WO1999054479A2

WO1999054479A2 - INHIBITION OF ALKYL HYDROPEROXIDE REDUCTASE, SUBUNIT C (AhpC) TO IMPART SUSCEPTIBILITY TO ANTIMICROBIAL REACTIVE NITROGEN INTERMEDIATES

Info

Publication number: WO1999054479A2
Application number: PCT/US1999/008704
Authority: WO
Inventors: Carl F. Nathan; Qiao-Wen Xie; Lei Chen
Original assignee: Cornell Research Foundation, Inc.
Priority date: 1998-04-21
Filing date: 1999-04-21
Publication date: 1999-10-28
Also published as: WO1999054479A9; WO1999054479A3

Abstract

The present invention relates to the use of alkyl hydroperoxide reductase subunit C (AhpC) proteins or polypeptides to confer resistance against antimicrobial reactive nitrogen intermediates (RNI). Also disclosed is a DNA molecule encoding alkyl hydroperoxide reductase subunit C from Mycobacterium tuberculosis and conferring on Mycobacterium tuberculosis resistance to antimicrobial RNI. Such DNA encoding AhpC can be used to screen for drugs that inhibit the ability of AhpC to confer resistance to antimicrobial RNI. Such drugs can be used to treat tuberculosis by sensitizing M. tuberculosis to RNI produced by host cells or delivered therapeutically by inhalation or by oral or other parenteral routes. Alternatively, therapeutics can be developed to treat gastric infection. The protein encoded by this DNA molecule is useful in vaccines to prevent infection by Mycobacterium tuberculosis, while the antibodies raised against this protein can be employed in passively immunizing those already infected by the organism. These proteins, antibodies, and DNA molecules may be utilized in diagnostic assays to detect Mycobacterium tuberculosis in tissue or bodily fluids. The protein or polypeptide is also useful as a therapeutic in treating conditions mediated by the production of reactive nitrogen intermediates.

Description

INHIBITION OF ALKYL HYDROPEROXIDE REDUCTASE, SUBUNIT C (AhpC) TO IMPART SUSCEPTIBILITY TO ANTIMICROBIAL REACTIVE

NITROGEN INTERMEDIATES

This application claims the benefit of U.S. Provisional Patent

Application Serial No. 60/082,573, filed April 21, 1998.

This invention arose out of research sponsored by the National

Institutes of Health (Grant No. HL51967). The U.S. Government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to the inhibition of alkyl hydroperoxide reductase subunit C (AhpC) to impart susceptibility to antimicrobial reactive nitrogen intermediates. A DNA molecule encoding AphC from Mycobacterium tuberculosis is also disclosed.

BACKGROUND OF THE INVENTION

Mycobacterium tuberculosis. Helicobacter pyrori. and Acid Nitrite.

Mycobacterium tuberculosis and Helicobacter pylori share remarkable statistics. Each infects about one-third of the human population, persists for decades, and causes disease in a small fraction of those infected. Despite the low disease rate, Mycobacterium tuberculosis is the single leading cause of death from infection, while Helicobacter pylori is the leading cause of gastric ulcers and a major contributor to duodenal ulcers, gastric cancer, and gastric lymphoma. Thus, these two bacteria together account for an extraordinary proportion of the chronic infectious morbidity and mortality of humankind. Both organisms provoke inflammation that leads human macrophages to express the high output isoform of nitric oxide synthase (iNOS or NOS2).

From the point of view of pathogenesis, what most unites these disparate organisms is that both must cope with reactive nitrogen intermediates ("RNI") in the context of acid. Mycobacterium tuberculosis is a facultative intracellular parasite of macrophages that encounters RNI and acid (pH > 4.5) in the phagolysosome of activated macrophages. Although some mycobacteria frustrate phagosome acidification in nonactivated macrophages (Sturgill-Koszicki ARI 151), activation of the macrophage overcomes this effect and acidification is preserved. Helicobacter pylori is a Gram Negative extracellular pathogen specialized to endure the harsh environment of the stomach, where the pH can be far lower ( > 1) and where RNI arise via a recently recognized enterosalivary cycle. In this enterosalivary cycle of RNI. nitrate ingested in the diet (for example, in green vegetables) is absorbed into the blood plasma to join the nitrate accumulating from the action of the three isoforms of NOS. Salivary glands extract plasma nitrate up a 10-fold gradient and secrete it into the mouth. Commensal bacteria deep in the posterior sulci of the tongue reduce the nitrate to nitrite, which dissolves in the oral saliva and is swallowed. In the stomach, protonation produces HNO₂ (nitrous acid), which spontaneously dismutates to generate nitric oxide (NO), nitrogen dioxide (NO₂), N7O₃, N O , which very likely form S-nitrosothiols (RSNO) upon encountering thiol-containing moieties in the gastric mucus. These RNI collectively serve to render the diet nearly sterile. The sterilization of gastric contents was a function generally ascribed to gastric acid alone before its synergy with nitrite was discovered.

There are two basic clinical patterns that follow infection with Mycobacterium tuberculosis. In the majority of cases, inhaled tubercle bacilli ingested by phagocytic alveolar macrophages are either directly killed or grow intracellularly to a limited extent in local lesions called tubercles. Infrequently in children and immunocompromised individuals, there is early hematogenous dissemination with the formation of small miliary (millet-like) lesions or life -threatening meningitis. More commonly, within 2 to 6 weeks after infection, cell-mediated immunity develops, and infiltration into the lesion of immune lymphocytes and activated macrophages results in the killing of most bacilli and the walling-off of this primary infection, often without symptoms being noted by the infected individual. Skin-test reactivity to a purified protein derivative ("PPD") of tuberculin and, in some cases, X-ray evidence of a healed, calcified lesion provide the only evidence of the infection. Nevertheless, to an unknown extent, dormant but viable Mycobacterium tuberculosis bacilli persist. - j -

The second pattern is the progression or breakdown of infection to active disease. Individuals with normal immune systems who are infected with Mycobacterium tuberculosis have a 10% lifetime risk of developing the disease.

In either case, the bacilli spread from the site of initial infection in the lung through the lymphatics or blood to other parts of the body, the apex of the lung and the regional lymph node being favored sites. Extrapulmonary tuberculosis of the pleura, lymphatics, bone, genito-urinary system, meninges, peritoneum, or skin occurs in about 15% of tuberculosis patients. Although many bacilli are killed, a large proportion of infiltrating phagocytes and lung parenchymal cells die as well, producing characteristic solid caseous (cheese-like) necrosis in which bacilli may survive but not flourish. If a protective immune response dominates, the lesion may be arrested, albeit with some residual damage to the lung or other tissue. If the necrotic reaction expands, breaking into a bronchus, a cavity is produced in the lung, allowing large numbers of bacilli to spread with coughing to the outside. In the worst case, the solid tissue, perhaps a result of released hydrolases from inflammatory cells, may liquefy, which creates a rich medium for the proliferation of bacilli, perhaps reaching 10⁹ per milliliter. The pathologic and inflammatory processes produce the characteristic weakness, fever, chest pain, cough, and, when a blood vessel is eroded, bloody sputum.

RNI Resistance

RNI generated by NOS2 are essential for the temporary control of tuberculosis in mice (Chan, J., et al., "Effects of Nitric Oxide Synthase Inhibitors on Murine Infection with Mycobacterium tuberculosis," Infect. Immun., 63:736-40 (1995); MacMicking, J. D.. "Identification of NOS2 as a Protective Locus Against Tuberculosis," Proc. Natl. Acad. Sci. USA. 94:5243-48 (1997)). Enzymatically active NOS2 is expressed in the tuberculous human lung within macrophages, the cells ultimately responsible for controlling the infection (Nicholson, S., et al., "Inducible Nitric Oxide Synthase in Pulmonary Alveolar Macrophages From Patients with

Tuberculosis," J. Exp. Med., 183:2293-302 (1996)), and can control the replication of mycobacteria in human pulmonary macrophases in vitro (Nozaki, Y., et al., "Mechanism of Nitric Oxide-Dependent Killing of Mycobacterium BCG in Human Alveolar Macrophages," Infect. Immun., 65:3644-47 (1997)). Human macrophages from lungs of patients with tuberculosis release very large amounts of nitric oxide (Wang, et al.. "Increased Exhaled Nitric Oxide in Active Pulmonary Tuberculosis due to Inducible NO Synthase Upregulation in Alveolar Macrophages," Eur. Respir. J. 1 1 :809-815 (1998)).

Among all bacteria that are ingested, Helicobacter pylori appears to be the most capable of resisting the synergistic antibacterial action of gastric acid plus gastric RNI. Yet. there has been no understanding of how Helicobacter pylori may survive RNI. The resistance of Helicobacter pylori to acid, which remains incompletely explained, has been studied in isolation, not taking into account that acidity appears to be only one part of a synergistic interaction of the antimicrobial system of the stomach, RNI representing the other partner.

Medical importance of new treatments for infection by Mycobacterium tuberculosis and Helicobacter pylori.

For Mycobacterium tuberculosis, the rapid emergence of multidrug resistance is associated with mortality rates near 50% even in optimally treated patients with mycobacterial disease. The intersection of the tuberculosis pandemic with the HIV epidemic threatens even higher rates of active tuberculosis in the infected population, which in turn may increase the rate of infection among all people in contact, regardless of their medical or economic status. New anti -tuberculous drugs are urgently needed. The problem presented by Helicobacter pylori is less urgent, yet vast, in view of the numbers infected, at risk of sequelae, or already afflicted. The therapy of peptic ulcer disease has been revolutionized by the discovery of Helicobacter pylori 's pathogenic role, but problems remain with the resultant recourse to antibiotics, including drug cost, patient compliance, side effects, bacterial resistance, and recrudescence of infection.

Alkyl hydroperoxide reductase was first cloned and purified from S. typhimurium and E. coli as the product of genes induced by oxidative stress under the positive control of the oxyR gene (Storz, G., et al., "An Alkyl Hydroperoxide Reductase Induced by Oxidative Stress in Salmonella typhimurium and Escherichia coli: Genetic Characterization and Cloning of ahp," J. BacterioL, 181 :2049-55 (1989); Jacobson, F. S., et al., "An Alkyl Hydroperoxide Reductase From Salmonella typhimurium Involved in the Defense of DNA Against Oxidative Damages. Purification and Properties." J. Biol. Chem.. 264: 1488-96 (1989); Tartaglia, L. A., et al., "Alkyl Hydroperoxide Reductase from Salmonella tymphimurium. Sequence and Homology to Thioredoxin Reductase and Other Flavoprotein Disulfide

Oxidoreductases," J. Biol. Chem.. 265:10535-40 (1990)). Hydroperoxides are mutagenic in bacteria (Farr, S. B., "Oxidative Stress Responses in Escherichia coli and Salmonella typhimurium '^'' Microbiol. Rev., 55:561-85 (1991)). Overexpression of alkyl hydroperoxide reductase activity suppressed spontaneous mutagenesis associated with aerobic metabolism in AoxyR mutants of S. typhimurium and E. coli (Storz. G.. et al., "Spontaneous Mutagenesis and Oxidative Damage to DNA to Salmonella typhimurium,^'" Proc. Natl. Acad. Sci. USA, 84:917-21 (1987); Greenberg, J. T., "Overexpression of Peroxide-Scavenging Enzymes in Escherichia coli Suppresses Spontaneous Mutagenesis and Sensitivity to Redox-Cycling Agents in oxy7?-mutants." EMBO J. 7:2611-17 (1988)). The isolated enzyme uses NAD(P)H to reduce alkyl hydroperoxides to the corresponding alcohols. This activity is manifest by a tetramer comprised of two 57-kDa monomers of the NAD(P)H-oxidizing flavoprotein AhpF, and two 21-kDa monomers of its peroxide-reducing partner. AhpC. Only a few homologs of AhpF have been identified (Chae, H. Z.. et al., "Cloning and Sequencing of Thiol-Specific Antioxidant From Mammalian Brain: Alkyl Hydroperoxide Reductase and Thiol-Specific Antioxidant Define a Large Family of Antioxidant Enzymes," Proc. Natl. Acad. Sci. USA, 91 :7017-21 (1994)). In contrast, AhpC homologs are widely distributed among prokaryotes (Chae, H. Z., et al., "Thioredoxin-Dependent Peroxide Reductase From Yeast," J. Biol. Chem.. 269:27670-678 (1994)), and AhpC is ~40%> identical to thioredoxin peroxidase from yeast (Chae, H. Z.. et al., "Cloning and Sequencing of Thiol-Specific Antioxidant From Mammalian Brain: Alkyl Hydroperoxide Reductase and Thiol-Specific Antioxidant Define a Large Family of Antioxidant Enzymes," Proc. Natl. Acad. Sci. USA, 91 :7017-21 (1994)), rat (Chae. H. Z., et al., "Cloning and Sequencing of Thiol- Specific Antioxidant From Mammalian Brain: Alkyl Hydroperoxide Reductase and Thiol-Specific Antioxidant Define a Large Family of Antioxidant Enzymes," Proc. Natl. Acad. Sci. USA, 91 :7017-21 (1994)), plants amoebae, nematodes. rodents, and humans (Chae, H. Z., et al., "Cloning and Sequencing of Thiol-Specific Antioxidant From Mammalian Brain: Alkyl Hydroperoxide Reductase and Thiol-Specific Antioxidant Define a Large Family of Antioxidant Enzymes." Proc. Natl. Acad. Sci. USA, 91 :7017-21 (1994); Lim, Y. S., et al., "The Thiol-Specific Antioxidant Protein from Human Brain: Gene Cloning and Analysis of Conserved Cysteine Region." Gene, 140:279-84 (1994); Jin, D. Y., et al., "Regulatory Role for a Novel Human Thioredoxin Peroxidase in NF-κB activation," J. Biol. Chem.. 272:30952-61 (1997)). Therefore, homologs of AphC define a large family of antioxidants present in organisms from all kingdoms. Herein a new function is demonstrated for ahpC from M. tuberculosis, H. pylori, and S. typhimurium: the ability to protect cells from RNI.

SUMMARY OF THE INVENTION

There are two general aspects of the present invention. One relates to the discovery that alkyl hydroperoxide reductase subunit C proteins or polypeptides imparts susceptibility to antimicrobial reactive nitrogen intermediates. The other aspect of the present invention is to the alkyl hydroperoxide reductase subunit C protein or polypeptide from Mycobacterium tuberculosis as well as the DNA molecule encoding that protein or polypeptide. The discovery that the inhibition of alkyl hydroperoxide reductase subunit C proteins or polypeptides impart susceptibility to reactive nitrogen intermediates can be utilized in a number of different ways. For example, proteins or polypeptides of that type can be administered, with or without a pharmaceutically- acceptable carrier, to a mammal under conditions effective to treat septic shock or stroke, or quenching overproduction of nitric oxides in response to infection by bacterial pathogens. The DNA molecule encoding an alkyl hydroperoxide reductase subunit C protein or polypeptide can be used to screen therapeutics for antibacterial effect by providing a growth medium containing nitric oxide and a test therapeutic, preparing host cells transformed with the DNA molecule, placing the cells in a growth medium, and determining whether the cells survive.

The present invention relates to isolated DNA molecules encoding alkyl hydroperoxide reductase subunit C useful in conferring on Mycobacterium tuberculosis resistance against antimicrobial reactive nitrogen intermediates (e.g.^', nitric oxide (NO), nitrite (NO?^"), nitrosonium (NO⁺), S-nitrosothiols (RSNO), nitrogen dioxide (NO₂), dinitrogen trioxide (N O₃), and dinitrogen tetraoxide (N₂O₄)) as well as isolated proteins or polypeptides encoded by these isolated DNA molecules. The molecule can be inserted as heterologous DNA in an expression vector forming a recombinant DNA expression system for producing the proteins or polypeptides. Likewise, the heterologous DNA, usually inserted in an expression vector to form a recombinant DNA expression system, can be incorporated in a cell to achieve this objective. The isolated DNA in a plasmid or isolated protein or polypeptide of the present invention can be combined with a pharmaceutically-acceptable carrier to form a vaccine or used alone for administration to mammals, particularly humans, for preventing infection by Mycobacterium tuberculosis. Alternatively, the protein or polypeptide of the present invention can be used to raise an antibody or a binding portion thereof. The antibody or binding portion thereof may be used alone or combined with a pharmaceutically-acceptable carrier to treat mammals, particularly humans, already exposed to Mycobacterium tuberculosis to induce a passive immunity to prevent disease occurrence.

The proteins or polypeptides of the present invention or the antibodies or binding portions thereof raised against them can also be utilized in a method for detection of Mycobacterium tuberculosis in a sample of tissue or body fluids. When the protein or polypeptide is utilized, it is provided as an antigen. Any reaction with the antigen or the antibody is detected using an assay system which indicates the presence of Mycobacterium tuberculosis in the sample. Alternatively, Mycobacterium tuberculosis can be detected in such a sample by providing a nucleotide sequence of the gene conferring on Mycobacterium tuberculosis resistance against antimicrobial reactive nitrogen intermediates as a probe in a nucleic acid hybridization assay or a gene amplication detection procedure (e.g., using a polymerase chain reaction procedure). Any reaction with the probe is detected so that the presence of Mycobacterium tuberculosis in the sample is indicated.

Isolation of the DNA molecules of the present invention constitutes a significant advance in the treatment and detection of such bacteria. It also provides the basis for a vaccine to prevent infection by Mycobacterium tuberculosis and a^' pharmaceutical agent for passive immunization for those exposed to Mycobacterium tuberculosis. The proteins utilized in the vaccine or to produce the pharmaceutical agent can be produced at high levels using recombinant DNA technology. In diagnostic applications, the proteins or polypeptides of the present invention as well as antibodies and binding portions thereof against them permit rapid determination of whether a particular individual is infected with Mycobacterium tuberculosis. Moreover, such detection can be carried out without requiring an examination of the individual being tested for an antibody response. For both tuberculosis and Helicobacter pylori, RNI resistance genes in pathogens could serve as targets for drugs that would act to sensitize the pathogen to the natural antimicrobial actions of the host. For example, tuberculosis might become more susceptible to iNOS expressed by macrophages, and Helicobacter pylori might become more susceptible to the combination of gastric acid and nitrite, the latter furnished by the normal enterosalivary cycle or ingested as a pharmaceutical additive. Rendering Helicobacter pylori as susceptible to natural gastric sterilization as E. coli is envisioned.

The present invention includes the cloning and characterization of a gene or genes from the bacterium Helicobacter pylori that confer(s) on this organism its resistance to a major natural antibacterial defense system of the stomach. The invention includes the idea of treating gastric ulcer disease caused by Helicobacter pylori (and preventing malignancy related thereto) by inhibiting the process by which the bacterium resists the host's ability to eliminate it. The natural antibacterial defense system of the stomach relevant to the present invention is the reaction of salivary or ingested nitrite with gastic acid. Salivary nitrite arises from bacterial reduction of salivary nitrate in the mouth. Salivary nitrate derives from dietary nitrate and endogenous sources such as nitric oxide synthases. In the stomach, the reaction of nitrite with acid generates nitric oxide, nitrogen dioxide, dinitrogen trioxide, and, probably, S-nitrosothiols, any or all of which alone or in combination with acid are broadly microbicidal. Helicobacter pylori possesses an unusual and as yet unexplained degree of resistance to this microbicidal system. The invention includes combining a pharmacologic agent that inhibits the major RNI-resistance mechanism(s) of Helicobacter pylori with an oral dose of nitrite in order to eradicate Helicobacter pylori from the stomach. The pharmacologic agent that inhibits the major RNI-resistance mechanism(s) of Helicobacter pylori need not be absorbed into the bloodstream to have this effect. A nonabsorbable compound would be unlikely to have systemic side effects in the body. The pharmacologic agent that inhibits the major RNI-resistance mechanism(s) of Helicobacter pylori need not be specific for the RNI-resistance mechanisms of Helicobacter pylori, but could also inhibit the RNI-resistance mechanisms of other bacteria without the danger of exerting broad-spectrum antibacterial activity in the gastrointestinal tract. Such an agent would effectively be restricted to the treatment of Helicobacter pylori infection, because it would have no antibacterial effect by itself; it would only sensitize bacteria to RNI. Among sites in the gastrointestinal tract that are colonized by bacteria, only the stomach normally contains RNI. Any nitrite that passed from the stomach into the rest of the gastrointestinal tract would be neutralized as the gastric contents are alkalinized by the pancreatic secretions. Thus, the normal commensal flora of the gastrointestinal tract should be spared, and some of the major side effects associated with current antimicrobial treatment of Helicobacter pylori infection would be avoided.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the genomic environment of ahpC in M. tuberculosis contrasted with that in S. typhimurium. Clone pMtb-aphC contains 3934 bp of DNA from M. tuberculosis including the coding region of ahpC and four additional putative coding regions (I-IV) with the indicated number of codons (aa) in the orientation shown by the arrows (also see GenBank Z81451). Gene III was earlier termed ahpD (GenBank U44840), but bears no relationship to ahpC or ahpF. The latter comprise a bicistronic operon in S. typhimurium (GenBank J05478). Figures 2A and B show the expression of AhpCivi_tb in recombinant E. coli and native mycobacteria. In Figure 2A. AhpCivitb is purified. Lysates of E. coli Ml 5 (pAhpC-3) treated with IPTG (lane 2) or not (lane 1) were loaded onto a 12% gel. Lanes 3-7 carry recombinant AhpCivi_tb eluted with 100 mM EDTA from Ni⁺- NTA resin onto which the IPTG-treated Ml 5 (pAhpC-3) lysate had been passed. Lane M contains molecular markers. The arrow indicates AhpCivitb- Identity and purity of the AhpCivi_tb band were confirmed by amino acid sequencing. In Figure 2B, AhpCivi_tb is detected. Following SDS-PAGE on a 12% gel, immunoblot was performed with antiserum raised against antigen purified in (A) (1 :2,000 dilution). Upper panel: lysates of E. coli Ml 5 (pAhpC-3) (lane 1), M. tuberculosis H37Ra (lane 2. 25 μg) and M. smegmatis mc²155 (lane 3, 22 μg). Arrows indicate AhpC translated from native start codon ("native") and recombinant AhpC fusion protein ("rec"). Lower panel: clinical isolates of M tuberculosis characterized as isoniazid sensitive (S) or resistant (R) and as catalase positive (+), negative (-) or not determined (ND). Lane 8 represents H37Rv (ATCC 25618). Protein per lane varied depending on availability: lane 1, 20 μg; lane 2, 20 μg; lane 3, 9.6 μg; lane 5, 20 μg; lane 6. 12 μg; lane 7, 12 μg; lane 8, 10 μg. In Figures 3A-C, S. typhimurium. strain LT2, AhpC was disrupted by insertion of a TnlO transposon into the 33^rd codon, creating strain TA4190. These figures show S. typhimurium ahpC::Tn70 strain (TA4190) is more susceptible to RNI than parental wild type strain (LT2). In Figure 3A, canonical phenotype is confirmed: disruption of ahpC (with associated decrease in aphF expression) causes hypersensitivity to cumene hydroperoxide in S. typhimurium. Discs were placed on agar containing LT2 or TA4190 and impregnated with 15 μl of 5% cumene hydroperoxide. Zones of inhibition were photographed after 18 hours incubation at 37°C. In Figure 3B, a new phenotype with respect to RNI is demonstrated — studies with acidified nitrite. Survival of S. typhimurium LT2 (■) and TA4190 (•) exposed to indicated concentrations of nitrite at pH 5 for 14 hours at 37°C in LB (panel a), or to 3 mM nitrite in LB at pH 5 for the indicated times (panel b). Panel c: controls with nitrate at pH 5 (0, LT2; Δ, TA4190) and nitrite at pH 7 (♦, LT2; A, TA4190) as indicated by cell growth measured by OD₆oo- Surviving organisms were determined as colony forming units on agar (solid symbols, panels a, b), while cell growth was measured by OD₆oo (u,LT2; O, TA4190) in panel b. Results are means ±SE of triplicates or quadruplicates; a and b are representative experiments out of 4. Most error bars fall within the symbols. In Figure 3C. a new phenotype with respect to RNI is demonstrated — studies with GSNO. S. typhimurium LT2 (■) or TA4190 (•) was exposed to indicated concentration of GSNO at pH 5 for 7 hours at 37°C in LB (panel a), or to 5 mM GSNO at pH 5 for the indicated times (panel b). Surviving organisms were determined as colony forming units on agar (solid symbols, panels a and b), while cell growth was measured by OD₆oo in panel b (3, LT2; O, TA4190). Results are means ± SE of triplicates or quadruplicates in one of 3-4 such experiments. Most error bars fall within the symbols.

Figures 4A-C show complementation of aphCF deficiency in S typhimurium ahpC::Tn\0 strain TA4190 by ahpC_{\ h} with and without ahpF_Sly. In Figure 4A. native and recombinant protein in S. typhimurium is expressed. Parental wild type strain LT2 (lane 1) and untransformed TA4190 (ahpC::TnlO) (lane 2) as controls were compared to TA4190 transformed with recombinant plasmids pStahpC-RB (lane 3), pStahpCF-RB (lane 4), pPs-MtahpC-RB (lane 5), and pPs-MtahpCF-RB (lane 6). 100 μg of lysate protein (upper and middle rows) and 150 μg (lower row) from each strain was subjected to SDS-PAGE and immunoblotted with antibodies specific for AhpCst> (1 :4000 dilution), AhpCivitb (1 :2000), or AhpFsty (1 : 1000). In Figure 4B. AhpCivi_tb is shown partially to restore resistance to cumene hydroperoxide. S. typhimurium LT2 (pRB3-273C), TA4190 (pRB3-273C), TA4190 (pStahpC-RB), TA4190 (pPs-MtahpC-RB) and TA4190 (pPs-MtahpCF-RB) were treated with 0 (black bars), 100 (gray bars), or 150 μM (hatched bars) cumene hydroperoxide in LB for 14 hours at 37°C (panel a) or with 100 μM cumene hydroperoxide in LB for 8 hours at 37°C (panel b; symbols: ■, LT2 (pRB3-273C); •, TA4190 (pRB3-273C); A, TA4190 (pStahpC-RB); ♦, TA4190 (pPs-MtahpC-RB); , TA4190 (pPs-MtahpCF-RB)). In Figure 4C, AhpC _tb is shown to restore resistance to RNI. S. typhimurium LT2 (pRB3-273C), TA4190 (pRB3-273C),

TA4190 (pStahpC-RB), TA4190 (pStahpCF-RB), TA4190 (pPs-MtahpC-RB), and TA4190 (pPs-MtahpCF-RB) were treated without (black bars) or with 2 mM NaNO₂ (hatched bars) in LB (pH 5) for 24 hours (panel a), and without (black bars) or with 4 (white bars) or 5 mM (hatched bars) GSNO in LB (pH 5) for 27 hours (panel b) at 37°C. In Figures 4B and 4C, surviving organisms were determined as colony forming units on LB agar containing ampicillin. Results are means ± SE triplicates or quadruplicates in a representative experiment. Figures 5A-B show the expression of AhpCivit_b in stably transfected human cells. An immunoblot is depicted in Figure 5 A. Lysates of human 293, 293/neo^r, 293/AhpC-L and 293/AhpC-2 cells (100 μg each) were subjected to SDS-PAGE in a 12% gel and immunoblotted with anti-AhpC_M,_h antiserum (1 :4000). Immunocytochemistry is shown in Figure 5B. 293/neo^r cells (a,b), 293/AhpC-l cells (c,d), and 293/AhpC-2 cells (e,f) were stained with pre-immune serum (a,c.e) or anti-AhpCjvitb antiserum (b,d,f). Original magnification is xl OOO.

Figure 6 shows that the expression of AhpCivitb increases resistance of human cells to GSNO. 293/neo^r (O) or 293/AhpC-l (•) cells were exposed to the indicated concentrations of GSNO in DMEM medium with 10% FBS for 48 h.

Viability was assayed by reduction of a tetrazolium salt with the value for untreated cells set to 100%. Results are means ± SE for triplicates and are representative of 3 experiments.

Figures 7A-D show that the expression of AhpC _tb increases resistance of human cells to cytotoxicity and apoptosis caused by expression of NOS2.

Figure 7A relates to viability. 293/neo^r cells (open bars), 293/AhpC-l (closed bars), or 293/AhpC-2 (hatched bars) were transiently transfected with NOS2 on pL8Amp (for brevity, NOS2) or with the vector pcDNAI/Amp (for brevity, vector). At indicated times, cell viability was determined by trypan blue exclusion. This panel summarizes results from 6 independent experiments. Figure 7B depicts expression of NOS2 and AhpCivi_tb- Immunoblots were performed with antisera specific for mouse NOS2 (upper row, 200 μg/sample after SDS-PAGE on a 7.5% gel) or AhpC_Mtb (lower row, 100 μg/sample, 12% gel). Lanes: 1 , 293/neo^r transfected with vector; 2, 293/neo^r transfected with NOS2; 3, 293/AhpC-l transfected with vector; and 4, 293/AhpC-l transfected with NOS2. Expression of NOS2 in transfected 293/AhpC-2 was confirmed in a separate experiment. Figure 7C relates to morphology of cultures prepared as in Figure 7A. Panel (a), 293/neo^r transfected with vector; Panel (b), 293/neo^r transfected with NOS2; Panel (c), 293/AhpC-l transfected with vector; Panel (d), 293/AhpC-l transfected with NOS2; Panel (e), 293/AhpC-2 transfected with vector; and Panel (f), 293/AhpC-2 transfected with NOS2. DNA fragmentation is depicted in Figure 7D. Genomic DNA was prepared from harvested cells as discussed with respect to Figure 7A and equal amounts were subjected to agarose gel electrophoresis and ethidium bromide staining. Lanes: 1. 293/neo^r transfected with vector: 2. 293/neo^r transfected with NOS2; 3, 293/AhpC-l transfected with vector; 4, 293/AhpC-l transfected with NOS2; 5. 293/AhpC-2 transfected with vector; and 6, 293/AhpC-2 transfected with NOS2. Figure 8 shows the reconstitution of TA4190 with Hpy Sty AhpC:

Survival in GSNO (pH 5) or ASN (pH 5) in a 6 hour time period.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to the discovery that inhibition of alkyl hydroperoxide reductase subunit C (AhpC) proteins or polypeptides imparts susceptibility to antimicrobial reactive nitrogen intermediates. A number of DNA molecules encoding such proteins and polypeptides have already been sequenced. These DNA molecules with their GenBank accession numbers in parentheses, which are hereby incorporated by reference, are as follows:

Mycobacterium tuberculosis (U16243, U18264, Z81451); Mycobacterium bovis (U24084); Mycobacterium leprae (L01095); Mycobacterium avium (U18263, M74232); Mycobacterium smegmatis (U43719); Salmonella typhimurium (J05478); Escherichia coli (D13187); Amphibacillus xylanus (D 13563); Bacillus alcalophilus (D 10701); Bacillus subtili (D78193); Clostridium pasteurianum (M60116);

Corynebacterium diphtheri (U18620); Entamoeba histolytica (D00871, M35635); Enterococcus faecalis (AF016233); Helicobacter pylori (M55507); Methanobacterium thermoautotrophicum (X74264); Rattus norvegicus (U06099); Stapphyloccus aureus (U92441); Streptococcus mutans (D21803) Xanthomonas campestris (U94336); Trypanosoma brucei (U26666); Legionella pneumophila (L46863); Sulfolobus metallicu (AF007757, U36479); Saccharomyces cerevisiae (L14640); Caenorhabditid elegans (T00682, Z32683, U37429); Bromo secalinas (X63202); and Mus musculus (U51679, X82067, U20611 , M28723, AF004670, Y12883). These DNA molecules can be used to produce alkyl hydroperoxide reductase subunit C proteins or polypeptides recombinantly. This procedure for protein production is discussed more fully infra. All AhpC homologs from the various species of life forms, including plants, bacteria, fungi, yeasts, protozoa, nematodes, insects, and vertebrates, are now collectively termed "peroxiredoxins." The terms AhpC and peroxiredoxin are used interchangeably in this application. In some instances, the proteins or polypeptides of the present invention can be used to develop drugs for treating diseases caused by intracellular pathogen infection. This can be achieved by looking at the mechanism by which the proteins or polypeptides of the present invention resist reactive nitrogen intermediates. Such a mechanism may be conserved across other intracellular pathogens. If so, this knowledge can be used to design drugs that will target this resistance mechanism. Drugs to target such mechanisms may not have an in vitro activity. That is, such drugs may not inactivate or kill the organism outside of the host cells. But, such drugs may allow the macrophages or other sources of reactive nitrogen intermediates to kill efficiently the intracellular organism, if the organism's ability to resist killing by reactive nitrogen intermediates is inhibited by such drugs. Thus, these drugs can be designed to allow the normal macrophage antimicrobial molecules to exert their effects that may otherwise be resisted by the organism. This would be a new way to target bacterial killing.

Alternatively, the proteins or polypeptides of the present invention can be used to kill pathogens which reside outside host cells. For example, these proteins can be used to treat Helicobacter pylori which is in the gastric contents and resists being killed by the stomach^'s antimicrobial mechanism, which chemically resembles the antimicrobial mechanism of macrophages.

Further, the DNA molecules of the present invention (or a portion thereof) can used as a probe to find other similar DNA molecules. The efficacy of such DNA molecules can be tested by producing recombinant bacteria, such as recombinant E. coli, which are deficient in endogenous AhpC protein or polypeptide encoding gene. These hosts are transformed with this DNA molecule and the recombinant and control bacteria are placed in a medium containing a reactive nitrogen intermediate and a therapeutic to be tested. Under these conditions, the control bacteria should always perish and the recombinant bacteria will perish only if the therapeutic has antibacterial effect. Reactive nitrogen intermediates, particularly nitric oxide, are well- known to mediate a number of adverse physiological conditions, including hypotension which accompanies sepsis. See Lowenstein, et. al., "Nitric Oxide: A Physiologic Messenger." Ann. Intern. Med. 120: 227-37 (1994). which is hereby incorporated by reference. All of these conditions can be treated in accordance with the present invention.

The vasculature is in a constant state of active dilation mediated by nitric oxide. Endothelial cells continuously release small amounts of nitric oxide, producing a basal level of vascular smooth muscle relaxation. When nitric oxide is produced, vascular smooth muscle relaxes and blood pressure decreases. There are, however, adverse conditions mediated by overproduction of nitric oxides. For example, septic hypotension occurs when bacterial infection causes the massive release of nitric oxide, which overwhelms the arterial smooth muscle and causes excess dilation and hypotension. When such a condition occurs, the proteins or polypeptides of the present invention can be administered to inhibit actions of nitric oxide and, as a result, to increase blood pressure.

Excessive production of nitric oxides is also known to be triggered by strokes. Neurons release nitric oxide that diffuses into adjacent neurons in a series of steps. The presynaptic neuron is triggered by glutamate binding to the N-methyl-D- aspartate subtype receptor. This receptor possesses a calcium channel that opens, and the resulting influx of calcium binds to calmodulin to activate neuronal nitric oxide synthase. Nitric oxide is produced and diffuses out of the presynaptic neuron into the postsynaptic neuron, where it binds to the heme group of guanylate cyclase, activating the enzyme to produce cGMP. Small amounts of nitric oxide allow glutamate to increase cGMP levels in the brain. However, massive releases of glutamate during stroke trigger formation of large amounts of nitric oxide that are neurotoxic to adjacent neurons. Administration of the proteins or polypeptides of the present invention can be used to treat stroke victims.

As noted above, nitric oxides are produced by the body^'s immune system to kill various pathogens. However, the overproduction of nitric oxides for this purpose can have adverse effects. In some situations, the production of nitric oxides may damage normal cells in the body. It is not desirable to prevent production of nitric oxide, because this would permit growth of this infectious pathogen. However, some quenching of the nitric oxide product would be desirable. Thus, administration of the proteins or polypeptides of the present invention to titrate the produced nitric oxides would be desirable to quench overproduction of nitric oxides in 5 response to infection by bacterial pathogens. This is different than the administration of agents which inhibit production of reactive nitrogen intermediates, because, here, there is no effort to control the enzymes producing nitric oxides (i.e.. nitric oxide synthases); it is the material produced by the enzyme that is being controlled.

Another aspect of the present invention relates to an isolated DNA

10 molecule encoding a hydroperoxide reductase subunit C and conferring on

Mycobacterium tuberculosis resistance against antimicrobial reactive nitrogen intermediates. The term "isolated" is intended to define molecules which are separated from their naturally-present components (i.e., Mycobacterium tuberculosis). This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID.

15 No. 1 as follows:

atgccactgc taaccattgg cgatcaattc cccgcctacc agctcaccgc tctcatcggc 60 ggtgacctgt ccaaggtcga cgccaagcag cccggcαact acttcaccac tatcaccagt 120

20 gacgaacacc caggcaagtg gcgggtggtg ttcttttggc cgaaagactt cacgttcgtg 180 tgccctaccg agatcgcggc gttcagcaag ctcaatgacg agttcgagga ccgcgacgcc 240

25 cagarcctgg gggtttcgat tgacagcgaa ttcgcgcatt tccagtggcg tgcacagcac 300 aacgacctca aaacgttacc cttcccgatg ctctccgaca tcaagcgcga actcagccaa 360 gccgcaggtg tcctcaacgc cgacggtgtg gccgaccgcg tgacctttat cgtcgacccc 420

30 aacaacgaga tccagttcgt ctcggccacc gccggttcgg tgggacgcaa cgtcgatgag 480 gtactgcgag tgctcgacgc cctccagtcc gacgagctgt gcgcatgcaa ctggcgcaag 540 ⁾5 ggcgacccga cgctagacgc tggcgaactc ctcaaggctt cggcctaa 588

The DNA molecule of SEQ. ID. No. 1 encodes for a protein or polypeptide having a deduced amino acid sequence corresponding to SEQ. ID. No. 2 40 as follows:

Met Pro Leu Leu Thr lie Gly Asp Gin Phe Pro Ala Tyr Gin Leu Thr 5 10 15 Ala Leu He Gly Gly Asp Leu Ser Lys Val Asp Ala Lys Gin Pro Gly

20 25 30

Asp Tyr Phe Thr Thr He Thr Ser Asp Glu His Pro Gly Lys Trp Arg

35 40 45

Val Val Phe Phe Trp Pro Lys Asp Phe Thr Phe Val Cys Pro Thr Glu 50 55 60

He Ala Ala Phe Ser Lys Leu Asn Asp Glu Phe Glu Asp Arg Asp Ala 65 70 75 80

Gin He Leu Gly Val Ser He Asp Ser Glu Phe Ala His Phe Gin Trp 85 90 95

Arg Ala Gin His Asn Asp Leu Lys Thr Leu Pro Phe Pro Met Leu Ser 100 105 110

Asp He Lys Arg Glu Leu Ser Gin Ala Ala Gly Val Leu Asn Ala Asp 115 120 125

Gly Val Ala Asp Arg Val Thr Phe He Val Asp Pro Asn Asn Glu He

130 135 140

Gin Phe Val Ser Ala Thr Ala Gly Ser Val Gly Arg Asn Val Asp Glu

145 150 155 160

Val Leu Arg Val Leu Asp Ala Leu Gin Ser Asp Glu Leu Cys Ala Cys 165 170 175

Asn Trp Arg Lys Gly Asp Pro Thr Leu Asp Ala Gly Glu Leu Leu Lys 180 185 190 Ala Ser Ala

195

Production of this isolated protein or polypeptide is preferably carried out using recombinant DNA technology. The protein or polypeptide is believed to have one or more antigenic determinants conferring on Mycobacterium tuberculosis resistance against antimicrobial reactive nitrogen intermediates.

Fragments of the above polypeptides or proteins are also encompassed by the method of the present invention. Suitable fragments can be produced by several means. In the first, subclones of the gene encoding a known protein are produced by conventional molecular genetic manipulation by subcloning gene fragments. The subclones then are expressed in vitro or in vivo in bacterial cells to yield a smaller protein or peptide that can be tested for activity in conferring resistance to reactive nitrogen intermediates. As an alternative, protein fragments can be produced by digestion^" of a full-length protein with proteolytic enzymes like chymotrypsin or trypsin. Different proteolytic enzymes are likely to cleave proteins at different sites based on the amino acid sequence of the protein. Some of the fragments that result from proteolysis may be active in conferring resistance to reactive nitrogen intermediates.

In another approach, based on knowledge of the primary structure of the proteins, fragments of the protein encoding gene may be synthesized by using the PCR technique together with specific sets of primers chosen to represent particular portions of the protein. These then would be cloned into an appropriate vector for expression of a truncated peptide or protein.

Chemical synthesis can also be used to make suitable fragments. Such a synthesis is carried out using known amino acid sequences for the proteins being produced. Alternatively, subjecting a full length protein to high temperatures and pressures will produce fragments. These fragments can then be separated by conventional procedures (e.g., chromatography, SDS-PAGE).

Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the properties, secondary structure, and hydropathic nature of the polypeptide. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide.

Suitable DNA molecules are those that hybridize to a DNA molecule comprising a nucleotide sequence of 50 continuous bases of SEQ. ID. Nos. 1 or 3 under stringent conditions characterized by a hybridization buffer comprising 0.9M sodium citrate ("SSC") buffer at a temperature of 37°C, more preferably comprising 20% formamide in 0.9M saline/0.09M SSC buffer at a temperature of 42°C, and remaining bound when subject to washing at 42°C, more preferably with 0.2x SSC buffer at 42°C. The proteins or polypeptides of the present invention are preferably produced in purified form (preferably at least about 80%, more preferably 90%>, pure) by conventional techniques. The proteins or polypeptides of the present invention are preferably produced in purified form by conventional techniques, of which the ^• following is one example. To isolate the proteins, the E. coli host cell carrying a recombinant plasmid is propagated, homogenized, and the homogenate is centrifuged to remove bacterial debris. The supernantant is then subjected to sequential ammonium sulfate precipitation. The fraction containing the proteins of the present invention are subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein fraction may be further purified by HPLC.

Any one of the DNA molecules conferring on Mycobacterium tuberculosis resistance to antimicrobial reactive nitrogen intermediates can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the selected DNA molecule into an expression system to which that DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.

U.S. Patent No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture.

Recombinant genes may also be introduced into viruses, such as vaccina virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.

Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtl 1, gt WΕS.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby incorporated by reference), pQΕ, pIH821, pGΕX, pΕT series (see F.W. Studier et. al., "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Gene Expression Technology vol. 185 (1990). which is hereby incorporated by - reference) and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982), which is hereby incorporated by reference.

A variety of host-vector systems may be utilized to express the protein- encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include, but are not limited to, the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); and insect cell systems infected with virus (e.g., baculovirus). The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.

Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA) translation). Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promoters differ from those of procaryotic promoters. Furthermore, eucaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eucaryotic cells.

Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno (SD) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3 '-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology, 68:473 (1979). which is hereby incorporated by reference. Promoters vary in their "strength" (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P_R and Pi promoters of coliphage lambda and others, including but not limited to /αcUV5, ompF, bla, Ipp. and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacOV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG

(isopropylthio-beta-D-galactoside). A variety of other operons. such as trp, pro. etc., are under different controls.

Specific initiation signals are also required for efficient gene transcription and translation in procaryotic host cells. These transcription and translation initiation signals may vary in "strength" as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various "strong" transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (SD) sequence about 7-9 bases 5' to the initiation codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Additionally. any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.

Once the desired isolated DNA molecule conferring on Mycobacterium tuberculosis resistance to antimicrobial reactive nitrogen intermediates has been cloned into an expression system, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation, transfection, or infection noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to. bacteria, virus, yeast, mammalian cells, and the like. From the present invention's determination of nucleotide sequences conferring on Mycobacterium tuberculosis resistance to antimicrobial reactive intermediates, a wide array of therapeutic and/or prophylactic agents and diagnostic procedures for, respectively, treating and detecting Mycobacterium tuberculosis can be developed. For example, an effective amount of the proteins or polypeptides of the present invention can be administered alone or in combination with a pharmaceutically-acceptable carrier to humans, as a vaccine, for preventing infection by Mycobacterium tuberculosis. Alternatively, it is possible to administer to individuals exposed to Mycobacterium tuberculosis an effective amount of an antibody or binding portion thereof against these proteins or polypeptides as a passive immunization. Such antibodies or binding portions thereof are administered alone or in combination with a pharmaceutically-acceptable carrier to effect short term treatment of individuals who may have been recently exposed to Mycobacterium tuberculosis. An additional therapeutic aspect of the present invention involves the administration of the subject DNA molecules to subjects requiring immunization against Mycobacterium tuberculosis. This is known as naked DNA vaccination where such DNA is injected into the muscles of subjects and enters cells, causing expression of the encoded protein. Ulmer, et. al., "Heterologous Protection Against Influenza by Injection of DNA Encoding a Viral Protein," Science 259: 1745-49 (1993), which is hereby incorporated by reference. The expressed protein has the same effect as if it were itself injected into the patient. Antibodies suitable for use in inducing passive immunity can be - monoclonal or polyclonal.

Monoclonal antibody production may be effected by techniques which are well-known in the art. Basically, the process involves first obtaining immune cells (lymphocytes) from the spleen of a mammal (e.g., mouse) which has been previously immunized with the antigen of interest (i.e., one of the proteins or peptides of the present invention) either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody. A description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, Nature, 256:495 (1975), which is hereby incorporated by reference.

Mammalian lymphocytes are immunized by in vivo immunization of the animal (e.g., a mouse) with one of the proteins or polypeptides of the present invention. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. The antigen is carried in appropriate solutions or adjuvants. Following the last antigen boost, the animals are sacrificed and spleen cells removed.

Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol (PEG) or other fusing agents (See Milstein and Kohler, Eur. J. Immunol., 6:51 1 (1976), which is hereby incorporated by reference). This immortal cell line, which is usually murine, but may also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described. Procedures for raising polyclonal antibodies are also well known. - Typically, such antibodies can be raised by administering one of the proteins or polypeptides of the present invention subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The antigens can be injected at a total volume of 100 μl per site at six different sites. Each injected material may contain synthetic surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. This and other procedures for raising polyclonal antibodies are disclosed in E. Harlow, et. al., Editors, Antibodies: A Laboratory Manual (1988), which is hereby incorporated by reference. In addition to utilizing whole antibodies, the processes of the present invention encompass use of binding portions of such antibodies. Such antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in J. Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-1 18 (N.Y. Academic Press 1983), which is hereby incorporated by reference.

The vaccines and passive immunization agents of this invention can be administered orally, parenterally, for example, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, or by application to mucous membranes, such as that of the nose, throat, and bronchial tubes. They may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.

The solid unit dosage forms can be of the conventional type. The solid form can be a capsule, such as an ordinary gelatin type containing the proteins or peptides of the present invention or the antibodies or binding portions thereof of the present invention and a carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch. In another embodiment, these compounds are tableted with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin, disintegrating agents such as cornstarch. potato starch, or alginic acid, and a lubricant like stearic acid or magnesium stearate.

The DNA molecules of the present invention or the proteins or polypeptides of the present invention or the antibodies or binding portions thereof of this invention may also be administered in injectable dosages by solution or suspension of these materials in a physiologically acceptable diluent with a pharmaceutical carrier. Such carriers include sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, olive oil, peanut oil, soybean oil. or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. For use as aerosols, the DNA molecules of the present invention or the proteins or polypeptides of the present invention or the antibodies or binding portions thereof of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The materials of the present invention also may be administered in a non- pressurized form such as in a nebulizer or atomizer.

In yet another aspect of the present invention, the proteins or polypeptides of the present invention can be used as antigens in diagnostic assays for the detection of Mycobacterium tuberculosis in body fluids. Alternatively, the detection of that bacillus can be achieved with a diagnostic assay employing antibodies or binding portions thereof raised by such antigens. Such techniques permit detection of Mycobacterium tuberculosis in a sample of the following tissues or body fluids: blood, spinal fluid, sputum, pleural fluids, urine, bronchial alveolar lavage, lymph nodes, bone marrow, or other biopsied materials. In one embodiment, the assay system has a sandwich or competitive format. Examples of suitable assays include an enzyme-linked immunosorbent assay, a radioimmunoassay. a gel diffusion precipitin reaction assay, an immunodiffusion assay. an agglutination assay, a fluorescent immunoassay, a protein A immunoassay, or an immunoelectrophoresis assay.

In an alternative diagnostic embodiment of the present invention, the nucleotide sequences of the isolated DNA molecules of the present invention may be used as a probe in nucleic acid hybridization assays for the detection of

Mycobacterium tuberculosis in various patient body fluids. The nucleotide sequences of the present invention may be used in any nucleic acid hybridization assay system known in the art, including but not limited to, Southern blots (Southern. J. Mol. Biol.. 98:503-17 (1975), which is hereby incorporated by reference), Northern blots (Thomas et al, Proc. Nat'l Acad. Sci. USA. 77:5201-05 (1980), which is hereby incorporated by reference), and Colony blots (Grunstein et al., Proc. Nat^'l Acad. Sci. USA, 72:3961-65 (1975), which is hereby incorporated by reference). Alternatively, the isolated DNA molecules of the present invention can be used in a gene amplification detection procedure (e.g., a polymerase chain reaction). See H.A. Erlich et. al., "Recent Advances in the Polymerase Chain Reaction," Science 252:1643-51 (1991), which is hereby incorporated by reference.

In addition to its use as a vaccine or in raising antibodies for passive immunization against Mycobacterium tuberculosis, the protein or polypeptide of the present invention has application as a therapeutic in treating conditions mediated by the production of reactive nitrogen intermediates. In this aspect of the present invention, advantage is taken of the ability of the subject protein or polypeptide to confer on Mycobacterium tuberculosis resistance against reactive nitrogen intermediates. Such compounds are part of the body's defense system against most infectious pathogens; however, by virtue of its ability to express the DNA molecule of the present invention, Mycobacterium tuberculosis is resistant to reactive nitrogen intermediates. It is also known, however, that reactive nitrogen intermediates have certain adverse effects on various physiological conditions, so the administration of the proteins or polypeptides of the present invention can be used to treat them. Here, the proteins or polypeptides of the present invention are administered to titrate the amounts of reactive nitrogen intermediates, thereby relieving a particular adverse condition. Administration of the proteins or polypeptides of the present invention can be carried out using the formulations and procedures discussed above. EXAMPLES

Example 1 - Materials

Chemicals were from Sigma Chemical Co. (St. Louis, MO) except as indicated. G418. X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) and trypsin were from GIBCO/BRL Life Technologies (Grand Island, NY). IPTG

(isopropyl-β-thiogalactopyranoside) was from Boehringer Mannheim (Indianapolis, IN). Restriction endonucleases, DNA polymerase I large fragment (Klenow) and calf intestinal alkaline phosphatase were from New England Biolabs (Beverly, MA). Pfu DNA polymerase was from Stratagene (La Jolla, CA); AmpliTaq DNA polymerase, MuLV reverse transcriptase polymerase, dNTPs and reagents for PCR and RT-PCR were from Perkin Elmer (Foster City, CA). Oligonucleotide primers were from Oligo Etc., Inc. (Guilford, CT).

Example 2 - Cloning of ahpC from M. tuberculosis

Portions of the ahpC coding region conserved in M avium (GenBank U18263) and M. leprae (GenBank L01095) were used to design the primer pair ACCAGTTCCCCGCCTAC (SEQ. ID. No. 3) (sense) and

ACCCTTGCGCCAGTTGCA (SEQ. ID. No. 4) (antisense). With M. tuberculosis H37Rv genomic DNA as template (gift of Lee Riley, Cornell Univ. Medical College), PCR amplification yielded a product with the expected size of 521 bp whose sequence was homologous to that of M. avium and M. leprae. This was used to prepare a probe with which we screened a λgtl 1 library of M. tuberculosis Erdman DNA (gift of Richard Young, Whitehead Institute, Cambridge, MA). Twenty clones were selected from -5 x 10 phage plaques. Two overlapping clones were combined and subcloned into the pT7-Blue vector (Novagen. Madison, WI) to yield pMtb-ahpC, which was sequenced.

Example 3 - Bacterial Strains

S. typhimurium TA4190 (ahpC::Tn!0) and its congenic wild type strain LT2 were from the Salmonella Genetic Stock Center at the University of Calgary, Calgary, Alberta, Canada. E. coli strain DH5α (GIBCO/BRL, Gaithersburg, MD) and XL 1 -Blue (Stratagene) were used for general genetic manipulation. E. coli Ml 5 (pRΕP4) (QIAGEN Inc., Chatsworth, CA) was used for overproduction of recombinant AhpCivi_tb- S. typhimurium and E. coli strains were cultured in LB broth or on LB agar (Sambrook, J., et al., "Molecular Cloning. A Laboratory Manual- Second Edition: Cold Spring Harbor Laboratory Press, (1989), which is hereby incorporated by reference) with antibiotics as appropriate: ampicillin (lOOμg/ml), tetracycline (15 μg/ml), or kanamycin (25 μg/ml). Lysates of M. smegmatis mc 155 and M. tuberculosis H37Ra were kindly provided by Sabine Ehrt (Cornell Univ. Medical College). M. tuberculosis H37Rv (ATCC 25618) was from Pablo Bifani (Public Health Research Institute, New York. NY). Dr. John T. Belisle (Colorado State University) supplied γ-irradiated clinical isolates of M. tuberculosis with defined isoniazid sensitivity and catalase status under NIAID contract NO1-AI-75320. Strains were considered isoniazid-sensitive if they manifest 100% suppression of CFU in 100 μg/ml isoniazid.

Example 4 - Plasmids

The ahpCF operon was cloned from S. typhimurium LT2 by PCR with the primers: forward, 5'-GGCGGCCTTTTTACTTTAGATC-3' (SEQ. ID. No. 5); reverse, 5'-AGGCCCGAATAGCTTACACTA-3' (SEQ. ID. No. 6), designed according to GenBank sequence J05478. The amplified 2.6-kb fragment was cloned into pT7-Blue (R), resulting in pStahpCF. Plasmid pRB3-273C, a gift from Dr. Ferric C. Fang (Univ. of Colorado Health Sciences Center, Denver, CO), served to introduce ahpC_Mtb, aphCsty,, and ahpF_Sty into S. typhimurium TA4190. A Smal fragment from pStahpCF containing ahpCs_ty was cloned into pRB3-273C in the opposite orientation to the vector's lacZ promoter, resulting in pStahpC-RB. Plasmid pStahpCF-RB was generated by subcloning an Xbal-Nhel fragment carrying ahpF_Sly into the Xbal site in pStahpC-RB, and ahpF_St . was placed downstream of ahpCs_ty to allow both genes to be expressed from the upstream promoter of ahpCFs_ty (Ps).

In order to achieve a level

expression similar to that of ah sty on pStahpC-RB, the entire promoter oϊahpCFs_ty (P_s) was first amplified by PCR using the forward primer described above and a reverse primer (5'-GAATTCCATATGTACTTCCTCCGTGTTTT-3^'(SEQ. ID. No. 7)) that engineered an Ndel site around the ATG start codon for use in subsequent cloning. The amplified P_s-containing fragment was cloned into pT7-Blue (R), generating pPs-T7. Another pair of primers were used to PCR-amplify the ORF of ahp tb from pMtb-ahpC. Similarly, the forward primer

(5'-GAGGAGACATATGCCACTGCTAACCATTG-3'(SEQ. ID. No. 8)) contained an Ndel site without changing codons of ahpCMt_b ORF, while the reverse primer (5'-CCTCTAGATTATGCCGAAGCCTTGAG-3'(SEQ. ID. No. 9)) incorporated an Xbal site after the ahpC_Mtb ORF. The 606-bp amplified product was digested with Ndel and Xbal and cloned into pPs-T7 digested by the same enzymes, resulting in pPs-MtahpC. A Sacl-Xbal fragment from pPs-MtahpC, which contained both the Ps element and the downstream ahpCMtb, was cloned into the compatible sites on pRB3-273C, resulting in pPs-MtahpC-RB. To construct a chimeric bi-cistronic &\φC_\ι,_b-ahpFs,_y operon whose transcription was directed by P_s, an ahpFs,_y containing Xbal-Nhel fragment was subcloned into Xbal -digested pPs-MtahpC-RB to generate pPs-MtahpCF-RB.

To express ahpC_M,b in mammalian cells, a 612-bp Hifl fragment containing the ORF of ah C_Mtb was end-filled and cloned into the EcoRV site of pcDNAI/Amp (Invitrogen, Carlsbad, CA) such that ahpC_Mtb was expressed from the vector-borne CMV promoter, resulting in pAhpC-mtbl. Ligation was performed using the Rapid DNA Ligation Kit (Boehringer Mannheim). Plasmids were transformed into S. typhimurium by electroporation.

Example 5 - Production of Antibody Against Recombinant AhpCivi_tb

A BamHI-XhoI fragment (679 bp) from pAhpC-mtbl was subcloned in frame downstream of an IPTG-inducible promoter in pQE-30 (QIAGEN). The resulting plasmid, pAhpC-3, was transformed into E. coli Ml 5 (pREP4) (QIAGEN). Upon induction with IPTG, a fusion protein was overexpressed with a hexahistidine tag at its N-terminus and purified on Ni⁺-NTA resin according to the manufacturer's instructions, as monitored by SDS-PAGE (12% gel). The protein was transferred to a PVDF membrane (Millipore, Bedford. MA) and subjected to N-terminal sequencing (NYS Center for Advanced Technology. Cornell Univ., Ithaca. NY). Purified recombinant AhpC_Mt was injected subcutaneously into female New Zealand rabbits (100 μg per injection, 7 injections, 2-4 week intervals). Freund^'s adjuvant was complete for the first injection and incomplete thereafter.

Example 6 - Disk Inhibition

Salmonella were grown in LB with antibiotics at 37°C for 8 hours and diluted 10-fold. Aliquots (0.1 ml; ~10 cells) were mixed with 2 ml of soft agar and immediately plated onto M9 plates (Storz, G., et al., "An Alkyl Hydroperoxide Reductase Induced by Oxidative Stress in Salmonella typhimurium and Escherichia coli: Genetic Characterization and Cloning of ahp," J. Bacteriol. 181 :2049-55 (1989), which is hereby incorporated by reference). Cumene hydroperoxide (15 μl, 5%) (Sigma) was applied to an 0.250-inch paper disc (BBL Microbiology Systems, Cockeysville, MD) centered on the surface of the agar.

Example 7 - Western Blot

Clinical isolates of M. tuberculosis were suspended in sonication buffer (50 mM Na-phosphate pH 7.8, 300 mM NaCl) and agitated with fine glass beads. The supernatant was concentrated (Microcon-10, Millipore, Bedford, MA) and protein measured by the Bradford method (Bio-RAD Laboratories, Hercules, CA). Overnight cultures of S. typhimurium were inoculated (1 :100) into 100 ml LB with or without antibiotics and incubated at 37°C for 4 hours with vigorous shaking. Cells were resuspended in 5 ml of sonication buffer, frozen at -80°C, thawed, and sonicated. Supernatant proteins were separated by SDS-PAGE and electroblotted onto a 0.2 μm pore nitrocellulose membrane (Schleicher & Shuell, Keene, NH). The membrane was blocked with the indicated antiserum, washed with TBST, and incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Life Sciences, Arlington Heights, IL). Bound antibody was detected by enhanced chemiluminescence (NEN™ Life Science Products, Boston, MA).

Example 8 - Mammalian Cells

The 293 human renal epithelial cell line (ATCC) was cultured in Dulbecco^'s modified Eagle's medium (Sigma) with 10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, UT), 200 units/ml penicillin and 200 μg/rnl streptomycin (complete medium) at 37°C in 5% CO₂-95% air. Cells were detached with a buffered salt solution containing 0.5% (w/v) trypsin and 0.2% (w/v) EDTA. For stable transfectants, cells were cotransfected with pcDNA3 (Invitrogen) containing neo^r and pAhpC-mtbl carrying ahp M_tb in the presence of calcium phosphate (Ruan, J., et al., "The Putative Calmodulin-Binding Region of Murine Inducible Nitric Oxide Synthase is Necessary but not Sufficient to Sustain Calmodulin Binding and Nitric Oxide Production at Trace Levels of Free Ca²⁺," J. Biol. Chem.. 271 :22679-86 (1996), which is hereby incorporated by reference). After 14 hours, cells were washed; complete medium containing G418 (500 μg/ml) was added and replaced every 2 days for another 10 days. Surviving cells were serially diluted with G418 (600 μg/ml). Individual colonies were isolated, expanded, verified by RT-PCR and immunoblot with antiserum against AhpCM_tb, and maintained in complete medium with G418 (500 μg/ml).

Example 9 - Immunocytochemistry

293 cells (~2xl 0⁴) were sedimented on a glass slide (Cytospin, Shandon Instruments, Sewickly, PA) and fixed with 1 % paraformaldehyde. 75 mM cacodylic acid, 0.12% sucrose, pH 7.4 followed by 3.1% formaldehyde in PBS, each for 10 minutes at room temperature. Slides were permeabilized with 0.05%o Triton X- 100, stained with preimmune serum or antiserum against AhpC_Mtb (1 :300), and developed as described (VECTASTAIN ABC-AP kit, Vector Labs) (Nicholson, S., et al., "Inducible Nitric Oxide Synthase in Pulmonary Alveolar Macrophages from Patients with Tuberculosis," J. Exp. Med., 183:2293-2302 (1996), which is hereby incorporated by reference).

Example 10 - Viability Assays 293 cells stably transfected with ahpC tb or the vector (neo^r) were transiently transfected in the presence of calcium phosphate with the vector pcDNAI/Amp or mouse NOS2 cDNA carried on this vector in pL8Amp (Ruan, J., et al., "The Putative Calmodulin-Binding Region of Murine Inducible Nitric Oxide Synthase is Necessary but not Sufficient to Sustain Calmodulin Binding and Nitric Oxide Production at Trace Levels of Free Ca"⁺," J. Biol. Chem., 271 :22679-86 (1996), which is hereby incorporated by reference). Approximately 40 hours later, cultures were trypsinized and trypan blue-excluding cells counted in a hemocytometer. Viability in NOS2-transfected cultures were expressed as a percentage of viable vector-transfected cultures derived from the same starting population. Other experiments used the CellTiter 96 AQueous assay (Promega, Madison, WI). in which viable cells convert a tetrazolium salt into a water-soluble formazan. 293/neo^r and 293/AhpC-l cells were cultured for 48 hours in triplicate in a

96-well plate (1x10 cells in 100 μl per well) in complete medium with G418 (500 μg/ml) and GSΝO at indicated concentrations. The tetrazolium assay was performed according to the manufacturer^'s instructions. After 1 hour at 37°C, OD ₉o was recorded (MR5000 microplate reader, Dynatech Laboratories, Chantilly, VA). Values for medium controls were deducted. Viability was determined as a percentage of dye reduction by untreated cells. To assess DΝA fragmentation, DΝA was collected from transfected 293 cells (2x10⁶) by the method of Liu, et al., "Induction of Apoptotic Program in Cell-Free Extracts: Requirement for dATP and Cytochrome C," Cell, 86:147-57 (1996), which is hereby incorporated by reference), electrophoresed on a 2% agarose gel at 50 V for 2 hours in 0.5xTBE buffer (Sambrook, J., et al., "Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, (1989), which is hereby incorporated by reference) stained with 2 μg/ml ethidium bromide and visualized under UV light.

Example 11 - Cloning of ahpC and Neighboring Genes from M. tuberculosis: Lack of a Bicistronic aphCF Operon

From M. tuberculosis Erdman, we cloned and sequenced 3934 bp of DNA that included aphC_Mtb flanked by four additional genes (Figure 1). Cloning of ahpC _tb was subsequently reported (Deretic, V., et al., "Mycobacterium tuberculosis is a Natural Mutant With an Inactivated Oxidative-Stress Regulatory Gene: Implications for Sensitivity to Isoniazid," Mol. MicrobioL. 17:889-900 (1995);

Sherman, D. R., et al., "Disparate Responses to Oxidative Stress in Saprophytic and Pathogenic Mycobacteria," Proc. Natl. Acad. Sci. USA. 92:6625-29 (1995), which are hereby incorporated by reference), and all five genes were contained in cosmid Y428 - jj -

(GenBank Z81451). Just 5^' to ahpC lies a disrupted oxyR (Deretic, V., et al., "Mycobacterium tuberculosis is a Natural Mutant With an Inactivated Oxidative- stress Regulatory Gene: Implications for Sensitivity to Isoniazid," Mol. Microbiol., 17:889-900 (1995); Sherman, D. R.. et al., "Disparate Responses to Oxidative Stress in Saprophytic and Pathogenic Mycobacteria," Proc. Natl. Acad. Sci. USA, 92:6625- 29 (1995), which are hereby incorporated by reference). At the protein level, AhpC tb is 32%o identical to the canonical AhpC from S. typhimurium (AhpCsty) (Tartaglia, L. A., et al., "Alkyl Hydroperoxide Reductase from Salmonella Tymphimurium. Sequence and Homology to Thioredoxin Reductase and other Flavoprotein Disulfide Oxidoreductases," J. Biol. Chem., 265: 10535-40 (1990), which is hereby incorporated by reference), with conservation of the two Cys and surrounding residues. In contrast, ahpF, encoding the flavoprotein component of alkyl hydroperoxide reductase within a bicistronic operon in S. typhimurium (Figure 1), was lacking from the vicinity of ahpC_Mtb, nor has an α/zpE homolog been reported elsewhere in the M. tuberculosis genome. Gene I is homologous to γ- glutamyl phosphate reductase in E. coli, while genes II-IV are of unknown function. The lack of any known ahpF in M. tuberculosis suggested two possibilities for ah Mtb- it might be vestigial, or it might subserve the same or related functions as in enterobacteria, but with a different mechanism for its own reduction.

Example 12 - Expression of AhpC in M. tuberculosis

That ahpC is vestigial in most clinical isolates of M. tuberculosis was suggested by negative immunoblots with cross-reactive anti-AhpC antibodies (Dhandayuthapani, S., et al., "Oxidative Stress Response and its Role in Sensitivity to Isoniazid in Mycobacteria: Characterization and Inducibility of ahpC by Peroxides in Mycobacterium Smegmatis and Lack of Expression in M. Avium and M. tuberculosis " J. Bacteriol.. 178:3641-49 (1996); Zhang, Y., et al, "Molecular Basis for the Exquisite Sensitivity of Mycobacterium tuberculosis to Isoniazid," Proc. Natl. Acad. Sci. USA. 93:13212-16 (1996), which are hereby incorporated by reference), this being attributed to the disruption of oxyR (Deretic, V., et al., "Mycobacterium tuberculosis is a Natural Mutant With an Inactivated Oxidative-stress Regulatory Gene: Implications for Sensitivity to Isoniazid," Mol. Microbiol, 17:889-900 (1995); Sherman, D. R., et al.. "Disparate Responses to Oxidative Stress in Saprophytic and Pathogenic Mycobacteria," Proc. Natl. Acad. Sci. USA, 92:6625-29 (1995), which are hereby incorporated by reference). However, expression of AhpC was demonstrated by microsequencing in catalase-deficient isolates of M. tuberculosis in association with upregulatory mutations in the ahpC promoter, mutations that were presumably fixed because they afforded compensation for the lack of catalase (Sherman, D. R., et al., "Compensatory ahpC Gene Expression in Isoniazid-Resistant Mycobacterium tuberculosis " Science, 272:1641-43 (1996), which is hereby incorporated by reference). Many isoniazid-resistant isolates are catalase-deficient. because catalase helps activate the pro-drug isoniazid to its active form (Zhang, Y., et al.. "The

Catalase-Peroxidase Gene and Isoniazid Resistance of Mycobacterium tuberculosis " Nature, 358:591 -593 (1992), which is hereby incorporated by reference). Conversely, most isoniazid-sensitive isolates are catalase-replete (Stoeckle, M. Y., et al., "Catalase-Peroxidase Gene Sequences in Isoniazid-sensitive and -resistant Strains of Mycobacterium tuberculosis from New York City," J. Infect. Pis.. 168:1063-65

(1993), which is hereby incorporated by reference). Based on these studies, isoniazid- sensitive isolates of M. tuberculosis have been regarded as predominantly AhpC- negative.

Since the foregoing impression was based in part on negative results with antibodies raised against AhpC from other species, recombinant AhpC_Mtb was purified (Figure 2A) and a specific antibody raised (Figure 2B, upper panel). This antibody detected no polypeptides in E. coli Ml 5, but stained two bands in E. coli Ml 5 (pAhpC-3) (Figure 2B, upper, lane 1). The band with apparent Mr -21.5 kD most likely corresponded to AhpC_Mtb translated from the authentic start codon of the a pC_Mtb ORF, while the higher M_r species represented the recombinant protein with its 2.8-kD N-terminal tag. This antibody immunoblotted a single polypeptide with the apparent M_r of native AhpC tb in M. smegmatis (lane 3) and M. tuberculosis H37Ra (lane 2), as well as in clinical isolates of M. tuberculosis characterized as isoniazid- sensitive or -resistant and as catalase-positive or -negative (Figure 2B, lower). Thus, expression of ahpC_Mtb is not precluded by disruption of oxyR, expression of catalase or sensitivity to isoniazid, and strains that express AhpC_Mtb do not appear to be rare. Examnle 13 - Inactivation of ahpC m^' S. typhimurium Causes Hypersensitivity to RNI

Disruption of ahpC would be the most direct means to test its function. In M. tuberculosis, targeted gene disruption has met with limited success (Pelicic, V., et al., "Efficient Allelic Exchange and Transposon Mutagenesis in Mycobacterium tuberculosis:^' Proc. Natl. Acad. Sci. USA, 94: 10955-60, (1997), which is hereby incorporated by reference), and has not yet been accomplished for ahpC_Mt_b- In contrast. ahpC has been inactivated in S. typhimurium strain LT2 by insertion of TnlO. generating strain TA4190 (Storz, G.. et al., "An Alkyl Hydroperoxide

Reductase Induced by Oxidative Stress in Salmonella typhimurium and Escherichia coli: Genetic Characterization and Cloning ofahp " J. Bacteriol. 181 :2049-55 (1989). which is hereby incorporated by reference). The transposon disrupts the 33rd codon of the ahpC ORF. Therefore, this organism was used to explore the function of ahpC. First, the reported phenotype of TA4190, namely, its increased sensitivity to cumene hydroperoxide compared to LT2, was confirmed (Figure 3A).

Next, it was determined whether disruption of ahpC rendered salmonella more sensitive to RNI. For this, two forms of RNI were used, both of which are physiologic products of the activated macrophage: mildly acidified nitrite and S-nitrosoglutathione (GSNO). Mildly acidified nitrite, often used to preserve foods, has long been recognized as bactericidal. At the pH encountered in phagolysosomes. a small proportion of nitrite is protonated. The protonated form dismutates to generate NO, NO , N₂O₃, and N 0 (reviewed in Ehrt, S., et al., "An Antioxidant Gene From Mycobacterium tuberculosis " J. Exp. Med., 186:1885-96 (1997), which is hereby incorporated by reference). When both LT2 and TA4190 were exposed to sodium nitrite (4 mM, pH 5) for 14 hours, ~10⁶-fold fewer TA4190 survived than LT2 (Figure 3B, panel a). Similarly, sodium nitrite (3 mM, pH 5) killed E5 logs of TA4190 cells within 24 hours, but was not bactericidal to LT2 (panel b). As controls, neither LT2 nor TA4190 was sensitive to sodium nitrate at pH 5 nor to sodium nitrite at pH 7 (panel c). The mildly acidic condition alone had no effect on the growth of TA4190. which matched that of LT2 (that is, the growth curves were the same as in panel c when nitrate was omitted). GSNO is bactericidal at neutrality (e.g., Ehrt, S., et al., "An Antioxidant Gene From Mycobacterium tuberculosis " J. Exp. Med., 186: 1885-96 (1997) . which is hereby incorporated by reference), but more stable under mildly acidic conditions (Feelisch, M.. et al., "Donors of Nitrogen Oxides," In Methods in Nitric Oxide Research, Feelisch. M., and Stamler, J.S., Editors, John Wiley and Sons, New York, 71-1 15 (1996). which is hereby incorporated by reference). Because the bactericidal activity of GSNO requires its uptake (De Groote, M. A., et al., "Homocysteine Antagonism of Nitric Oxide-Related Cytostasis in Salmonella typhimurium " Science, 272:414-17 (1996), which is hereby incorporated by reference), enhanced stability is accompanied by enhanced potency. Hence, GSNO was also used at pH 5. Disruption of ahpC in TA4190 led to marked hypersensitivity to GSNO compared to LT2 (Figure 3C, panels a and b).

Example 14 - ahpC from M. tuberculosis Complements ahpCF Deficiency in S. typhimurium for Resistance to RNI It was then determined whether ahpCMtb could functionally replace ahpCsty. To introduce these genes into strain TA4190, pRB3-273C, a medium-copy plasmid that is stable in salmonella (Berggren, R.E., et al., "HIV gpl20-Specific Cell- Mediated Immune Responses in Mice After Oral Immunization With Recombinant Salmonella," J. Acquir. Immune Defic. Syndr. Hum. Retrovirol.. 10:489-495 (1995), which is hereby incorporated by reference) was used. The ORFs of ahpC _tb, ahpCs_ty, and ahpCF_si_y operon were individually subcloned onto pRB3-273C under the ahpCFs,y promoter to yield, respectively, plasmids pPs-MtahpC-RB, pStahpC-RB, and pStahpCF-RB. In TA4190, ahpF expression is thought to be eliminated by polarity as a result of TnlO insertion (Storz, G., et al., "An Alkyl Hydroperoxide Reductase Induced by Oxidative Stress in Salmonella typhimurium and Escherichia coli: Genetic Characterization and Cloning of ahp " J. Bacteriol., 181 :2049-55 (1989), which is hereby incorporated by reference). To investigate the role of AhpF s^,, ahpFsty was also placed downstream of ahpCMt_b to form a chimeric bi-cistronic operon in pPs-MtahpCF-RB. Expression of the proteins AhpCsty, AhpCMtb, and AhpF sty after transformation of TA4190 was confirmed by immunoblotting (Figure 4A). As expected, anti-AhpCs_ty antibody did not immunoblot TA4190, whose αhpC has been disrupted by Tn70 insertion (upper row, lane 2), but it did detect a single polypeptide species in the wild type strain LT2 (lane 1) and in TA4190 carrying either pStahpC- RB (lane 3) or pStahpCF-RB (lane 4). Immunoblots with anti-AhpC_Mtb antibody were negative with lysates of LT2, TA4190. and TA4190 (pStahpC-RB) (middle row, lanes 1 -3), indicating that this antibody does not cross-react with AhpCsty ■ When TA4190 was transformed with pPs-MtahpC-RB or pPs-MtahpCF-RB, anti-AhpCMtb antibody immunoblotted Ahp _Mtb (lanes 5 and 6). Immunoblots confirmed expression of AhpF sty (~51 kD) from the chromosome in LT2 (lower row, lane 1) and from pStahpCF-RB (lane 4) and pPs-MtahpCF-RB (lane 6) in transformed TA4190. Anti- AhpFsty also reacted faintly with TA4190, suggesting that Tn70 insertion into αhpCs,_v greatly reduced, but did not totally abolish, the transcription of downstream αhpF (lane 2).

Next, LT2 and TA4190. both transformed with the vector pRB3-273C to comprise positive and negative controls, respectively, were compared to TA4190 transformed with pStahpC-RB, pPs-MtahpC-RB, and pPs-MtahpCf-RB for their resistance to cumene hydroperoxide. Vector-transformed TA4190 was the most susceptible, succumbing to micromolar cumene hydroperoxide, while LT2 (pRB3- 273C) was not sensitive to the same concentrations (Figure 4B). Surprisingly, transformation of TA4190 with pStahpC-RB alone restored resistance to cumene hydroperoxide to the same level displayed by wild type control LT2 (pRB3-273C) (panels a and b). Given that purified AhpCsty requires AhpF to reduce cumene hydroperoxide (Jacobson, F. S., et al., "An Alkyl Hydroperoxide Reductase from Salmonella typhimurium Involved in the Defense of DNA against Oxidative Damages. Purification and Properties," J. Biol. Chem., 264:1488-96 (1989), which is hereby incorporated by reference), this suggests that the residual level of AhpF in TA4190 (see Figure 4A, lower row, lane 2) was sufficient to cooperate with AhpCsty to constitute a functional alkyl hydroperoxide reductase. The gene ahpCMtb, expressed from pPs-MtahpC-RB, was less efficient than ahpCs_ty in affording partial protection against cumene hydroperoxide. AhpF expression in trans in conjunction with expression of ahpC _tb provided TA4190 with additional protection (Figure 4B, panels a and b). The impaired efficiency of AhpC_Mtb operating in anti-ROI mode with salmonella AhpF presumably reflected the phylogenetic distance between the donor and recombinant host. Viable TA4190 (pRB3-273C) were reduced by 4-5 orders of magnitude by nitrite (2 mM, pH 5) (Figure 4C, panel a) or GSNO (5 mM, pH 5) (panel b), while LT2 was resistant (panel a and b). The deficiency in TA4190^'s ability to survive in RNI was fully complemented in trans not only by expression of ahpCsty. but also by

Complementation conferred by either species^' ahpC for resistance to RNI did not require the help of ahpFs,_} (Figure 4C), in contrast to the situation with cumene hydroperoxide (Figure 4B).

Example 15 - Expression of a pCMtb in Stably Transfected Human Cells and Protection from GSNO

Macrophages can control the replication of M. tuberculosis through expression of NOS2 (Chan, J., et al.. "Killing of Virulent Mycobacterium tuberculosis by Reactive Nitrogen Intermediates Produced by Activated Murine Macrophages," J Exp. Med. 175: 11 1 1-22 (1992); MacMicking, J. D., "Identification of NOS2 as a Protective Locus against Tuberculosis," Proc. Natl. Acad. Sci. USA, 94:5243-48 (1997), which are hereby incorporated by reference). However, activated macrophages also produce large amounts of ROI (Nathan, C. F., et al., "Hydrogen Peroxide Release from Mouse Peritoneal Macrophages: Dependence on Sequential Activation and Triggering," J. Exp. Med., 146:1648-62 (1977), which is hereby incorporated by reference), making them unsuitable as a test cell in which to assess the effect of AhpC selectively against NOS2-derived RNI. As a substitute, a transfectable human epithelial cell line, 293, that expresses neither the respiratory burst oxidase nor NOS2 was used. Transient transection of NOS2 or vector alone into 293 cells permitted the effects of physiologically relevant (macrophage-like) amounts of RNI to be tested in the presence of no more than basal levels of ROI.

To this end, two independent lines of 293 cells that stably expressed transfected ahpCMtb, termed 293/AhpC-l and 293/AhpC-2, were cloned. Both lines expressed full-length AhpC_Mtb protein as judged by immunoblot (Figure 5A). Nearly every cell in each line was positive, as assessed by immunocytochemistry with specific antiserum (Figure 5B). 293 cells stably transfected with vector alone (293/neo^r) (Figure 5A. B) as well as parental 293 cells were nonreactive by both assays (Figure 5A). Addition of GSNO to the cell culture medium killed 293 cells in a dose-dependent fashion, as monitored with a tetrazolium reduction assay. However, the concentration of GSNO required to kill 50% of 293/AhpC-l was about 5 fold higher than that needed to kill 50% of 293/neo^r (Figure 6).

Example 16 - Expression of A pCM_tb Protected Human Cells from Cytotoxicity and Apoptosis Induced by Expression of NOS2

The two independent 293 cell lines stably expressing AhpCMtb (293/AhpC-l and 293/AhpC-2), along with 293/neo^r as negative control, were transiently tranfected with either control vector (pcDNAI/Amp) or NOS2 carried on this vector (pL8Amp). About 40 hours later, cells excluding trypan blue were counted microscopically. 293/AhpC-l and 293/AhpC-2 consistently survived better than 293/neo^r. the apparent survival advantage ranging from 35 to 60% (Figure 7A). Improved viability was not explained by differential expression of NOS2 at the level of enzyme protein (Figure 7B) or product (nitrite) accumulating in the medium.

Inspection of the cultures revealed more striking differences. Figure 7C shows representative fields. Without NOS2, vector-transformed 293/neo^r cells remained adherent and nearly confluent (panel a). After transfection with NOS2, many 293/neo^r cells seemed to disappear. Most of the remainder detached from the plate and rounded up (panel b). In contrast, expression of NOS2 had little impact on the morphology of 293/AhpC-l or 293/AhpC-2 (panels c-f).

The greater destruction caused by NOS2 as evaluated morphologically compared to that evident with trypan blue was consistent with the possibility that many of the cells were undergoing apoptosis, since apoptotic cells can exclude trypan blue (e.g., Lazebnik, Y. A., et al., "Nuclear Events of Apoptosis In vitro in Cell-Free Mitotic Extracts: A Model System for Analysis of the Active Phase of Apoptosis," J. Cell Biol., 123:7-22 (1993), which is hereby incorporated by reference). RNI generated by NOS2 can induce apoptosis in macrophages, neurons, pancreatic β cells, thymocytes. and tumor cells (Xie, K., et al., "Transfection with the Inducible Nitric Oxide Synthase Gene Suppresses Tumorigenicity and Abrogates Metastasis by K- 1735 Melanoma Cells." J. Exp. Med., 181 :1333-44 (1995); Sandau. K., et al., "The Balance Between Nitric Oxide and Superoxide Determines Apoptotic and Necrotic Death of Rat Mesangial Cells," J. Immunol., 158:4938-46 (1997) and references^" cited therein, which are hereby incorporated by reference). Internucleosomal DNA cleavage is a biochemical marker of apoptosis (Enari, M., et al., "A Caspase- Activated DNase That Degrades DNA During Apoptosis, and Its Inhibitor ICAD,^" Nature, 391 :43-50 (1998), which is hereby incorporated by reference). Genomic

DNA isolated from cultures like those in Figure 7D revealed such DNA fragmentation only in NOS2-transfected 293/neo^r cells (lane 2). Chromatin DΝA from NOS2- transfected 293/AhpC-l and 293/AhpC-2 remained intact (lanes 4 and 6). Hence, expression of ahpC_Mtb prevented 293 cells from undergoing apoptotic cell death induced by expression of NOS2.

AhpC protected both bacterial and human cells against RΝI. Protection was effective against RΝI generated chemically (by acidified nitrite or GSΝO) or biochemically (by ΝOS2), and was evident against levels of injury ranging from stasis to lysis (for bacteria) and from apoptosis to necrosis (for mammalian cells). The gene ahpC, which is as diverse as those from S. typhimurium (a purple bacterium) and M. tuberculosis (an actinomycete). had anti-RNI functions. The degree of protection against RNI conferred on Salmonella by its own ahpC was comparable to. if not greater than, the protection conferred against alkyl hydroperoxides. Considering the ubiquity of RNI as products of soil commensals (Conrad, R.. "Soil Microorganisms as Controllers of Atmospheric Trace Gases H₂, CO, CH₄, OCS, N₂0, and NO," Microbiol. Rev.. 60:609-640 (1996), which is hereby incorporated by reference) as well as animal hosts (Nathan, C.„ et al., "Nitric Oxide Synthases: Roles. Tolls and Controls," Cell, 79:915-18 (1994), which is hereby incorporated by reference), the physiologic function of ahpC may consist in resisting RNI as much as in reducing alkyl hydroperoxides. These findings cast ahpC in a new light, as the most widely distributed RNI resistance gene known.

Example 17 - Other RNI Resistance Genes Several other microbial RNI resistance genes have recently been identified, one from M. tuberculosis and the others from enterics. A gene called noxrl (for nitrogen oxides and oxygen intermediates resistance- 1) was cloned from M. tuberculosis and appears confined to the M. tuberculosis complex. Expression of noxrl in E. coli and S. typhimurium conferred resistance to acidified nitrite. GSNO, H₂0₂, HOC1 and H⁺ (Εhrt, S., et al., "An Antioxidant Gene From Mycobacterium tuberculosis " J. Εxp. Med., 186:1885-96 (1997), which is hereby incorporated by reference). In E. coli, NO shares the ability of intracellularly generated (but not exogenous) O₂ ^" to induce soxRS, a regulon of 10 genes (Nunoshiba, T., et al., "Activation by Nitric Oxide of an Oxidative-Stress Response that Defends Escherichia coli against Activated Macrophages," Proc. Natl. Acad. Sci. USA, 90:9993-97 (1993). which is hereby incorporated by reference). In E. coli. but not S. typhimurium (Fang. F. C, "Mechanisms of Nitric Oxide-Related Antimicrobial Activity," J. Clin. Invest., 99:2812-25 (1997). which is hereby incorporated by reference), the soxRS system confers modest resistance to NO (Nunoshiba, T., et al.. "Activation by Nitric Oxide of an Oxidative-Stress Response that Defends Escherichia coli Against Activated Macrophages," Proc. Natl. Acad. Sci. USA. 90:9993-97 (1993); Nunoshiba, T.. et al., "Roles of Nitric Oxide in Inducible Resistance of Escherichia coli to Activated Macrophages." Infect. Immun., 63:794-98 (1995), which are hereby incorporated by reference) via induction of Mn-superoxide dismutase (sodA), glucose 6-phosphate dehydrogenase (zwf), endonuclease IV (nfo), and micF. a suppressor of OmpF porin. SOD may confer resistance to RNI by diverting O₂ ^" from reaction with NO and thus forestalling the production of peroxynitrite (De Groote, M. A., et al.. "Periplasmic Superoxide Dismutase Protects Salmonella from Products of Phagocyte Oxidase and Nitric Oxide Synthase," Proc. Natl. Acad. Sci. USA. 94: 13997-14001 (1997), which is hereby incorporated by reference), a mechanism distinct from the protection against pre-formed RNI seen with AhpC. The mechanisms by which other genes in the soxRS regulon contribute to RNI resistance are unknown. In S. typhimurium, mutations in genes encoding enzymes involved in the repair of DNA (De Groote, M. A., et al., "Genetic and Redox Determinants of Nitric Oxide Cytotoxicity in a Salmonella typhimurium Model," Proc. Natl. Acad. Sci. USA. 92:6399-403 (1995). which is hereby incorporated by reference) or the synthesis of homocysteine (meth) (De Groote, M. A., et al., "Homocysteine Antagonism of Nitric Oxide-Related Cytostasis in Salmonella typhimurium^ Science. 272:414-17 (1996), which is hereby incorporated by reference) enhance susceptibility to S-nitrosothiols. Finally, in E. coli. both H₂O₂ (Farr. S. B.. "Oxidative Stress Responses in Escherichia coli and Salmonella typhimurium." Microbiol. Rev., 55:561-85 (1991), which is hereby incorporated by reference) and S-nitrosothiols (Hausladen, A., et al., "Nitrosative Stress— Activation of the Transcription Factor OxyR," Cell, 86:719-29 (1996), which are hereby incorporated by reference), induce oxyR. which controls a regulon of 9 genes, including those encoding catalase, glutathione reductase, and AhpCF. When oxidized or S-nitrosylated, the transcription factor OxyR induces the regulon, whose products confer resistance to H₂0₂, alkyl hydroperoxides, and S-nitrosothiols (Hausladen, A., et al., "Nitrosative Stress-Activation of the Transcription Factor OxyR.^" Cell 86:719- 29 (1996). which is hereby incorporated by reference). Which gene(s) in the oxyR regulon are responsible for resistance to S-nitrosothiols was previously unknown. The present findings suggest that ahpC is (one of) the responsible gene(s). RNI can inactivate glutathione peroxidase (Asahi, M., et al., "Inactivation of Glutathione Peroxidase by Nitric Oxide. Implication for Cytotoxicity," J. Biol. Chem., 270:21035-39 (1995), which is hereby incorporated by reference), an important component of mammalian cell defenses against ROI (Nathan, C. F., et al., "Tumor Cell Antioxidant Defenses: Inhibition of the Gluthathione Redox Cycle Enhances Macrophage-Mediated Cytolysis," J. Exp. Med., 153:766-82 (1981), which is hereby incorporated by reference). Cells less able to redox-cycle glutathione might be more sensitive to endogenous ROI, against which AhpC might protect them, giving the appearance that AhpC protects directly from exogenous RNI. Such a scenario is exceedingly unlikely for several reasons. First, the foregoing postulate could not explain why the protection afforded to S. typhimurium by AhpC was independent of AhpF in the case of RNI, but dependent on AhpF in the case of ROI. Second, depletion of any element of an ROI resistance path has never been found to lead to rapid, extensive cell death from endogenous oxidants; such depletion only serves to sensitize cells to exogenous oxidants. A germane illustration is provided by S. typhimurium strain TA4190, which is completely deficient in AhpC and extensively deficient in AhpF. TA4190 grows normally in the absence of an exogenous oxidant stress, even though it is highly sensitive when such a stress is supplied. Thus, in contrast to the situation with SOD (De Groote, M. A., et al., "Periplasmic Superoxide Dismutase Protects Salmonella from Products of Phagocyte Oxidase and Nitric Oxide Synthase." Proc. Natl. Acad. Sci. USA. 94: 13997-14001 (1997), which is hereby incorporated by reference), the anti-RNI effect of AphC is distinct from its anti-ROI effect. In short, AhpC appears to be bifunctional.

AhpCF can act as a lipid hydroperoxide reductase. Glutathione peroxidase serves as a precedent for a mammalian lipid hydroperoxide reductase that can metabolize RNI. While glutathione peroxidase normally uses glutathione to reduce lipid hydroperoxides to the corresponding alcohols or hydrogen peroxide to water, it can use GSNO in place of glutathione, releasing an unidentified form of RNI (Freedman. J. E., "Glutathione Peroxidase Potentiates the Inhibition of Platelet Function by S-nitrosothiols," J. Clin. Invest.. 96:394-400 (1995), which is hereby incorporated by reference). However, because the product of this reaction is more potent than GSNO as an inhibitor of platelet aggregation (Freedman, J. E., "Glutathione Peroxidase Potentiates the Inhibition of Platelet Function by S- nitrosothiols," J. Clin. Invest., 96:394-400 (1995), which is hereby incorporated by reference), the reaction does not appear to constitute a detoxification. Alternatively, glutathione peroxidase can use glutathione to catabolize peroxynitrite to nitrite, a reaction dependent on the enzyme's distinctive selenocysteine residue (Sies, FI., et al., "Gluthathione Peroxidase Protects Against Peroxynitrite-Mediated Oxidations. A New Function for Selenoproteins as Peroxynitrite Reductase," J. Biol. Chem., 272:27812-17 (1997), which is hereby incorporated by reference). Neither of the foregoing mechanisms is likely to explain the anti-RNI function of AhpC: AhpC alone lacks activity as a lipid hydroperoxide reductase; AhpC is not known to contain selenium; mycobacteria do not contain glutathione (Newton, G. L., et al., "Distribution of Thiols in Microorganisms: Mycothiol is a Major Thiol in Most Actinomycetes," J. BacterioL, 178:1990-95 (1996), which is hereby incorporated by reference); and nitrite itself is one of the RNI against which ahpC conferred protection.

Given the relative inefficiency of AhpC _tb operating against ROI in salmonella, it was striking how proficiently AhpC_Mtb protected both bacterial and human cells against RNI. Because a small number of molecules of AhpC can protect cells against a vast molar excess of RNI, the action of AhpC is probably catalytic. AhpC^'s catalytic mechanism against RNI may involve another protein that serves as a reducing cofactor. AhpC in S. typhimurium requires AhpF to detoxify alkyl hydroperoxides. Defense against RNI does not involve AhpF, and M. tuberculosis appears to lack AhpF (see also Wilson, T. M., et al.. "ahpC. a Gene Involved in Isoniazid Resistance of the Mycobacterium tuberculosis Complex," Mol. Microbiol.. 19: 1025-34 (1996), which is hereby incorporated by reference). Therefore, for anti-RNI defense, another protein may shuttle electrons from NAD(P)H to AhpC in place of AhpF. A candidate for such an AhpF equivalent is thioredoxin reductase. The thioredoxin reductase cloned from M. tuberculosis (GenBank X95798) is 32% identical to the C-terminal half of AhpF from S. typhimurium. Thioredoxin, which has also been identified in M. tuberculosis (Wieles, B., et al., "Identification and

Functional Characterization of Thioredoxin of Mycobacterium tuberculosis " Infect. Immun., 63:4946-48 (1995), which is hereby incorporated by reference), can accelerate the decomposition of S-nitrosothiols in vitro (Nikitovic, D., et al., "S- Nitrosogluththione is Cleaved by Thioredoxin System with Liberation of Glutathione and Redox Regulating Nitric Oxide," J. Biol. Chem., 271 :19180-85 (1996), which is hereby incorporated by reference). Thioredoxin may be part of the RNI resistance mechanism that operates through AhpC.

Isoniazid is the mainstay of antituberculous therapy. It is for isoniazid- resistant tuberculosis that new therapeutic approaches are mostly sorely needed. Many isoniazid-resistant isolates are catalase-deficient (Zhang, Y., et al., "The

Catalase-Peroxidase Gene and Isoniazid Resistance of Mycobacterium tuberculosis " Nature, 358:591-93 (1992), which is hereby incorporated by reference). In such organisms, inhibition of AhpC might cripple a major back-up path for ROI resistance, while also rendering the bacteria susceptible to RNI. Mycobacteria whose AhpC has been inhibited may be more readily killed when exposed to RNI or ROI that are delivered by the host's immune system, RNI delivered by inhalation (Frostell, C. G., "Inhaled Nitric Oxide, Clinical Rationale and Applications," Adv. Pharmacol.. 34:439-56 (1995). which is hereby incorporated by reference), or NO-donating organochemicals (Hanson, S. R., et al., "Nitric Oxide Donors: A Continuing Opportunity in Drug Design," Adv. Pharmacol., 34:383-98 (1995), which is hereby incorporated by reference). Conversely, disruption of ahpC in M. tuberculosis or inhibition of its protein product might lead to overproduction of catalase, as observed in B. subtilis (Bsat, N., et al., "Mutation of the Bacillus Subtilis Alkyl Hydroperoxide Reductase (ahpCF) Operon Reveals Compensatory Interactions Among Hydrogen Peroxide Stress Genes,^" J. BacterioL, 178:6569-86 (1996), which is hereby incorporated by reference), which in turn might restore sensitivity to isoniazid. Although microbial RNI resistance has been focused on, these results have implications for eukaryotic cells. In the absence of ahpC ι,_b, epithelial cells underwent marked apoptosis and necrosis after they were transfected to express NOS2 at levels typical of activated macrophages. When transfected with

the epithelial cells were substantially protected. Macrophages themselves can tolerate similar levels of NOS2 without evident autotoxicity (Vodovotz, Y., et al..

"Inactivation of Nitric Oxide Synthase Following Prolonged Incubation of Mouse Macrophages with Interferon-Gamma and Bacterial Lipopolysaccharide,^" J. Immunol., 152:4110-18 (1994), which is hereby incorporated by reference). The divergent fates of various NOS2-expressing mammalian cells may reflect differential expression of endogenous anti-RNI genes homologous to ahpC. Indeed, the anti- apoptotic function of a human thioredoxin peroxidase has very recently been demonstrated (Zhang, P., et al., "Thioredoxin Peroxidase is a Novel Inhibitor of Apoptosis with a Mechanism Distinct From That of Bcl-2," J. Biol. Chem., 272:30615-18 (1997), which is hereby incorporated by reference). Oxidant-induced apoptosis is a mechanism shared by such diverse processes as p53-dependent tumor suppression, ischemia-reperfusion, radiation therapy, and antineoplastic chemotherapy (Polyak, K., et al., "A Model for p53-induced Apoptosis." Nature. 389:300-05 (1997); Hug. H., et al., "Reactive Oxygen Intermediates are Involved in the Induction of CD95 Ligand mRNA Expression by Cytostatic Drugs in Hepatoma Cells," J. Biol. Chem., 272:28191-93 (1997), which are hereby incorporated by reference). Inhibition or induction of such enzymes may facilitate therapeutic regulation of the extent of oxidant-induced apoptosis.

Example 18 - Complementation of a pC Deficiency in Salmonella typhimurium (Sty) ahpC: Tn 10 Strain TA4190 With AhpC from Helicobacter pylori (Hpy)

The Sty strain LT2 is wild type at the ahpC locus. This gene is completely disrupted in the TA4190 strain by the insertion of transposon TnlO (ahpC:TnlO) into this gene locus. This gene disruption renders the mutant strain^' highly susceptible to reactive nitrogen intermediates generated from the stresses used in this experiment. This phenotype is rescued when the mutant strain is supplied with either the ahpC gene from Sty or Hpy. Sty TA4190 (TA) and its cogenic wild type strain LT2 were transformed with control vector pPs-T7. The TA-Sty-AhpC and TA- Hpy- AhpC strains have the vector containing either the ahpC gene of Sty or Hpy, respectively. The pPs-T7 vector carries an ampicillin resistance gene as well as the promoter sequence of the Sty ahpC gene. The promoter expresses the inserted ahpC gene at comparable levels to the Sty ahpC gene on the bacterial chromosome of LT2. Once in the bacterial cell, this vector replicates independently of the bacterial chromosomal DNA and leads to the protein expression of the gene introduced. Overnight cultures were grown in Luria Bertani medium (LB) supplemented with ampicillin 100 μg/ml (LbAmp) (for LT2) or Amp 100 plus Tetracycline 15 μg/ml (for TA, TA-Sty-AhpC and TA-H/?y-AhpC). Bacteria from 14 hour overnight cultures were diluted in LbAmp pΗ 5.0 to 3-5 x 10⁷/ml. 100 microliter (μl) aliquots of each diluted strain were plated in triplicate in 96 well flat bottom polystyrene plates (Corning) containing 10 μl of the indicated nitric oxide generating compound or not. S-nitroso-glutathione (GSNO) was reconstituted in sterile deionized distilled water (d₂Η₂O) and plated accordingly for the indicated final concentrations. Sodium nitrite (Sigma) was prepared by diluting 1 molar NaNO₂ stock into sterile d₂H₂O and, then, plated accordingly for the indicated final concentrations. This compound is designated acidified nitrite (ASN) due to the slightly acidified conditions used in this assay.

The 96 well plate was incubated at 37°C while shaking at 75 rpm. At 3. 6, and 9 hour time points, 10 μl of each well's contents were diluted into 100 μl of LbAmp- 10%) AlamarBlue (Accumed) and stored at 4°C overnight. As bacteria grow in this medium, the redox sensitive dye of AlamarBlue, resazurin, is reduced to resorfin which emits fluorescence. The next day, these plates were incubated at 37°C while shaking at 75 rpm, and the reduction of the AlamarBlue was recorded in arbitrary units (FSU). Each plate was read every hour for ten hours with a Cytofluor 2350 plate reading fluorometer (Millipore) set to excitation 530 nm, emission 590 nm, and sensitivity 2. Fluorescence data from each well and time point were compiled in MicroSoft Excel and normalized by subtraction of the reagent ^' blank. Where the curves of fluorescence units versus time were linear, they were analyzed by linear regression. By using the slope and y-intercept, the time to half maximal fluorescence (4000 FSU) was calculated for each well. This was then used to extrapolate the calculated CFU based on linear regression analysis of log CFU (determined by LbAmp plating) versus time to half maximal fluorescence predetermined for each strain (Shiloh et al.. Infection and Immunity 65:3193-3198 (1997), which is hereby incorporated by reference). The data shown are from a representative experiment and expressed as the mean of triplicate samples + standard deviation.

The graph provided depicts the 6 hour time point of this assay and gives evidence for the susceptible phenotype of the TA strain in both stresses assayed. For example, at 5 millimolar (mM) GSNO, the TA strain is killed over four logs when compared to the LT2 strain. However, the TA-Sty-AhpC and TA-Hpy-AhpC strains partially rescue this extreme sensitivity. The same phenomenon is observed in the ASN treated cells at 5 mM, where the TA strain survives 100 fold less than LT2 and the complementing strains, regardless of the origin of ahpC (Sty or Hpy), show full resistance. In summary, it has been observed that the Hpy ahpC gene can partially or fully complement the AhpC deficiency of the TA4190 strain comparably to the Sty ahpC gene with respect to conferring resistance to nitrosative stress.

Although the invention has been described in detail for the purpose of illustration, it is understood that such details are solely for that purpose and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims

WHAT IS CLAIMED:

1. A method of screening therapeutics for their ability to sensitize bacteria to antibacterial effects of reactive nitrogen intermediates comprising: providing a growth medium containing a reactive nitrogen intermediate and a test therapeutic; preparing recombinant host cells deficient in endogenous alkyl hydroperoxide reductase subunit C protein or polypeptide encoding genes and transformed or not with a DNA molecule encoding an alkyl hydroperoxide reductase subunit C protein or polypeptide; placing the recombinant host cells in the growth medium: and determining whether the host cells perish or survive in the growth medium.

2. A method according to claim 1 , wherein host cells that are not recombinant are also placed in the growth medium as a control.

3. A method according to claim 1, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is a Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium avium, Mycobacterium smegmatis, Salmonella typhimurium, Escherichia coli, Amphibacillus xylanus, Bacillus alcalophilus, Bacillus subtilis, Clostridium pasteurianum, Corynebacterium diphtheriae, Entamoeba histolytica, Enter ococcus faecalis, Helicobacter pylori, Methanobacterium thermoautotrophicum, Rattus norvegicus, Stapphyloccus aureus, Streptococcus mutans, Xanthomonas campestris, Trypanosoma brucei, Legionella pneumophila, Sulfolobus metallicus, Saccharomyces cerevisiae, Caenorhabditis elegans, Bromo secalinas, Mus musculus, rice, wheat, bovine, rat, or human protein or polypeptide.

4. A method according to claim 3, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is an Helicobacter pylori protein or polypeptide.

5. A method according to claim 3, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is a Mycobacterium tuberculosis protein or polypeptide.

6. A method of treating septic hypotension in mammals comprising: administering an effective amount of an alkyl hydroperoxide reductase subunit C protein or polypeptide to mammals.

7. A method according to claim 6, wherein said administering is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.

8. A method according to claim 6, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is a Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium avium, Mycobacterium smegmatis, Salmonella typhimurium, Escherichia coli, Amphibacillus xylanus, Bacillus alcalophilus, Bacillus subtilis, Clostridium pasteurianum, Corynebacterium diphtheriae, Entamoeba histolytica, Enter ococcus faecalis, Helicobacter pylori, Methanobacterium thermoautotrophicum, Rattus norvegicus,

Stapphyloccus aureus, Streptococcus mutans, Xanthomonas campestris, Trypanosoma brucei, Legionella pneumophila, Sulfolobus metallicus, Saccharomyces cerevisiae, Caenorhabditis elegans, Bromo secalinas, Mus musculus, rice, wheat, bovine, rat, or human protein or polypeptide.

9. A method according to claim 8, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is an Helicobacter pylori protein or polypeptide.

10. A method according to claim 8 wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is a Mycobacterium tuberculosis protein or polypeptide. - so i l . A method of treating stroke in mammals comprising: administering an effective amount of an alkyl hydroperoxide reductase subunit C protein or polypeptide to mammals.

12. A method according to claim 1 1 , wherein said administering is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.

13. A method according to claim 1 1 wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is a Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium avium, Mycobacterium smegmatis, Salmonella typhimurium, Escherichia coli, Amphibacillus xylamis, Bacillus alcalophilus, Bacillus subtilis, Clostridium pasteurianum, Corynebacterium diphtheriae, Entamoeba histolytica, Enter ococcus faecalis, Helicobacter pylori, Methanobacterium thermoautotrophicum, Rattus norvegicus,

14. A method according to claim 13, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is an Helicobacter pylori protein or polypeptide.

15. A method according to claim 13, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is a Mycobacterium tuberculosis protein or polypeptide.

16. A method of quenching overproduction of nitric oxides in response to infection by bacterial pathogens comprising: administering an effective amount of an alkyl hydroperoxide reductase subunit C protein or polypeptide to mammals.

17. A method according to claim 16. wherein said administering is oral, intradermal. intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.

18. A method according to claim 16, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is a Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium avium, Mycobacterium smegmatis, Salmonella typhimurium, Escherichia coli, Amphibacillus xylanus, Bacillus alcalophilus, Bacillus subtilis, Clostridium pasteurianum, Corynebacterium diphtheriae, Entamoeba histolytica, Enter ΌCOCCUS faecalis,

Helicobacter pylori, Methanobacterium thermoautotrophicum, Rattus norvegicus, Stapphyloccus aureus, Streptococcus mutans, Xanthomonas campestris, Trypanosoma brucei, Legionella pneumophila, Sulfolobus metallicus, Saccharomyces cerevisiae, Caenorhabditis elegans, Bromo secalinas, Mus musculus, rice, wheat, bovine, rat, or human protein or polypeptide.

19. A method according to claim 18, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is an Helicobacter pylori protein or polypeptide.

20. A method according to claim 18, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is a Mycobacterium tuberculosis protein or polypeptide.

21. A method of treating septic hypotension in mammals comprising: administering to mammals an effective amount of the pharmaceutical composition comprising: an alkyl hydroperoxide subunit C protein or polypeptide and a pharmaceutically-acceptable carrier.

22. A method according to claim 21 , wherein said administering is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.

23. A method according to claim 21 , wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is a Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium avium, Mycobacterium smegmatis, Salmonella typhimurium, Escherichia coli, Amphibacillus xylanus. Bacillus alcalophilus, Bacillus subtilis, Clostridium pasteurianum, Corynebacterium diphtheriae, Entamoeba histolytica, Enterococcus faecalis,

24. A method according to claim 23, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is an Helicobacter pylori protein or polypeptide.

25. A method according to claim 23, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is a Mycobacterium tuberculosis protein or polypeptide.

26. A method of treating stroke in mammals comprising: administering to mammals an effective amount of the pharmaceutical composition comprising: an alkyl hydroperoxide subunit C protein or polypeptide and a pharmaceutically-acceptable carrier.

27. A method according to claim 26, wherein said administering is oral, intradermal. intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.

28. A method according to claim 26, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is a Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae. Mycobacterium avium, Mycobacterium smegmatis, Salmonella typhimurium, Escherichia coli, Amphibacillus xylanus, Bacillus alcalophilus, Bacillus subtilis, Clostridium pasteurianum, Corynebacterium diphtheriae, Entamoeba histolytica, Enterococcus faecalis, Helicobacter pylori, Methanobacterium thermoautotrophicum, Rattus norvegicus, Stapphyloccus aureus, Streptococcus mutans, Xanthomonas campestris. Trypanosoma brucei, Legionella pneumophila, Sulfolobus metallicus, Saccharomyces cerevisiae, Caenorhabditis elegans. Bromo secalinas, Mus musculus, rice, wheat, bovine, rat, or human protein or polypeptide.

29. A method according to claim 28, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is an Helicobacter pylori protein or polypeptide.

30. A method according to claim 28, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is a Mycobacterium tuberculosis protein or polypeptide.

31. A method of quenching overproduction of nitric oxides in response to infection by bacterial pathogens comprising: administering to mammals an effective amount of the pharmaceutical composition comprising: an alkyl hydroperoxide subunit C protein or polypeptide and a pharmaceutically-acceptable carrier.

32. A method according to claim 31 , wherein said administering is oral, intradermal. intramuscular, intraperitoneal. intravenous, subcutaneous, or intranasal.

33. A method according to claim 31, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is a Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium avium, Mycobacterium smegmatis, Salmonella typhimurium, Escherichia coli, Amphibacillus xylanus, Bacillus alcalophilus, Bacillus subtilis, Clostridium pasteurianum, Corynebacterium diphtheriae, Entamoeba histolytica, Enterococcus faecalis, Helicobacter pylori, Methanobacterium thermoautotrophicum, Rattus norvegicus, Stapphyloccus aureus. Streptococcus mutans, Xanthomonas campestris, Trypanosoma brucei, Legionella pneumophila, Sulfolobus metallicus, Saccharomyces cerevisiae, Caenorhabditis elegans, Bromo secalinas, Mus musculus, rice, wheat, bovine, rat, or human protein or polypeptide.

34. A method according to claim 33, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is an Helicobacter pylori protein or polypeptide.

35. A method according to claim 33, wherein the alkyl hydroperoxide reductase subunit C protein or polypeptide is a Mycobacterium tuberculosis protein or polypeptide.

36. An isolated DNA molecule encoding a Mycobacterium tuberculosis alkyl hydroperoxide reductase subunit C protein or polypeptide and conferring resistance to antimicrobial reactive nitrogen intermediates.

37. An isolated DNA molecule according to claim 36 wherein said

DNA molecule encodes a protein or polypeptide with an amino acid sequence comprising SEQ. ID. No. 2 or nucleic acid molecules which hybridize to the nucleotide sequence of SEQ. ID. No. 1 under stringent conditions.

38. An isolated DNA molecule according to claim 36, wherein said

DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 1 or nucleic acid molecules which hybridize to the nucleotide sequence of SEQ. ID. No. 1 under stringent conditions.

39. An isolated protein or polypeptide encoded by a DNA molecule according to claim 36.

40. An isolated protein or polypeptide according to claim 39, wherein the protein or polypeptide has an amino acid sequence corresponding to SEQ. ID. No. 2.

41. An isolated protein or polypeptide according to claim 39, wherein said protein or polypeptide is recombinant.

42. An isolated protein or polypeptide according to claim 39, wherein said protein or polypeptide is purified.

43. An isolated protein or polypeptide according to claim 39, wherein said protein or polypeptide has one or more antigenic determinants and confers on Mycobacterium tuberculosis resistance to antimicrobial reactive nitrogen intermediates.

44. A method of vaccinating mammals against infection by

Mycobacterium tuberculosis comprising: administering an effective amount of an isolated

Mycobacterium tuberculosis protein or polypeptide according to claim 39 to mammals.

45. A method according claim 44, wherein said administering is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.

46. A recombinant DNA expression system comprising an expression vector into which is inserted a heterologous DNA according to claim 36.

47. A recombinant DNA expression system according claim 46, wherein said heterologous DNA comprises the nucleotide sequence corresponding to SEQ. ID. No. 1 or nucleic acid molecules which hybridize to the nucleotide sequence of SEQ. ID. No. 1 under stringent conditions.

48. A recombinant DNA expression system according to claim 46. wherein said heterologous DNA is inserted into said vector in proper sense orientation and correct reading frame.

49. A recombinant DNA expression system according to claim 46, wherein said heterologous DNA is inserted into said vector in antisense orientation and correct reading frame.

50. A host cell incorporating a heterologous DNA according to claim 36.

51. A host cell according to claim 50, wherein said heterologous

DNA comprises the nucleotide sequence corresponding to SEQ. ID. No. 1 or a nucleic acid which hybridizes to the nucleotide sequence of SEQ. ID. No. 1 under stringent conditions.

52. A host cell according to claim 50, wherein said heterologous

DNA is inserted in a recombinant DNA expression system comprising an expression vector.

53. A pharmaceutical composition comprising: an isolated protein or polypeptide according to claim 39; and a pharmaceutically-acceptable carrier.

54. A pharmaceutical composition according to claim 53, wherein said protein or polypeptide is purified.

55. A method of vaccinating mammals against infection by Mycobacterium tuberculosis comprising: administering an effective amount of the pharmaceutical composition according to claim 53 to mammals.

56. A method according claim 55, wherein said administering is oral, intradermal. intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.

57. An isolated antibody or binding portion thereof against a protein or polypeptide according to claim 39.

58. An isolated antibody or binding portion thereof according to claim 57, wherein said antibody is monoclonal or polyclonal.

59. An isolated antibody or binding portion thereof according to claim 57, wherein said antibody is specific for an antigenic determinant of said protein or polypeptide encoded by a gene fragment conferring on Mycobacterium tuberculosis resistance to antimicrobial reactive nitrogen intermediates.

60. A method of passively immunizing mammals infected with Mycobacterium tuberculosis comprising: administering an effective amount of said antibody or binding portion thereof according to claim 57 to mammals infected with Mycobacterium tuberculosis.

61. A method according to claim 60, wherein said administering is oral, intradermal. intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.

62. A composition for passively immunizing mammals infected with Mycobacterium tuberculosis comprising: an isolated antibody or binding portion thereof according to claim 57 and a pharmaceutically-acceptable carrier.

63. A composition according to claim 62, wherein said antibody is monoclonal or polyclonal.

64. A composition according to claim 62 wherein said antibody is specific for an antigenic determinant of said protein or polypeptide encoded by a gene fragment conferring on Mycobacterium tuberculosis resistance to antimicrobial reactive nitrogen intermediates.

65. A method of passively immunizing mammals infected with Mycobacterium tuberculosis comprising: administering an effective amount of said composition according to claim 62 to mammals infected with Mycobacterium tuberculosis.

66. A method according to claim 65, wherein said administering is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.

67. A method for detection of Mycobacterium tuberculosis in a sample of tissue or body fluids comprising: providing a protein or polypeptide according to claim 39 as an antigen; contacting the sample with the antigen; and detecting any reaction which indicates that Mycobacterium tuberculosis is present in the sample using an assay system.

68. A method according to claim 67, wherein the assay system is selected from the group consisting of an enzyme-linked immunosorbent assay, a radioimmunoassay, a gel diffusion precipitin reaction assay, an immunodiffusion assay, an agglutination assay, a fluorescent immunoassay, a protein A immunoassay, and an immunoelectrophoresis assay.

69. A method for detection of Mycobacterium tuberculosis in a sample of tissue or body fluids comprising: providing an antibody or binding portion thereof according to claim 57; contacting the sample with the antibody or binding portion thereof; and detecting any reaction which indicates that Mycobacterium tuberculosis is present in the sample using an assay system.

70. A method according to claim 69, wherein the assay system is selected from the group consisting of an enzyme-linked immunosorbent assay, a radioimmunoassay, a gel diffusion precipitin reaction assay, an immunodiffusion assay, an agglutination assay, a fluorescent immunoassay, a protein A immunoassay, and an immunoelectrophoresis assay.

71. A method for detection of Mycobacterium tuberculosis in a sample of tissue or body fluids comprising: providing a nucleotide sequence of the DNA molecule according to claim 36 as a probe in a nucleic acid hybridization assay; contacting the sample with the probe; and detecting any reaction which indicates that Mycobacterium tuberculosis is present in the sample.

72. A method for detection of Mycobacterium tuberculosis in a sample of tissue or body fluids comprising: providing a nucleotide sequence of the DNA molecule according to claim 36 as a probe in a gene amplification detection procedure; contacting the sample with the probe; and detecting any reaction which indicates that Mycobacterium tuberculosis is present in the sample.

73. A method of vaccinating mammals against infection by Mycobacterium tuberculosis comprising: administering an effective amount of the DNA molecule according to claim 36.

74. A method according to claim 73, wherein said administering is oral, intradermal. intramuscular, intraperitoneal. intravenous, subcutaneous, or intranasal.

75. A pharmaceutical composition comprising: an isolated DNA molecule according to claim 39 and a pharmaceutically-acceptable carrier.

76. A method of vaccinating mammals against infection by Mycobacelerium tuberculosis comprising: administering an effective amount of the pharmaceutical composition according to claim 75.

77. A method according to claim 76. wherein said administering is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.