Silencing of Oxidative Stress Response in Mycobacterium tuberculosis: Expression Patterns of ahpC in Virulent and Avirulent Strains and Effect of ahpC Inactivation

Intracellular pathogens such as Mycobacterium tuberculosis are able to survive in the face of antimicrobial products generated by the host cell in response to infection. The product of the alkyl hydroperoxide reductase gene (ahpC) of M. tuberculosis is thought to be involved in protecting the organism against both oxidative and nitrosative stress encountered within the infected macrophage. Here we report that, contrary to expectations, ahpC expression in virulent strains of M. tuberculosis and Mycobacterium bovis grown in vitro is repressed, often below the level of detection, whereas expression in the avirulent vaccine strain M. bovis BCG is constitutively high. The repression of the ahpC gene of the virulent strains is independent of the naturally occurring lesions of central regulator oxyR. Using a green fluorescence protein vector (gfp)-ahpC reporter construct we present data showing that repression of ahpC of virulent M. tuberculosis also occurred during growth inside macrophages, whereas derepression in BCG was again seen under identical conditions. Inactivation of ahpC on the chromosome of M. tuberculosis by homologous recombination had no effect on its growth during acute infection in mice and did not affect in vitro sensitivity to H2O2. However, consistent with AhpC function in detoxifying organic peroxides, sensitivity to cumene hydroperoxide exposure was increased in the ahpC::Km(r) mutant strain. The preservation of a functional ahpC gene in M. tuberculosis in spite of its repression under normal growth conditions suggests that, while AhpC does not play a significant role in establishing infection, it is likely to be important under certain, as yet undefined conditions. This is supported by the observation that repression of ahpC expression in vitro was lifted under conditions of static growth.     

Mycobacteria lacking the RD1 region do not induce necrosis in the lungs of mice lacking interferon-gamma

The genetic region of difference 1 (RD1) in Mycobacterium tuberculosis has recently been hypothesized to encode for proteins that are cytotoxic to the host cell in nature. We demonstrate here that while M. tuberculosis grew progressively in the lungs of gene disrupted mice (GKO) unable to produce interferon-gamma (IFN-gamma), similar mice infected instead with M. bovis bacillus Calmette-Guérin (BCG) reproducibly exhibited an obvious slowing of the disease after about 20 days. Closer examination of BCG-infected GKO mice showed a florid granulomatous inflammation in the lungs, whereas similar mice infected with M. tuberculosis exhibited wholesale progressive necrosis. In the BCG-infected GKO mice large numbers of activated effector T cells, some strongly positive for the cytokine tumour necrosis factor, as well as activated natural killer cells accumulated in the lungs. To further test the hypothesis that the differences observed were directly associated with the loss of the RD1 region, it was then shown that a mutant of M. tuberculosis lacking RD1 grew progressively in both normal and GKO mice but failed to induce any degree of necrosis in either animal despite reaching similar levels in the lungs. However, when mice were infected with this mutant, in which the RD1 region had been restored by complementation, wholesale necrosis of the lungs again occurred. These data support the hypothesis that proteins encoded in the RD1 region are a major cause of necrosis and contribute significantly to the pathogenesis of the disease.     

Anaerobic arginine metabolism of Mycobacterium tuberculosis is mediated by arginine deiminase (arcA), but is not essential for chronic persistence in an aerogenic mouse model of infection

In many pathogens, degradation of arginine via the arginine deiminase pathway supports anaerobic metabolism. Here we show by deletion of Rv1001 (arcA) in Mycobacterium tuberculosis that this gene functions as an arginine deiminase. Arginine metabolism in the presence of oxygen was not affected by the mutation, indicating a separate pathway for arginine degradation under aerobic conditions. Following aerosol infection in mice, the DeltaarcA mutant and wild-type strain of M. tuberculosis multiplied and persisted in infected organs in a similar fashion.     

Immunological responses and protective immunity against tuberculosis conferred by vaccination of Balb/C mice with the attenuated Mycobacterium tuberculosis (phoP) SO2 strain

The Mycobacterium tuberculosis phoP mutant strain SO2 has been shown previously to be more attenuated than Mycobacterium bovis bacillus Calmette-Guérin (BCG) and confers protective immunity against tuberculosis in mice and guinea pig models. In this study we have investigated the survival and immunological responses of Balb/c mice infected with the M. tuberculosis SO2 strain. All Balb/C mice survived intratracheal infection with M. tuberculosis SO2 strain under conditions where all the mice infected with the parental M. tuberculosis MT103 had died after 9 weeks. Infection of Balb/c mice with M. tuberculosis SO2 was associated with comparatively lower levels of interferon (IFN)-gamma, interleukin (IL)-4 and tumour necrosis factor (TNF)-alpha and higher levels of inducible nitric oxide synthase (iNOS) during the late stage of infection, when compared with M. tuberculosis MT103 infection. The delayed-type hypersensitivity (DTH) response against M. tuberculosis culture filtrates was similar in mice infected with either the M. tuberculosis phoP SO2 strain or M. tuberculosis MT103. The protective efficacy of M. tuberculosis SO2 was compared with M. bovis BCG when delivered subcutaneously to groups of Balb/C mice. Following intratracheal challenge with M. tuberculosis H37Rv, protection was generated by 60 days post-challenge in mice vaccinated with either vaccine. At day 120 post-challenge the levels of protection were still significantly greater when compared with the non-vaccinated control group. The levels of protection conferred by vaccination with M. tuberculosis SO2 or with M. bovis BCG were similar, as measured by granuloma coalescence and pneumonia in addition to growth reduction of M. tuberculosis H37Rv.     

Ribonucleotide reductase class III, an essential enzyme for the anaerobic growth of Staphylococcus aureus, is a virulence determinant in septic arthritis

Staphylococcus aureus is the most common cause of joint infections. It also contributes to several other diseases such as pneumonia, osteomyelitis, endocarditis, and sepsis. Bearing in mind that S. aureus becomes rapidly resistant to new antibiotics, many studies survey the virulence factors, with the aim to find alternative prophylaxis/treatment regimens. One potential virulence factor is the bacterial ability to survive at different oxygen tensions. S. aureus expresses ribonucleotide reductases (RNRs), which help it to grow under both aerobic and anaerobic conditions, by reducing ribonucleotides to deoxyribonucleotides. In this study, we investigated the role of RNR class III, which is required for anaerobic growth, as a virulence determinant in the pathogenesis of staphylococcal arthritis. The wild-type S. aureus strain and its isogenic mutant nrdDG mutant were inoculated intravenously into mice. Mice inoculated with the wild-type strain displayed significantly more severe arthritis, with significantly more synovitis and destruction of the bone and cartilage versus mutant strain inoculated mice. Further, the persistence of bacteria in the kidneys was significantly more pronounced in the group inoculated with the wild-type strain. Together these results indicate that RNR class III is an important virulence factor for the establishment of septic arthritis.     

Construction and characterization of a Mycobacterium tuberculosis mutant lacking the alternate sigma factor gene, sigF

The alternate RNA polymerase sigma factor gene, sigF, which is expressed in stationary phase and under stress conditions in vitro, has been deleted in the virulent CDC1551 strain of Mycobacterium tuberculosis. The growth rate of the DeltasigF mutant was identical to that of the isogenic wild-type strain in exponential phase, although in stationary phase the mutant achieved a higher density than the wild type. The mutant showed increased susceptibility to rifampin and rifapentine. Additionally, the DeltasigF mutant displayed diminished uptake of chenodeoxycholate, and this effect was reversed by complementation with a wild-type sigF gene. No differences in short-term intracellular growth between mutant and wild-type organisms within human monocytes were observed. Similarly, the organisms did not differ in their susceptibilities to lymphocyte-mediated inhibition of intracellular growth. However, mice infected with the DeltasigF mutant showed a median time to death of 246 days compared with 161 days for wild-type strain-infected animals (P < 0.001). These data indicate that M. tuberculosis sigF is a nonessential alternate sigma factor both in axenic culture and for survival in macrophages in vitro. While the DeltasigF mutant produces a lethal infection of mice, it is less virulent than its wild-type counterpart by time-to-death analysis.     

Ribonucleotide reductase class III,an essential enzyme for the anaerobic growth of Staphylococcus aureus,is a virulence determinant in septic arthritis

Staphylococcus aureus is the most common cause of joint infections. It also contributes to several other diseases such as pneumonia, osteomyelitis, endocarditis, and sepsis. Bearing in mind that S. aureus becomes rapidly resistant to new antibiotics, many studies survey the virulence factors, with the aim to find alternative prophylaxis/treatment regimens. One potential virulence factor is the bacterial ability to survive at different oxygen tensions. S. aureus expresses ribonucleotide reductases (RNRs), which help it to grow under both aerobic and anaerobic conditions, by reducing ribonucleotides to deoxyribonucleotides. In this study, we investigated the role of RNR class III, which is required for anaerobic growth, as a virulence determinant in the pathogenesis of staphylococcal arthritis. The wild-type S. aureus strain and its isogenic mutant nrdDG mutant were inoculated intravenously into mice. Mice inoculated with the wild-type strain displayed significantly more severe arthritis, with significantly more synovitis and destruction of the bone and cartilage versus mutant strain inoculated mice. Further, the persistence of bacteria in the kidneys was significantly more pronounced in the group inoculated with the wild-type strain. Together these results indicate that RNR class III is an important virulence factor for the establishment of septic arthritis.     

The Conserved Hypothetical Protein Rv0574c Is Required for Cell Wall Integrity,Stress Tolerance,and Virulence of Mycobacterium tuberculosis

The virulence of Mycobacterium tuberculosis is intimately related to its distinctive cell wall. The biological significance of poly-α-l-glutamine (PLG), a component in the cell wall of virulent mycobacteria, has not been explored adequately. The focus of this study is to investigate the role of a locus, Rv0574c, coding for a polyglutamate synthase-like protein, in the synthesis of poly-α-l-glutamine in the context of mycobacterial virulence. Evaluation of Rv0574c gene expression in M. tuberculosis demonstrated its growth-phase-linked induction with concomitant accumulation of poly-α-l-glutamine in the cell wall. Rv0574c was activated under conditions prevalent in the tubercular granuloma, e.g., hypoxia, nitric oxide, and CO₂. For functional characterization, we produced a deletion mutant of the Rv0574c gene by allelic exchange. The mutant produced smaller amounts of poly-α-l-glutamine in the cell wall than did the wild-type bacterium. Additionally, the increased sensitivity of the mutant to antitubercular drugs, SDS, lysozyme, and mechanical stress was accompanied by a drastic reduction in the ability to form biofilm. Growth of the ΔRv0574c strain was normal under in vitro conditions but was retarded in THP-1 macrophages and in the lungs and spleen of BALB/c mice. This was in agreement with histopathology of the lungs showing slow growth and less severe pathology than that of the wild-type strain. In summary, this study demonstrates that the protein encoded by the Rv0574c locus, by virtue of modulating PLG content in the cell wall, helps in maintaining cellular integrity in a hostile host environment. Also, its involvement in protecting the pathogen from host-generated lethal factors contributes to the infectious biology of M. tuberculosis.     

Oxygen status of lung granulomas in Mycobacterium tuberculosis-infected mice

It is often assumed that Mycobacterium tuberculosis (Mtb)-induced granulomatous lesions, particularly those undergoing central caseation, are anoxic, and that the survival of Mtb in these lesions requires the integrity of its non-oxidative respiratory pathways. Using the hypoxia marker pimonidazole, we now provide immunohistochemical evidence that in the most frequently used animal model system of inbred mice Mtb-induced granulomas, even after more than one year of aerogenic infection, are not severely hypoxic. In contrast, chronic aerosol infection with M. avium strain TMC724 was associated with hypoxia surrounding necrotizing granuloma centres. Direct measurements of oxygen tension with a flexible microelectrode in mouse lungs chronically infected with Mtb disclosed a wide range of oxygen partial pressures in different parts of the lungs which, however, rarely approached the anoxic conditions consistently found in necrotizing tumours. We further show that an Mtb mutant, defective in nitrate reductase (narG) necessary for survival under anaerobic conditions in vitro, can persist in the lungs of chronically infected mice to a similar extent as wild-type Mtb. These findings have important implications for the use of the mouse model of Mtb infection in developing eradication chemotherapy and for evaluating putative mechanisms of chronic persistence and latency of Mtb.     

In vitro and in vivo characterization of a Mycobacterium tuberculosis mutant deficient in glycosyltransferase Rv1500

In Mycobacterium marinum, the homologue of Rv1500 of M. tuberculosis encodes a glycosyltransferase. Initially, it was suggested that this gene is involved in the synthesis of phosphatidylinositol mannosides (PIMs), generating Ac(2)PIM(7) from Ac(2)PIM(5). Phosphatidylinositol mannoside and its related compounds lipomannan (LM) and lipoarabinomannan (LAM) have been shown to modulate the host response to an infection with M. tuberculosis. Here, we generated a deletion mutant of Rv1500 in M. tuberculosis H37Rv, and analyzed the mutant using a biochemical approach as well as in vitro and in vivo infection models. Inactivation of Rv1500 did not lead to an altered expression pattern of PIMs in M. tuberculosis H37Rv. We found phosphatidylinositol (PI), PIM(2), AcPIM(2), Ac(2)PIM(2), and AcPIM(6) in both strains, but were unable to detect Ac(2)PIM(7) or Ac(2)PIM(5) either in the wild type or the mutant strain. Uptake and growth of H37Rv and Rv1500 mutant strains in murine bone marrow-derived macrophages was identical, and TNFalpha and IL-12p40 production in mouse macrophages and dendritic cells was induced to similar levels following infection with either strain. Aerosol challenge of mice showed that wild type and Rv1500 mutant strains had identical growth rates in infected organs over time. We verified mRNA expression of Rv1500 in H37Rv and conclude that Rv1500 must serve a redundant role in viability and virulence of M. tuberculosis.     

MHC class Ia-restricted T cells partially account for beta2-microglobulin-dependent resistance to Mycobacterium tuberculosis.

Recent studies have highlighted the heterogeneous nature of the CD8(+) T cell response during human Mycobacterium tuberculosis infection; MHC class Ia, MHC class Ib and CD1 have all been identified as significant restriction elements. Here we have attempted to define the role of MHC class Ia in resistance to M. tuberculosis infection in mice. The course of M. tuberculosis infection in mice deficient in a single MHC class Ia molecule, either H2-K(b) or H2-D(b), was essentially identical to that observed in wild-type mice. In contrast, mice fully deficient in MHC class Ia molecules (H2-K(b) / H2-D(b) double knockout mice) were substantially more susceptible to M. tuberculosis infection. However, the double knockout mice were not as susceptible as beta 2-microglobulin-deficient mice, which have a broader phenotypic deficit. Thus, antigen presentation via MHC class Ia is an important component in resistance to M. tuberculosis, but its absence only partially accounts for the increased susceptibility of beta 2-microglobulin-deficient mice.     

Disruption of the gene homologous to mammalian Nramp1 in Mycobacterium tuberculosis does not affect virulence in mice

Natural-resistance-associated macrophage protein 1 (Nramp1) is a divalent cation transporter belonging to a family of transporter proteins highly conserved in eukaryotes and prokaryotes. Mammalian and bacterial transporters may compete for essential metal ions during mycobacterial infections. The mycobacterial Nramp homolog may therefore be involved in Mycobacterium tuberculosis virulence. Here, we investigated this possibility by inactivating the M. tuberculosis Nramp1 gene (Mramp) by allelic exchange mutagenesis. Disruption of Mramp did not affect the extracellular growth of bacteria under standard conditions. However, the Mramp mutation was associated with growth impairment under conditions of limited iron availability. The Mramp mutant displayed no impairment of growth or survival in macrophages derived from mouse bone marrow or in Nramp1(+/+) and Nramp1(-/-) congenic murine macrophage cell lines. Following intravenous challenge in BALB/c mice, counts of parental and Mramp mutant strains were similar in the lungs and spleens of the animals at all time points studied. These results indicate that Mramp does not contribute to the virulence of M. tuberculosis in mice.     

Attenuation of late-stage disease in mice infected by the Mycobacterium tuberculosis mutant lacking the SigF alternate sigma factor and identification of SigF-dependent genes by microarray analysis

The Mycobacterium tuberculosis alternate sigma factor, SigF, is expressed during stationary growth phase and under stress conditions in vitro. To better understand the function of SigF we studied the phenotype of the M. tuberculosis DeltasigF mutant in vivo during mouse infection, tested the mutant as a vaccine in rabbits, and evaluated the mutant's microarray expression profile in comparison with the wild type. In mice the growth rates of the DeltasigF mutant and wild-type strains were nearly identical during the first 8 weeks after infection. At 8 weeks, the DeltasigF mutant persisted in the lung, while the wild type continued growing through 20 weeks. Histopathological analysis showed that both wild-type and mutant strains had similar degrees of interstitial and granulomatous inflammation during the first 12 weeks of infection. However, from 12 to 20 weeks the mutant strain showed smaller and fewer lesions and less inflammation in the lungs and spleen. Intradermal vaccination of rabbits with the M. tuberculosis DeltasigF strain, followed by aerosol challenge, resulted in fewer tubercles than did intradermal M. bovis BCG vaccination. Complete genomic microarray analysis revealed that 187 genes were relatively underexpressed in the absence of SigF in early stationary phase, 277 in late stationary phase, and only 38 genes in exponential growth phase. Numerous regulatory genes and those involved in cell envelope synthesis were down-regulated in the absence of SigF; moreover, the DeltasigF mutant strain lacked neutral red staining, suggesting a reduction in the expression of envelope-associated sulfolipids. Examination of 5'-untranslated sequences among the downregulated genes revealed multiple instances of a putative SigF consensus recognition sequence: GGTTTCX(18)GGGTAT. These results indicate that in the mouse the M. tuberculosis DeltasigF mutant strain persists in the lung but at lower bacterial burdens than wild type and is attenuated by histopathologic assessment. Microarray analysis has identified SigF-dependent genes and a putative SigF consensus recognition site.     

Mycobacterium tuberculosis uptake by recipient host macrophages is influenced by environmental conditions in the granuloma of the infectious individual and is associated with impaired production of interleukin-12 and tumor necrosis factor alpha

Transmission of Mycobacterium tuberculosis from one individual to another usually is associated with episodes of coughing. The bacteria leave the environment of the lung cavity of the infected person and travel in droplets to reach the recipient's respiratory tract. Therefore, at the time that the bacteria encounter alveolar cells (macrophages and epithelial cells) in the new host, they express virulence determinants that are regulated by the environmental conditions in the infected person. To determine if those environmental conditions encountered in the lung cavity (hyperosmolarity, acidic pH, and low oxygen tension, among others) would influence the uptake of M. tuberculosis by the recipient's alveolar macrophages, M. tuberculosis H37Rv was incubated under several conditions for different periods of time, washed at 4 degrees C, and used to infect human monocyte-derived macrophages. While increased osmolarity had no effect on M. tuberculosis uptake compared to the uptake of bacteria grown on 7H10 Middlebrook medium, both acidic pH and anaerobiosis increased the uptake of the H37Rv strain four- to sixfold. Using anti-CD11b receptor blocking antibodies or mannoside to inhibit the uptake of M. tuberculosis by macrophages, we determined that while uptake of M. tuberculosis cultured on 7H10 medium was inhibited 77% +/- 6% in the presence of anti-CD11b antibody, the antibody had no effect on the uptake of M. tuberculosis incubated at pH 6.0 and was associated with 27% inhibition of M. tuberculosis previously exposed to anaerobic conditions. The mannose receptor was also not involved with invasion after exposure to acidic conditions, and mannoside resulted in only 32% inhibition of uptake by macrophages of M. tuberculosis exposed to anaerobiosis. Uptake by macrophages also resulted in the secretion of significantly lower amounts of interleukin-12 and tumor necrosis factor alpha than that by macrophages infected with a strain cultured under laboratory conditions. M. tuberculosis cultured under the pH and oxygen concentration found in the granuloma expresses a large number of proteins that are different from the proteins expressed by bacteria grown under laboratory conditions. The results suggest that M. tuberculosis in vivo may be adapted to gain access to the intracellular environment in a very efficient fashion and may do so by using different receptors from the complement and mannose receptors.     

Glutamine synthetase GlnA1 is essential for growth of Mycobacterium tuberculosis in human THP-1 macrophages and guinea pigs

To assess the role of glutamine synthetase (GS), an enzyme of central importance in nitrogen metabolism, in the pathogenicity of Mycobacterium tuberculosis, we constructed a glnA1 mutant via allelic exchange. The mutant had no detectable GS protein or GS activity and was auxotrophic for L-glutamine. In addition, the mutant was attenuated for intracellular growth in human THP-1 macrophages and avirulent in the highly susceptible guinea pig model of pulmonary tuberculosis. Based on growth rates of the mutant in the presence of various concentrations of L-glutamine, the effective concentration of L-glutamine in the M. tuberculosis phagosome of THP-1 cells was approximately 10% of the level assayed in the cytoplasm of these cells (4.5 mM), indicating that the M. tuberculosis phagosome is impermeable to even very small molecules in the macrophage cytoplasm. When complemented by the M. tuberculosis glnA1 gene, the mutant exhibited a wild-type phenotype in broth culture and in human macrophages, and it was virulent in guinea pigs. When complemented by the Salmonella enterica serovar Typhimurium glnA gene, the mutant had only 1% of the GS activity of the M. tuberculosis wild-type strain because of poor expression of the S. enterica serovar Typhimurium GS in the heterologous M. tuberculosis host. Nevertheless, the strain complemented with S. enterica serovar Typhimurium GS grew as well as the wild-type strain in broth culture and in human macrophages. This strain was virulent in guinea pigs, although somewhat less so than the wild-type. These studies demonstrate that glnA1 is essential for M. tuberculosis virulence.     

Deletion of the Mycobacterium tuberculosis resuscitation-promoting factor Rv1009 gene results in delayed reactivation from chronic tuberculosis

Approximately one-third of the human population is latently infected with Mycobacterium tuberculosis, comprising a critical reservoir for disease reactivation. Despite the importance of latency in maintaining M. tuberculosis in the human population, little is known about the mycobacterial factors that regulate persistence and reactivation. Previous in vitro studies have implicated a family of five related M. tuberculosis proteins, called resuscitation promoting factors (Rpfs), in regulating mycobacterial growth. We studied the in vivo role of M. tuberculosis rpf genes in an established mouse model of M. tuberculosis persistence and reactivation. After an aerosol infection with the M. tuberculosis Erdman wild type (Erdman) or single-deletion rpf mutants to establish chronic infections in mice, reactivation was induced by administration of the nitric oxide (NO) synthase inhibitor aminoguanidine. Of the five rpf deletion mutants tested, one (deltaRv1009) exhibited a delayed reactivation phenotype, manifested by delayed postreactivation growth kinetics and prolonged median survival times among infected animals. Immunophenotypic analysis suggested differences in pulmonary B-cell responses between Erdman- and deltaRv1009-infected mice at advanced stages of reactivation. Analysis of rpf gene expression in the lungs of Erdman-infected mice revealed that relative expression of four of the five rpf-like genes was diminished at late times following reactivation, when bacterial numbers had increased substantially, suggesting that rpf gene expression may be regulated in a growth phase-dependent manner. To our knowledge, deltaRv1009 is the first M. tuberculosis mutant to have a specific defect in reactivation without accompanying growth defects in vitro or during acute infection in vivo.     

Superoxide dismutase-deficient mutants of Helicobacter pylori are hypersensitive to oxidative stress and defective in host colonization

Superoxide dismutase (SOD) is a nearly ubiquitous enzyme among organisms that are exposed to oxic environments. The single SOD of Helicobacter pylori, encoded by the sodB gene, has been suspected to be a virulence factor for this pathogenic microaerophile, but mutations in this gene have not been reported previously. We have isolated mutants with interruptions in the sodB gene and have characterized them with respect to their response to oxidative stress and ability to colonize the mouse stomach. The sodB mutants are devoid of SOD activity, based on activity staining in nondenaturing gels and quantitative assays of cell extracts. Though wild-type H. pylori is microaerophilic, the mutants are even more sensitive to O(2) for both growth and viability. While the wild-type strain is routinely grown at 12% O(2), growth of the mutant strains is severely inhibited at above 5 to 6% O(2). The effect of O(2) on viability was determined by subjecting nongrowing cells to atmospheric levels of O(2) and plating for survivors at 2-h time intervals. Wild-type cell viability dropped by about 1 order of magnitude after 6 h, while viability of the sodB mutant decreased by more than 6 orders of magnitude at the same time point. The mutants are also more sensitive to H(2)O(2), and this sensitivity is exacerbated by increased O(2) concentrations. Since oxidative stress has been correlated with DNA damage, the frequency of spontaneous mutation to rifampin resistance was studied. The frequency of mutagenesis of an sodB mutant strain is about 15-fold greater than that of the wild-type strain. In the mouse colonization model, only 1 out of 23 mice inoculated with an SOD-deficient mutant of a mouse-adapted strain became H. pylori positive, while 15 out of 17 mice inoculated with the wild-type strain were shown to harbor the organism. Therefore, SOD is a virulence factor which affects the ability of this organism to colonize the mouse stomach and is important for the growth and survival of H. pylori under conditions of oxidative stress.