in the cells of the subject being treated. Such selective inhibitors can be identified using various screening methods known in the art, e.g., as described in International Application Publication No. WO 2011/130181.
The methods of the present invention are useful for treating acute or chronic infections caused by various microorganisms, including, e.g., bacteria, fungi and protozoa. Non-limiting examples of encompassed bacterial genera include, e.g., Bacillus, Brucella, Clostridium, Enterococcus, Escherichia, Francisella, Helicobacter, Klebsiella, Legionella, Listeria, Mycobacterium, Pseudomonas, Salmonella, Shigella, Staphylococcus, Streptococcus, Trypanasoma, Vibrio, and Yersinia. In one specific embodiment, bacteria are from a species selected from the group consisting of E. coli, S. aureus, B. anthracis, and P. aeruginosa.
Non-limiting examples of microbial infectious diseases which can be treated by the methods of the present invention include, e.g., pneumonia, bronchitis, diphtheria, pertussis (whooping cough), tetanus, endocarditis, sepsis, bacterial gastroenteritis, cholera, tuberculosis, gonorrhea, chlamydia, syphilis, bacterial meningitis, malaria, trachoma, leishmaniasis, chagas disease, trichomoniasis, lyme disease, and leprosy.
The methods of the present invention can be used to treat infections in various animals, including, e.g., mammals, birds and fish. In one specific embodiment, the methods of the present invention are used to treat infections in humans.
Examples of antimicrobial compounds that can be used in the methods provided herein include, without limitation, quinolones, acridines, phenothiazines, aminoglycosides, macrolides, amphenicols, steroids, ansamycins, antifolates, polymyxins, glycopeptides, cephalosporins, and lactams. For example, a method as provided herein can include administration of an antimicrobial compounds selected from the group consisting of novobiocin, norfloxacin, nalidixic acid, oxolinic acid, acriflavine, 9-aminoacridine, proflavine, trifluoperazine, promethazine, chlorpromazine, streptomycin, apramycin, tylosin, oleandomycin, erythromycin, josamycin, spirathycin, troleandomycin, chloramphenicol, fusidic acid, rifamycin SV, trimethoprim, 2,4-diamino-6,7-diisopropylpteridine, polymyxin B, colistin, vancomycin, cefsulodin, cephalothin, cefoperazone, oxacillin, nafcillin, phenethicillin, penicillin G, cloxacillin, moxalactam, and carbenicillin. Other antimicrobial compounds that can be used include, for example, those listed in Tables 1 and 2, below.
In some embodiments, the methods provided herein also can include further administering an inhibitor of endogenous microbial NO production or an NO scavenger.
Non-limiting examples of useful inhibitors of endogenous NO production include L-arginine, NG-monomethyl-L-arginine (NMMA), NG-nitro-L-arginine methyl ester (NAME), NG-nitro-L-arginine (NNA), NG-amino-L-arginine (NAA), NG,NG-dimethylarginine (asymmetric dimethylarginine, called ADMA), L-Thiocitrulline, S-methyl-L-Thiocitrulline, diphenyleneiodonium chloride, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy 3-oxide, 7-nitroindazole, N(5)-(1-iminoethyl)-L-ornithine, aminoguanidine, canavanine, ebselen, S-methyl-L-citrulline, S-methylisourea, and 2-mercaptoethylguanidine. See, also, the inhibitors disclosed in Hobbs et al. (1999) Annu. Rev. Pharmacol. Toxicol. 39:191-220; and Salard et al. (2006) J. Inorg. Biochem. 100:2024-2033. In some embodiments, iNOS-specific inhibitors can be particularly useful.
NO scavengers include, without limitation, non-heme iron-containing peptides or proteins, porphyrins, metalloporphyrins, dithiocarbamates, dimercaptosuccinic acid, phenanthroline, desferrioxamine, pyridoxal isonicotinoyl hydrazone (PIH), 1,2-dimethyl-3hydroxypyrid-4-one (L1), [+] 1,2-bis(3,5-dioxopiperazine-lyl)propane (ICRF-187), 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (Carboxy-PTIO), and the like. A particularly useful NO scavenger may be a perfluorocarbon emulsion, as disclosed in Rafikova et al. (2004) Circulation 110(23):3573-3580.
In conjunction with the methods of the present invention, provided herein are various combination pharmaceutical compositions. In one embodiment, the invention provides a pharmaceutical composition comprising (i) an antimicrobial compound that becomes compromised by H2S or natural products of H2S metabolism in vivo and (ii) an inhibitor of microbial endogenous H2S production. In a specific embodiment, this composition further comprises (iii) an inhibitor of microbial endogenous nitric oxide (NO) production or a NO scavenger. In a separate embodiment, the invention provides a pharmaceutical composition comprising (i) an inhibitor of microbial endogenous H2S production and (ii) an inhibitor of microbial endogenous nitric oxide (NO) production or a NO scavenger.
The compounds described herein can be formulated into pharmaceutical compositions and formulations. Compounds therefore can be admixed, encapsulated, conjugated or otherwise associated with one or more pharmaceutically acceptable carriers and/or other molecules, molecular structures, or mixtures of compounds such as, for example, liposomes, polyethylene glycol, receptor targeted molecules, for oral, topical or other formulations, for assisting in uptake, distribution and/or absorption.
Methods for formulating and subsequently administering therapeutic compositions are well known to those skilled in the art. Dosing generally is dependent on the severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Persons of ordinary skill in the art routinely determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages can vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50 found to be effective in in vitro and in vivo animal models. Typically, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, biweekly, weekly, monthly, or even less often. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state.
Pharmaceutical compositions can be administered by a number of methods, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be, for example, topical (e.g., transdermal, sublingual, ophthalmic, or intranasal); pulmonary (e.g., by inhalation or insufflation of powders or aerosols); oral; or parenteral (e.g., by subcutaneous, intrathecal, intraventricular, intramuscular, or intraperitoneal injection, or by intravenous drip). Administration can be rapid (e.g., by injection) or can occur over a period of time (e.g., by slow infusion or administration of slow release formulations). For treating tissues in the central nervous system, compounds can be administered by injection or infusion into the cerebrospinal fluid, typically with one or more agents capable of promoting penetration of the polypeptides across the blood-brain barrier.
The term, “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably still up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value.
As used herein, the term “paralogs” (of CBS, CSE, and 3MST) refers to homologous sequences within a microbial genome which result from a gene duplication event. The CBS, CSE, and 3MST paralogs encompassed by the present invention may have altered functional properties as compared to CBS, CSE, and 3MST, but are still involved in or affect the endogenous microbial H2S production.
As used herein, the term “compromised by H2S or natural products of H2S metabolism” refers to antimicrobial compounds which become less effective in the presence of H2S or natural products of H2S metabolism (i.e., any sulfur-containing organic [e.g. cysteine, glutathione] or inorganic [e.g., FeS, NaHS] products of H2S metabolism). This term encompasses complete and partial loss of antimicrobial activity of a given compound.
In the context of the present invention insofar as it relates to any of the disease conditions recited herein (e.g., infection), the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.
The terms “administering” or “administration” are intended to encompass all means for directly and indirectly delivering a compound to its intended site of action. The compounds of the present invention can be administered locally to the affected site (e.g., by direct injection into the affected tissue) or systemically. The term “systemic” as used herein includes parenteral, topical, oral, spray inhalation, rectal, nasal, and buccal administration. Parenteral administration includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial administration.
As used herein, the term “therapeutically effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present invention, the term “therapeutically effective” refers to that quantity of a compound (e.g., an inhibitor of endogenous H2S production or its combination with a second antimicrobial compound and/or an inhibitor of endogenous microbial NO production or an NO scavenger) or pharmaceutical composition containing such compound that is sufficient to delay manifestation, arrest the progression, relieve or alleviate at least one aspect of an infection. Note that when a combination of active ingredients is administered the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually.
The phrase “pharmaceutically acceptable,” as used in connection with compositions of the invention, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to an animal (e.g., a human). Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
The term “subject” refers to any animal, including, e.g., mammals, birds and fish, and, in particular, humans.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.
In accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; DNA Cloning: A Practical Approach, Volumes I and II (Glover ed. 1985); Oligonucleotide Synthesis (Gait ed. 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1985); Transcription and Translation (Hames and Higgins eds. 1984); Animal Cell Culture (Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); and Ausubel et al. eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. 1994, among others.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Strains and Growth Conditions:
E. coli, S. aureus, and P. aeruginosa strains were grown in Luria-Bertani (LB) broth or on LB plates supplemented with 1.5% Bacto agar at 37° C. B. anthracis strains were grown in BHI media supplemented with glycerol at 37° C. Construction of sseA deletion and sseA overexpression strains of E. coli were produced according to Datsenko and Wanner (34). Briefly, an attL-KmR-attR cassette (35) was amplified with primers 5′-atgcgtgagaatttacgttatgtaattcagtatcaccgctcaagttagtataaaaaagct-3′ (SEQ ID NO: 1) and 5′-ttatttcactggctcaaccggtaaatctgcccgcgtgaagcctgcttttttatactaag-3′ (SEQ ID NO: 2) and transformed into an MG1655 strain containing pKD46. sseA mutants were selected on LB agar supplemented with Km and verified by PCR with primers 5′-attagcatgcaggcggctac-3′ (SEQ ID NO: 3) and 5′-tgctggaaaggccgcgaa-3′ (SEQ ID NO: 4).
To construct an sseA overexpression strain, the sseA promoter was substituted for PLtet-O1 (24). Briefly, an attL-CmR-attR cassette (35) was amplified with primers 5′-cgcagatcttgaagcctgcttttttatac-3′ (SEQ ID NO: 5) and 5′-cgctcaagttagtataaaaaagct-3′ (SEQ ID NO: 6) and ligated with PLtet-O1 obtained with primers 5′-cgcagatctcgagtccctatcagtg-3′ (SEQ ID NO: 7) and 5′-aatttctcctctttccatgg-3′ (SEQ ID NO: 8). A PLtet-O1- attL-CmR-attR cassette was then amplified with primers 5′-ccctgccacaatggcccgttagcaacgtcgaataacgctcaagttagtataaaaaagct-3′ (SEQ ID NO: 9) and 5′-gtgatactgaattacataacgtaaattctcacgcatggtacctttctcctattaatga-3′ (SEQ ID NO: 10). The first primer contains the upstream region of sseA and sequence of attR, while the second primer contains the coding region of sseA and sequence of PLtet-O1. The PCR fragment was transformed into MG1655 containing pKD46. CmR clones were tested in the presence of the PLtet-O1-attL-CmR-ttR cassette by PCR with primers 5′-attagcatgcaggcggctac-3′ (SEQ ID NO: 11) and 5′-gcaaaagaggctgatttggct-3′ (SEQ ID NO: 12).
E. coli metB, metC, malY, cysM, cysK, and tnaA deletion mutants were obtained from the E. coli Keio Knockout Collection (Thermo Scientific).
The B. anthracis nos deletion strain Sterne 34F2 was described previously (28). The conditional KO (CO) of cbs/cse in B. anthracis 34F2 was made using a protocol described in Fisher and Hanna, J. Bacteriol. 187, 8055 (2005). The 0.5 kb fragment of cbs from −20 to 480 with respect to the translational start site was amplified from 34F2 genomic DNA by using primers designed for use in the Gateway cloning system (Invitrogen). The upstream primer had the sequence 5′-ggggacaagtttgtacaaaaaagcaggctAAGGGGGAGAACACGATGAATG-3′ (SEQ ID NO: 19; RBS and start codon underlined), and the downstream primer had the sequence 5′-ggggaccactttgtacaagaaagctgggtgtgccgaccaaagttcaggac-3′ (SEQ ID NO: 20). The resulting amplicon was transferred to pDONRtet using standard BP reaction conditions (Invitrogen), followed by transformation of competent DH10B E. coli and selection on LB plates containing 10 μg/ml of tetracycline. Cloned amplicons were transferred to pNFd13 (Ts pE194 ori) under the spac promoter using the standard LR reaction (Invitrogen), followed by transformation of DH10B E. coli and selection on LB plates containing 50 μg/ml Km. Integration of the plasmid into the targeted B. anthracis locus was done at 39° C. essentially as described in Fisher and Hanna, J. Bacteriol. 187, 8055 (2005). The nos deletion in the resulting 34F2 cbs/cse::pNFd13 strain was obtained as described in Shatalin et al., Proc. Natl. Acad. Sci. U.S.A. 105, 1009 (2008), except that a spectinomycin cassette was used instead of Km.
The P. aeruginosa PA-14 wt and PA-14 cbs mutant strains were obtained from Massachusetts General Hospital's P. aeruginosa mutants collection (Department of Molecular Biology, Richard B. Simches Research Center.
Generation of Growth Curves:
Growth curves were obtained on a Bioscreen C automated growth analysis system (Oy Growth Curves Ab Ltd.). Subcultures of specified strains Were grown overnight at 30° C., diluted 1:100 in fresh media and grown to OD600 ˜0.8 at 37° C. Cultures were then diluted 1:100 in LB or BHI media with appropriate antibiotics or reagents as described in the text or figure legends. 300 μl of each mixture was inoculated into honeycomb wells in triplicate and grown at 37° C. with maximum shaking on the platform of the Bioscreen C instrument. OD600 values determined were recorded automatically at different times and the means of the triplicate cultures were plotted.
Generation of Survival Curves:
Overnight cultures were inoculated into LB supplemented with 0.5 mM Cys and grown at 37° C. to ˜2×107 cells per ml. Then cells were diluted 100 times in sterile 0.9% NaCl and treated by Gm (50 μg/ml) or Km (250 μg/ml) or Cm (70 μg/ml for P. aeruginosa strains and 35 μg/ml for other strains) at room temperature in, the presence or absence of 0.2 mM of NaHS. CBS/CSE inhibitors, 0.5 mM PAG and 50 μM AOAA, were used in experiments with WT S. aureus and P. aeruginosa. At different times samples of cells were placed on LB agar and incubated at 37° C. for 16-18 hours. Cell survival was determined by counting CFU and is shown as the mean±SD from three independent experiments.
H2S Detection:
To monitor H2S production in wild type (wt) and mutant cells, a lead acetate detection method was used (7). Paper strips saturated with 2% Pb(Ac)2 were affixed to the inner wall of a culture tube, above the level of a liquid culture of wild type or mutant bacteria. Overnight cultures were diluted 1:50 in LB or BHI without or with Cys (25-500 μM), and incubated 18-20 hours at 37° C. with aeration. Stained paper strips were scanned and quantified with an AlphaImager (Imgen Technologies; Alexandria, Va.). The results were normalized per ODs.
Assay of Chromosomal DNA Damage:
The assay was done as described previously (28). Cells were grown to OD600=1.0 in LB media and treated with H2O2 (1 mM—E. coli; 2 mM—B. anthracis; 2.5 mM—P. aeruginosa) or antibiotics Em (1 Mg/ml—B. anthracis); Ap (10 μg/ml—E. coli; 125 μg/ml—P. aeruginosa) for 4 hours. Cells were equalized to OD600=1.0 by addition of fresh media and the cells from 2 ml of the equalized cultures were harvested by centrifugation. Total genomic DNA was isolated from the bacteria pellets with a QIAamp DNA Mini Kit (Qiagen; Valencia, Calif.) or according the GenElute Bacterial Genomic DNA Kit protocol for Gram Positive Bacteria (Sigma; St. Louis, Mo.). DNA was extracted with phenol/chloroform and quantified with the PicoGreen dsDNA quantitation reagent (Molecular Probes®; Invitrogen; Carlsbad, Calif.) and λ phage DNA as a standard. An arbitrarily chosen, 10-kb, 6-kb, and 5.8-kb fragments of E. coli, B. anthracis and P. aeruginose genomes, respectively, were used for qPCR. Primer sequences were as follows: E. coli 5′-ttccattgggatgtagatgctg-3′ (forward; SEQ ID NO: 13) and 5′-ggtaaaagagtcaagggaagaacc-3′ (reverse; SEQ ID NO: 14), B. anthracis 5′-acgattgacttctctcacttcggt-3′ (forward; SEQ ID NO: 15) and 5′-aaacatttgctcttgatgtcctgga-3′ (reverse; SEQ ID NO: 16), P. aeruginose 5′-ctttgcaacctgtacatgccttg-3′ (forward; SEQ ID NO: 17) and 5′-catcgtagtagttgatcggatggac-3′ (reverse; SEQ ID NO: 18). PCR was performed with Phusion DNA polymerase (Finnenzymes). The 50-μl PCR mixture contained 0.5 ng of genomic DNA as a template, 1.5 μM primers, 200 μM dNTPs (Fermentas), Phusion GC PCR buffer, and 0.5 μl of DNA polymerase. DNA was subjected to 29 to Cycles of PCR (98° C. for 30 seconds, 53° C. for 30 seconds, and 72° C. for 3 or 9 minutes). PCR products were separated by electrophoresis in a 0.8% or 1% agarose gel, stained with ethidium bromide, scanned, and quantified with an AlphaImager (Imgen Technologies).
Electrophoresis Assay of DNA Damage In Vitro (Fenton Reaction):
The nicking reaction mixture contained 0.1 μg pBR322 plasmid DNA in 20 mM Tris HCl buffer, pH 8.0, 30 μM FeCl3, 4 mM Cys and 4 mM H2O2, in the presence or absence of 0.2 mM NaHS. After incubating the mixture at room temperature for 15 min the DNA samples were applied to an 0.8% agarose gel in a TAE buffer system, and electrophoresis was performed at 80 V for 30 min. Following electrophoresis, gels were stained with ethidium bromide for 30 min. After washing, the bands were visualized in a UV transilluminator. The modification of the fluorescence intensity of the bands is due to DNA strand breakage that leads to a decrease in the proportion of the supercoiled form and to an increase in the relaxed form produced by a nick in one strand. Pulse Field Gel Electrophoresis (PFGE): E. coli cells were grown to OD600-0.8 at 32° C. 200 μM of NaHS, 10 μg/ml of Ap, or 1 mM of peroxide were added as indicated and cells were allowed to grow at 37° C. for 4 more hours. Agarose plugs were prepared using 3×108 cells/plug and treated with lysozyme and Proteinase K according to the BioRad CHEF protocol (Bio-Rad Laboratories; Hercules, Calif.). Linearized 4.6 Mb E. coli chromosomal DNA marker was made by digesting the genomic DNA plug of SMR8476 strain with I-SceI endonuclease. DNA fragments were separated on a 1% agarose gel in 0.5×TBE at 14° C. for 24 hours at 6 V/cm using a Bio-Rad CHEF-DR II angle system with a 2.8-26.3 second linear switch time ramp. Gels were stained with EtBr and visualized with UV-trans-illumination. DNA was quantified (integrated density of the linear products) using Image) software.
CBS/CSE Expression Assay:
B. anthracic Stern 34F2 cbs/cse::pNFd13 cells (see above) carrying the lacZ reporter under the native cbs promoter were used. To monitor expression of cbs-lacZ fusions, bacteria were grown overnight at 37° C. in LB medium, washed and diluted 1:25 in fresh medium containing erythromycin or H2O2 at the specified concentrations. Cultures were incubated for 2-2.5 hr at 37° C. (OD600=0.5-0.6) before measurement of β-galactosidase activity. Shown β-galactosidase activities correspond to mean values from at least three independent experiments. Error bars correspond to the standard deviation.
Assays of the SOS Response:
Overnight cultures of strain 10973 harboring a recA′-gfp chromosomal fusion (18) was grown in Luria broth with kanamycin (20 μg/ml). The cultures were diluted in LB to OD600=0.1 and grown at 37° C. to an OD600 ˜1.0. Asp (3 mM), Gm (5 μg/ml) and NaHS (200 μM) were added at OD600=0.5. Fluorescence was measured (Ex. wave length: 480 nm and Em. wavelength: 520 nm) in a Perkin Elmer LS55 spectrofluorometer (Perkin Elmer; Waltham, Mass.). Fluorescence values were normalized to OD600 values and plotted.
Catalase Activity Assay:
Degradation of H2O2 was monitored in real time as a decrease in absorbance at 240 nm (37). Aliquots of extracts to be monitored or of pure catalase were mixed with 50 mM phosphate buffer (pH 7.0) and placed into a 1 ml quartz cuvette. 40 mM H2O2 solution was added and the kinetics of its degradation recorded. Total H2O2 degrading activity was measured as the decrease of H2O2 concentration per milligram of total protein per second. OD240 was converted to the concentration of H2O2 according to the calibration curve (10 mM H2O2=0.36 OD240).
Superoxide Dismutase (SOD) Activity Assay:
Superoxide dismutase activity of cell extracts was determined using the Cayman Chemical Company Superoxide Dismutase Assay Kit (Cat#706002; Cayman Chemical Co.; Ann Arbor, Mich.) according to the Manufacture's recommendations.
Chemicals and Reagents:
All chemicals were from Sigma, except PYO and the Superoxide dismutase assay kit, which were purchased from Cayman. The CuFL NO sensor was provided by Dr. S. Lippard, MIT. See, Shatalin et al., Proc. Natl. Acad. Sci. U.S.A. 105, 1009 (2008).
To determine whether CBS, CSE, or 3MST produces H2S in bacteria, each enzyme was inactivated genetically or chemically in four clinically relevant and evolutionarily distant pathogenic species: Bacillus anthracis (Sterne), Pseudomonas aeruginosa (PA14), Staphylococcus aureus (MSSA RN4220 and MRSA MW2), and Escherichia coli (MG1655). The first three species have the CBS/CSE operon, but not 3MST, whereas E. coli carries 3MST, but not CBS/CSE. The chromosomal organization of H2S genes (
Further experiments demonstrated that 3MST is the major source of H2S in E. coli. In addition to 3MST (sseA), the E. coli genome encodes several other enzymes that could potentially generate H2S. These include cystathionine-γ-synthase MetB, cystathionine-β-synthases MetC and MalY, and CysM, CysK, and TnaA, which all are cysteine desulfurases (27). Pb(Ac)2 analysis of individual strains harboring knockouts of each of these genes indicates that 3MST is the major source of H2S production under specified growth conditions (
To elucidate the physiological role of H2S, wt and 3MST-deficient E. coli were compared in a phenotype microarray (PMA) (
H2S-mediated cytoprotection resembles that of NO, which defends certain Gram-positive bacteria against some of the same antibiotics as does H2S (I. Gusarov, K. Shatalin, M. Starodubtseva, E. Nudler, Science 325, 1380 (2009)). NO-mediated protection relies, in part, on its ability to defend bacteria against oxidative stress imposed by antibiotics (I. Gusarov, E. Nudler, Proc. Natl. Acad. Sci. U.S.A. 102, 13855 (2005); K. Shatalin et al., Proc. Natl. Acad. Sci. U.S.A. 105, 1009 (2008); and 1. Gusarov, K. Shatalin, M. Starodubtseva, E. Nudler, Science 325, 1380 (2009)). To examine whether H2S acts by a similar mechanism, detailed analyses of its effect on bacterial killing by representative antibiotics (gentamicin (Gm), ampicillin (Ap), and nalidixic acid (NA)) were conducted (
Consistently, H2S-generating enzymes provided protection against antibiotics only under aerobic conditions. As shown in
The above results suggested that H2S bolsters the antioxidant capacity of bacterial cells. Indeed, H2S-deficient B. anthracis, E. coli, S. aureus, and P. aeruginosa displayed higher susceptibility to peroxide than their wt counterparts, whereas NaHS rendered them more resistant to the peroxide (
Formation of double-strandDNAbreaks (DSBs) is the primary cause of bacterial death from peroxide (O. I. Aruoma, B. Halliwell, E. Gajewski, M. Dizdaroglu, J. Biol. Chem. 264, 20509 (1989); and S. I. Liochev, I. Fridovich, IUBMB Life 48, 157 (1999)). These DSBs result from the Fenton reaction (J. A. Imlay, Annu. Rev. Microbiol. 57, 395 (2003)), which also can be triggered by antibiotics (I. Gusarov, K. Shatalin, M. Starodubtseva, E. Nudler, Science 325, 1380 (2009); M. A. Kdhanski, D. J. Dwyer, B. Hayete, C. A. Lawrence, J. J. Collins, Cell 130, 797 (2007); M. A. Kohanski, J. Dwyer, J. J. Collins, Nat. Rev. Microbiol. 8, 423 (2010); and B. W. Davies et al., Mol. Cell. 36, 845 (2009)). To examine whether H2S protects bacteria from the damaging Fenton reaction, chromosomal DNA integrity was monitored by pulsed-field gel electrophoresis (PFGE) (
The antioxidant effect of endogenous H2S can also be explained, in part, by its ability to augment the activities of catalase and superoxide dismutase (SOD) (
The extent of genomic DSBs can be assessed by PCR of an arbitrarily chosen segment of the bacterial chromosome (I. Gusarov, E. Nudler, Proc. Natl. Acad. Sci. U.S.A. 102, 13855 (2005)). In this assay, equal amounts of genomic DNA from wt and mutant cells grown with or without antibiotics or H2O2 were isolated for PCR. The number of reaction cycles was selected so that DSBs influenced the yield of the final PCR product. The yield of the PCR product from exponentially growing, H2S-deficient B. anthracis, E. coli, or P. aeruginosa treated with H2O2 or antibiotics was substantially lower than that from similarly treated wt cells (
To further implicate H2S in genome maintenance, its effect on the SOS response was monitored using a chromosomal recA-gfp gene fusion (M. Kostrzynska, K. T. Leung, H. Lee, J. T. Trevors, J. Microbiol. Methods 48, 43 (2002)). Addition of a sub-lethal amount of gentamicin induced significant GFP fluorescence (
Thus, H2S increases bacteria resistance to oxidative stress and antibiotics by a dual mechanism (
Taken together, these results directly implicate endogenous H2S in the mitigation of chromosomal damage inflicted by antibiotics, and indicate that endogenous H2S renders bacteria more resistant to oxidative stress and antibiotics by suppressing the DNA-damaging Fenton reaction.
This cytoprotective mechanism of H2S parallels that of NO (I. Gusarov, K. Shatalin, M. Starodubtseva, E. Nudler, Science 325, 1380 (2009)), which suggests that bacteria that produce both gases may benefit from their synergistic action. To test this hypothesis, the effect of simultaneously inhibiting H2S and NO on B. anthracis growth was examined. A strain of B. anthracis in which both bacterial nitric oxide synthase (bNOS) and CBS/CSE were genetically inactivated could not be generated, suggesting that the absence of both gases is incompatible with B. anthracis survival. Indeed, B. anthracis Δnos cells containing an isopropylb-D-thiogalactopyranoside (IPTG)-inducible CBS/CSE conditional knockout could grow only in the presence of IPTG (
Because endogenous H2S diminishes the effectiveness of many clinically used antibiotics, the inhibition of this “gaskeeper” should be considered as an augmentation therapy against a broad range of pathogens. Bacterial CBS, CSE, and 3MST have diverged substantially from their mammalian counterparts (
The results presented herein suggest several non-mutually exclusive mechanisms that could explain the H2S-mediated defense against oxidative stress (
To further investigate the validity of this conclusion, the effect of H2S on the Fenton reaction in vitro was examined. Hydrogen peroxide alone or together with Fe3+ did not produce significant strand brakes in pBR322DNA (
To examine the role of free Cys in oxidative stress associated with antibiotics, its effect on gentamicin (Gm) toxicity was studied in wt and 3MST-deficient cells. WT E. coli became more resistant to the antibiotic in Cys-enriched media (0.5 mM L-cysteine) than in regular LB (<20 μM L-cysteine) (
A study was conducted to demonstrate potentiation of antibiotics by AOAA. E. faecalis (ATCC 29212) cultures were prepared at initial inoculums of ˜5×105 CFU/ml in Mueller-Hinton II Broth (cation-adjusted). Aliquots of 190 μl were dispensed to each well of a fresh set of plates, and 10 μl of an antibiotic solution (levofloxacin, meropenem, gentamicin, piperacillin, linezolid, cefepime, daptomycin, vancomycin, or chloramphemicol) were transferred to the corresponding wells in each bacterial plate (
Minimum inhibitory concentration (MIC) determination was conducted as described in Example 7, above. To assess antibiotic potentiation by AOAA and PAG, MIC was determined for nine antibiotics (levofloxacin, meropenem, gentamicin, piperacillin, linezolid, cefepime, daptomycin, vancomycin, and chloramphemicol) and four bacterial strains (E. faecium (A2373), E. faecalis (ATCC 9212), S. pneumoniae (ATCC 49619), and P. aeruginosa (PAO1)) in the presence of antibiotic alone, 1 mM PAG, 0.1 mM AOAA, or 0.5 mM PAG+0.05 mM AOAA. The results are summarized in Table 3. The smaller the MIC value, the stronger the effect of antibiotic potentiation. As shown in Table 3, in most cases, AOAA has a stronger antibiotic potentiation effect than PAG or even AOAA+PAG. These data also suggest that inhibiting CBS (targeted by AOAA) may be more clinically relevant than inhibiting CSE (targeted by PAG) (
E. faecium A2373
E. faecalis ATCC29212
S. pneumoniae ATCC49619
P. aeruginosa PAO1
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.
This application claims the benefit of U.S. Provisional Patent Application No. 61/438,524, filed Feb. 1, 2011, which is incorporated herein by reference in its entirety.
This invention was made with government support under grant No. 5DP1OD000799, awarded by the National Institutes of Health. The government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/US2012/023542 | 2/1/2012 | WO | 00 | 11/15/2013 |
Number | Date | Country | |
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61438524 | Feb 2011 | US |