Role of Mycobacterium tuberculosisCopper-Zinc Superoxide Dismutase

Characterization of CuZnSOD-deficient M. tuberculosisand M. bovis BCG.The M. tuberculosis sodCgene was mutated by allelic exchange (17). A DNA fragment containing sodC and 500 bp of its flanking sequences was generated by PCR using primers SODC0.5-5′ (5′-ggtgctgttgtttctcgg-3′) and SODC0.5-3′ (5′-tcggcatcactttgtgcg-3′). The fragment was cloned into pCR2.1TOPO (Invitrogen) and subcloned intoPstI-digested and blunt-ended pSL1180 (Pharmacia), constructing pOD1. pOD4 was created by cloning thePstI-flanked aph gene (kanamycin resistance) of pUC4K into the sodC PstI site of pOD1. TheNotI-SpeI fragment of pOD4 containingsodC::aph was blunt ended and cloned into the SmaI site of pXYL4, a plasmid bearing thexylE gene (17), creating pOD6. The 4-kbBamHI fragment containingsodC::aph and xylE was isolated from pOD6 and ligated at the BamHI site of pPR27, a vector which contains the counterselectable sacB gene and the thermosensitive origin of replication of pAL5000 (17), constructing pOD7. To achieve allelic exchange, pOD7 was electroporated (17) into M. tuberculosis H37Rv (14 001 0001; Centre National de Référence des Mycobactéries, Institut Pasteur, France). Transformants were selected at 32°C on 7H11 medium containing kanamycin (20 μg/ml) and then grown in 7H9 broth containing kanamycin. Gene replacement accompanied by plasmid loss was selected for on 7H11-kanamycin–2% sucrose at 39°C (17). Loss of the plasmid was confirmed in 100% of the resultant colonies by spraying with catechol, a chromogenic substrate of XylE (6, 17). Gene replacement of sodC was verified by Southern blotting of genomic DNA from four colonies, using the sodC gene as a probe (Fig.1A). One mutant clone was designated MTsodC.

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Fig. 1.

Disruption of sodC. (A) Southern blot analysis of the M. tuberculosis H37Rv parental strain (lane 1) and sodC-defective mutants (lanes 2 to 5). Chromosomal DNAs were digested with EagI and analyzed by Southern blotting with a ³²P-labeled probe corresponding tosodC. sodC mutants gave a single 2.2-kb fragment, as expected from double crossover. (B) Southern blot analysis of theM. bovis BCG parental strain (lane 1) and thesodC mutant (lane 2). Chromosomal DNAs were digested withEcoRI and probed with the digoxigenin-labeled SODC1-SODC2 PCR product. The presence of hybridizing fragments of 1.9 and 1.1 kb is consistent with a double crossover and gene replacement. (C) Absence of CuZnSOD in mutant strains. Western blot analysis was performed with whole-cell extracts of M. tuberculosis H37Rv,sodC-defective mutant MTsodC, M. bovis BCG, andsodC-deficient mutant BCGsodC. Total protein (10 μg) from each extract was immunoblotted with an anti-M. tuberculosisSodC polyclonal antibody.

sodC was deleted from M. bovis BCG by delivery of a mutated gene on a suicide vector. The suicide plasmid pSMT100 is a pUC19-based vector carrying a hygromycin resistance gene (hyg) and sacB. A 2.1-kb region upstream ofsodC which included the initiation codon of sodCwas amplified by PCR using primers SODC1 (5′-ggactagtcgtccaagccaggttcgttc-3′) and SODC2 (5′-gctctagaggtgatcggcgggctttgg-3′) and Pwo DNA polymerase (Boehringer Mannheim). The fragment was digested withXbaI and SpeI and cloned into the SpeI site upstream of hyg in pSMT100. Then a 2.0-kb region downstream of sodC but including the sodCtermination codon was amplified using SODC3 (5′-ggactagtcgctacgtccaggtcaatggg-3′) and SODC4 (5′-gctctagacgcagtgaatgtggttcaggc-3′), digested withSpeI and XbaI, and cloned into theXbaI site downstream of hyg to make pSMT105. UV-irradiated plasmid (1 μg) (13) was electroporated into M. bovis BCG (1173P2; Institut Pasteur), and transformants arising from double-crossover gene replacement were selected in a single-step double selection on 7H11-hygromycin (50 μg/ml)–2% sucrose at 37°C. In 23 of 25 transformants screened by Southern hybridization, gene replacement was confirmed, and one of these was designated BCGsodC (Fig. 1B).

The absence of SodC from the mutants was confirmed by Western blotting using a rabbit polyclonal antibody raised against the M. tuberculosis H37Rv SodC. Crude protein extracts were obtained from the mutant and wild-type strains by disruption in a Mini-BeadBeater (BioSpecs) (16). After denaturing polyacrylamide gel electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes and probed with the SodC antibody diluted 1:2,500. Immunoreactivity was visualized with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (Biosys) and 5-bromo-4-chloro-3-indolylphosphate–nitroblue tetrazolium (BCIP-NBT) substrate (Sigma). The SodC protein was detected in lysates from both parental strains but was absent from the mutant strains (Fig. 1C). We also investigated the effect of sodC disruption on the expression of SodA. Western blotting using a polyclonal antibody against the M. tuberculosis SodA and SOD activity staining of native gels (3) revealed no change in the levels of SodA (data not shown).

Sensitivity of CuZnSOD-deficient M. tuberculosis andM. bovis BCG to ROI.The sodC disruption did not affect the growth rate of the bacteria in 7H9 broth. The sensitivity of mycobacterial strains to plumbagin and menadione, which are two superoxide-generating agents, and to hydrogen peroxide was assessed by metabolic labeling of mycobacteria with [³H]uracil (20). Mid-log-phase cultures of mycobacteria were diluted in 7H9 broth at 10⁸ CFU/ml. A 100-μl volume of this suspension was incubated in a 96-well plate at 37°C for 5 h with the addition of 0.5 μCi of [³H]uracil per ml and the stress reagents at a range of concentrations (0 to 25.6 mM plumbagin, 0 to 76.8 mM menadione, and 0 to 25.6 mM hydrogen peroxide). The assay was stopped and mycobacteria were killed by the addition of 50% ethanol. Cultures were recovered on fiberglass filters in a cell harvester, and radioactivity was measured using a liquid scintillation counter. The background radioactivity was subtracted from subsequent determinations. The inhibitory effect of each reagent was measured as a percentage of the [³H]uracil incorporation observed in wells without reagent. Wilcoxon test and t test analyses were performed, and the most significant result is indicated. Both MTsodC and BCGsodC mutant strains were more sensitive to the superoxide-generating agents plumbagin (P = 0.02 and P < 0.0007, respectively) and menadione (P < 0.0001 and P = 0.03, respectively) than their respective parental strains (Fig. 2A and B and 3A and B). The differences were statistically significant, although the data that were obtained at concentrations where plumbagin and menadione are toxic were similar (Fig. 2B and 3A and B). MTsodC and BCGsodC were also significantly more sensitive to hydrogen peroxide (P = 0.0007 and P < 0.0001, respectively) than were their parental strains (Fig. 2C and 3C). This ROI-sensitive phenotype was successfully complemented in MTsodC by reintroduction ofsodC at the attB site on the chromosome by using a hyg-containing derivative of pYUB295 (W. R. Jacobs, Albert Einstein College of Medicine, Bronx, N.Y.). ROI resistance was fully restored (data not shown).

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Fig. 2.

Sensitivity of the M. tuberculosis sodC-defective mutant to ROI. Mycobacteria were diluted in 7H9 medium at 10⁸ CFU/ml and incubated at 37°C for 5 h with various concentrations of menadione (A), plumbagin (B), or hydrogen peroxide (C). The inhibitory effect of these reagents onM. tuberculosis H37Rv (solid circles) and M. tuberculosis MTsodC (open circles) was measured as percentage of [³H]uracil incorporation in wells without reagent. TheP values (paired t test) were considered significant (0.02 for panel B) to extremely significant (<0.0001 for panel A and 0.0007 for panel C).

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Fig. 3.

Sensitivity of the M. bovis sodC-defective mutant to ROI. Mycobacteria were diluted in 7H9 medium at 10⁸ CFU/ml and incubated at 37°C for 5 h with various concentrations of menadione (A), plumbagin (B), and hydrogen peroxide (C). The inhibitory effect of these reagents on M. bovis BCG (solid circles) and M. bovis BCGsodC (open circles) was measured as a percentage of [³H]uracil incorporation in wells without reagent. The P values (Wilcoxon signed-rank test) were considered significant (0.03 for panel A) to extremely significant (0.0007 for panel B and <0.0001 for panel C).

It is believed that CuZnSODs protect bacteria from exogenous superoxide, since most are exported to the periplasmic space or secreted (25). Indeed, CuZnSOD deficiency increases the sensitivity to superoxide generated in vitro in bacteria such asCaulobacter crescentus (22), S. enterica serovar Typhimurium (7, 9), N. meningitidis (27), and Haemophilus ducreyi(21). The increased sensitivity to superoxide-generating agents of CuZnSOD-deficient M. tuberculosis and the location of the enzyme at the periphery of bacilli (28) suggest the potential for a similar protective role against exogenous oxidative stress. The increased sensitivity to exogenous hydrogen peroxide of CuZnSOD-deficient M. tuberculosis, likeS. enterica serovar Typhimurium and Escherichia coli sodC mutants (11), could be due to the Haber-Weiss reaction, in which iron reduced by superoxide reacts with peroxides to generate hydroxyl radicals (23, 25).

Survival of CuZnSOD-deficient M. bovis BCG in mouse bone marrow macrophages.To assess the role of SodC in intracellular growth and protection against killing by macrophages, the survival rates of the BCGsodC mutant strain and its parental strain were compared during infection of unstimulated and activated mouse bone marrow-derived macrophages. Bone marrow-derived macrophages were obtained from femoral bones of 6- to 8-week-old female C57BL/6 mice and cultivated for 8 to 10 days in Dulbecco modified Eagle medium (Gibco BRL, Glasgow, Scotland) supplemented with 2 mM l-glutamine (Gibco BRL), 10% fetal calf serum (Labtech), 5% horse serum (Labtech), and 15% L-929 culture supernatant. The macrophages were seeded in 24-well culture plates at 2 × 10⁵cells/well 24 h before infection. For experiments with activated macrophages, 100 U of gamma interferon per ml and 10 ng of E. coli lipopolysaccharide per ml were added 24 h before infection. The macrophage activation status was confirmed before each experiment by measurement of CD54 (ICAM-1) up-regulation and induction of inducible nitric oxide synthase. Macrophages were infected with the mutant and wild-type BCG strains at a multiplicity of infection of 0.5 to 1 bacterium/cell. After 2 to 3 h at 37°C, the cells were washed twice in phosphate-buffered saline, and fresh medium was added. The number of mycobacteria associated with the monolayers was assessed at 0, 1, 2, and 3 days postinfection for activated macrophages and 0, 1, 3, 5, 7, and 9 days for unstimulated macrophages. The cell monolayer was washed once with phosphate-buffered saline, and then 1 ml of 0.1% Triton X-100 was added to lyse the macrophages. Lysates were serially diluted and plated onto 7H11 medium, and CFU were counted after 17 to 21 days. The experiments were performed twice, with three determinations per time point. The BCGsodC strain and its parental strain showed similar kinetics of intracellular growth in nonstimulated bone marrow macrophages (Fig. 4A). In activated macrophages, approximately 90% killing was observed at 3 days for both the BCGsodC mutant and parental strains (Fig. 4B). These data suggest that SodC does not protect M. bovis BCG against killing by macrophages.

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Fig. 4.

Survival of sodC-defective mutants in macrophages. Unstimulated mouse bone marrow-derived macrophages (A) and macrophages activated by 100 U of gamma interferon per ml and 10 ng ofE. coli lipopolysaccharide per ml (B) were infected with theM. bovis BCG sodC mutant strain (open circles) and the parental strain (solid circles) at a multiplicity of infection of 0.5 to 1 mycobacterium per cell. Macrophages were lysed, and the number of mycobacteria associated with macrophages was assessed by plating on 7H11. p.i., postinfection.

Survival of CuZnSOD-deficient M. tuberculosis in guinea pigs.It has been reported that Brucella abortus andS. enterica serovars Typhimurium, Choleraesuis, and DublinsodC mutants behaved similarly to their respective parental strains within macrophages, although their CuZnSOD was shown to contribute to pathogenicity in vivo (9, 24). Therefore, it was of interest to test the effect of M. tuberculosis sodCdisruption on pathogenicity. Outbred female Hartley guinea pigs were injected subcutaneously with 10⁴ viable units of parental and mutant M. tuberculosis strains in 0.2 ml of saline solution (five replicates/strain). Animals were sacrificed 5 weeks after infection, and there were no visible differences in tuberculosis lesions in the spleen, lungs, lymph nodes, or liver or at the site of injection. The lymph nodes draining the site of injection and spleen were homogenized, and serial dilutions were plated onto 7H11 medium. There was no difference between strains in the number of CFU recovered from spleens or lymph nodes (Fig. 5). Thus, SodC does not make an obvious contribution to the pathogenicity of M. tuberculosis. However, its in vitro sensitivity to ROI suggests that it could protect the periphery of the bacilli against ROI at some stage of its life cycle. If CuZnSOD does not form a major component of defense against ROI, FeSOD may be important. Using an identical strategy to that used to interrupt sodC, we have been unable to disrupt sodA under aerobic or microaerophilic conditions. It is not known whether this is due to technical problems, to the fact that FeSOD is essential for the viability of M. tuberculosis, or to detrimental polar effects on the expression of downstream genes. The role of FeSOD in mycobacterial pathogenicity remains an open question.

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Fig. 5.

Survival of sodC-defective mutants in guinea pigs. Guinea pigs were injected with the M. tuberculosis sodC mutant strain (open bars) and the parental strain (solid bars). Lymph nodes and spleen were collected and homogenized after 5 weeks, and the number of mycobacteria was assessed by plating onto 7H11 medium.