INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI: 10.1128/IAI.69.1.529–533.2001

Jan. 2001, p. 529–533 Vol. 69, No. 1

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

NOTES

Role of Mycobacterium tuberculosis Copper-ZincSuperoxide Dismutase

OLIVIER DUSSURGET,1* GRAHAM STEWART,2 OLIVIER NEYROLLES,2

PASCALE PESCHER,1 DOUGLAS YOUNG,2 AND GILLES MARCHAL1

Unite de Physiopathologie de l’Infection, Institut Pasteur, 75724 Paris Cedex 15, France,1 andDepartment of Infectious Diseases and Microbiology, Imperial College School of Medicine,

St. Mary’s Campus, London W2 1PG, United Kingdom2

Received 10 April 2000/Returned for modification 15 May 2000/Accepted 21 September 2000

Superoxide dismutases (SODs) play an important role in protection against oxidative stress and have beenshown to contribute to the pathogenicity of many bacterial species. To determine the function of the myco-bacterial copper and zinc-cofactored SOD (CuZnSOD), we constructed and characterized Mycobacteriumtuberculosis and Mycobacterium bovis BCG CuZnSOD null mutants. Both strains were more sensitive tosuperoxides and hydrogen peroxide than were their respective parental strains. The survival of M. bovis BCGin unstimulated as well as activated mouse bone marrow-derived macrophages was not affected by the loss ofCuZnSOD. The survival of CuZnSOD deficient-M. tuberculosis in guinea pig tissues was comparable to that ofits parental strain. These results indicate that the mycobacterial CuZnSOD is not essential for intracellulargrowth within macrophages and does not detectably contribute to the pathogenicity of M. tuberculosis.

Superoxide dismutases (SODs) are metalloenzymes that cat-alyze the dismutation of superoxide radicals to hydrogen per-oxide and molecular oxygen. They are initial components ofthe cellular defense against reactive oxygen intermediates(ROI) resulting from univalent reduction of oxygen, and theycontribute to the survival of bacterial pathogens such as Shi-gella flexneri (10), Campylobacter jejuni (18), Salmonella en-terica serovar Typhimurium (8, 9, 26), Yersinia enterocolitica(19), and Neisseria meningitidis (27).

Mycobacterium tuberculosis produces a tetrameric iron-co-factored SOD (FeSOD or SodA) encoded by the sodA gene (5,29) and a copper and zinc SOD (CuZnSOD or SodC) encodedby the sodC gene (28). FeSOD is among the major extracellu-lar proteins released by M. tuberculosis during growth (2). It isexported in an active form via a signal peptide-independentpathway that has not been fully characterized (12, 29). TheCuZnSOD possesses a putative signal peptide and is localizedto the periphery of M. tuberculosis (28). It has been hypothe-sized that the presence of SODs at the periphery of M. tuber-culosis and in the extracellular milieu could protect bacteriafrom superoxides generated exogeneously, e.g., by host phago-cytes (12, 28, 29). The killing of M. tuberculosis by host-acti-vated phagocytic cells is mediated to some extent by ROI alongwith reactive nitrogen intermediates (1, 4, 14, 15).

To investigate the contribution of mycobacterial CuZnSODto the defense of bacteria against oxidative killing, we con-structed isogenic mutants of M. tuberculosis and M. bovis BCGand compared them with their parental strains for sensitivity to

ROI in vitro and for survival in murine bone marrow-derivedmacrophages and in a guinea pig model of infection.

Characterization of CuZnSOD-deficient M. tuberculosis andM. bovis BCG. The M. tuberculosis sodC gene was mutated byallelic exchange (17). A DNA fragment containing sodC and500 bp of its flanking sequences was generated by PCR usingprimers SODC0.5-59 (59-ggtgctgttgtttctcgg-39) and SODC0.5-39(59-tcggcatcactttgtgcg-39). The fragment was cloned intopCR2.1TOPO (Invitrogen) and subcloned into PstI-digestedand blunt-ended pSL1180 (Pharmacia), constructing pOD1.pOD4 was created by cloning the PstI-flanked aph gene (kana-mycin resistance) of pUC4K into the sodC PstI site of pOD1.The NotI-SpeI fragment of pOD4 containing sodC::aph wasblunt ended and cloned into the SmaI site of pXYL4, a plasmidbearing the xylE gene (17), creating pOD6. The 4-kb BamHIfragment containing sodC::aph and xylE was isolated frompOD6 and ligated at the BamHI site of pPR27, a vector whichcontains the counterselectable sacB gene and the thermosen-sitive origin of replication of pAL5000 (17), constructingpOD7. To achieve allelic exchange, pOD7 was electroporated(17) into M. tuberculosis H37Rv (14 001 0001; Centre Nationalde Reference des Mycobacteries, Institut Pasteur, France).Transformants were selected at 32°C on 7H11 medium con-taining kanamycin (20 mg/ml) and then grown in 7H9 brothcontaining kanamycin. Gene replacement accompanied byplasmid loss was selected for on 7H11-kanamycin–2% sucroseat 39°C (17). Loss of the plasmid was confirmed in 100% of theresultant colonies by spraying with catechol, a chromogenicsubstrate of XylE (6, 17). Gene replacement of sodC wasverified by Southern blotting of genomic DNA from four col-onies, using the sodC gene as a probe (Fig. 1A). One mutantclone was designated MTsodC.

sodC was deleted from M. bovis BCG by delivery of a mu-

* Corresponding author. Mailing address: Institut Pasteur, 28 rue duDr Roux, 75724 Paris Cedex 15, France. Phone: 33.1.40.61.30.31. Fax:33.1.45.68.87.06. E-mail: odussur@pasteur.fr.

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tated gene on a suicide vector. The suicide plasmid pSMT100is a pUC19-based vector carrying a hygromycin resistance gene(hyg) and sacB. A 2.1-kb region upstream of sodC which in-cluded the initiation codon of sodC was amplified by PCRusing primers SODC1 (59-ggactagtcgtccaagccaggttcgttc-39) andSODC2 (59-gctctagaggtgatcggcgggctttgg-39) and Pwo DNApolymerase (Boehringer Mannheim). The fragment was di-gested with XbaI and SpeI and cloned into the SpeI site up-stream of hyg in pSMT100. Then a 2.0-kb region downstreamof sodC but including the sodC termination codon was ampli-fied using SODC3 (59-ggactagtcgctacgtccaggtcaatggg-39) andSODC4 (59-gctctagacgcagtgaatgtggttcaggc-39), digested withSpeI and XbaI, and cloned into the XbaI site downstream ofhyg to make pSMT105. UV-irradiated plasmid (1 mg) (13) waselectroporated into M. bovis BCG (1173P2; Institut Pasteur),and transformants arising from double-crossover gene re-placement were selected in a single-step double selection on7H11-hygromycin (50 mg/ml)–2% sucrose at 37°C. In 23 of 25transformants screened by Southern hybridization, gene re-placement was confirmed, and one of these was designatedBCGsodC (Fig. 1B).

The absence of SodC from the mutants was confirmed byWestern blotting using a rabbit polyclonal antibody raisedagainst the M. tuberculosis H37Rv SodC. Crude protein ex-tracts were obtained from the mutant and wild-type strains bydisruption in a Mini-BeadBeater (BioSpecs) (16). After dena-turing polyacrylamide gel electrophoresis, proteins were trans-ferred onto polyvinylidene difluoride membranes and probedwith the SodC antibody diluted 1:2,500. Immunoreactivity wasvisualized with alkaline phosphatase-conjugated goat anti-rab-bit immunoglobulin G (Biosys) and 5-bromo-4-chloro-3-in-dolylphosphate–nitroblue tetrazolium (BCIP-NBT) substrate(Sigma). The SodC protein was detected in lysates from bothparental strains but was absent from the mutant strains (Fig.1C). We also investigated the effect of sodC disruption on theexpression of SodA. Western blotting using a polyclonal anti-body against the M. tuberculosis SodA and SOD activity stain-

ing of native gels (3) revealed no change in the levels of SodA(data not shown).

Sensitivity of CuZnSOD-deficient M. tuberculosis and M.bovis BCG to ROI. The sodC disruption did not affect thegrowth rate of the bacteria in 7H9 broth. The sensitivity ofmycobacterial strains to plumbagin and menadione, which aretwo superoxide-generating agents, and to hydrogen peroxidewas assessed by metabolic labeling of mycobacteria with [3H]u-racil (20). Mid-log-phase cultures of mycobacteria were dilutedin 7H9 broth at 108 CFU/ml. A 100-ml volume of this suspen-sion was incubated in a 96-well plate at 37°C for 5 h with theaddition of 0.5 mCi of [3H]uracil per ml and the stress reagentsat a range of concentrations (0 to 25.6 mM plumbagin, 0 to76.8 mM menadione, and 0 to 25.6 mM hydrogen peroxide).The assay was stopped and mycobacteria were killed by theaddition of 50% ethanol. Cultures were recovered on fiberglassfilters in a cell harvester, and radioactivity was measured usinga liquid scintillation counter. The background radioactivity wassubtracted from subsequent determinations. The inhibitory ef-fect 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 mostsignificant result is indicated. Both MTsodC and BCGsodCmutant strains were more sensitive to the superoxide-generat-ing agents plumbagin (P 5 0.02 and P , 0.0007, respectively)and menadione (P , 0.0001 and P 5 0.03, respectively) thantheir respective parental strains (Fig. 2A and B and 3A and B).The differences were statistically significant, although the datathat were obtained at concentrations where plumbagin andmenadione are toxic were similar (Fig. 2B and 3A and B).MTsodC and BCGsodC were also significantly more sensitiveto hydrogen peroxide (P 5 0.0007 and P , 0.0001, respec-tively) than were their parental strains (Fig. 2C and 3C). ThisROI-sensitive phenotype was successfully complemented inMTsodC by reintroduction of sodC at the attB site on thechromosome by using a hyg-containing derivative of pYUB295

FIG. 1. Disruption of sodC. (A) Southern blot analysis of the M. tuberculosis H37Rv parental strain (lane 1) and sodC-defective mutants (lanes2 to 5). Chromosomal DNAs were digested with EagI and analyzed by Southern blotting with a 32P-labeled probe corresponding to sodC. sodCmutants gave a single 2.2-kb fragment, as expected from double crossover. (B) Southern blot analysis of the M. bovis BCG parental strain (lane1) and the sodC mutant (lane 2). Chromosomal DNAs were digested with EcoRI and probed with the digoxigenin-labeled SODC1-SODC2 PCRproduct. The presence of hybridizing fragments of 1.9 and 1.1 kb is consistent with a double crossover and gene replacement. (C) Absence ofCuZnSOD in mutant strains. Western blot analysis was performed with whole-cell extracts of M. tuberculosis H37Rv, sodC-defective mutantMTsodC, M. bovis BCG, and sodC-deficient mutant BCGsodC. Total protein (10 mg) from each extract was immunoblotted with an anti-M.tuberculosis SodC polyclonal antibody.

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(W. R. Jacobs, Albert Einstein College of Medicine, Bronx,N.Y.). ROI resistance was fully restored (data not shown).

It is believed that CuZnSODs protect bacteria from exoge-nous superoxide, since most are exported to the periplasmicspace or secreted (25). Indeed, CuZnSOD deficiency increasesthe sensitivity to superoxide generated in vitro in bacteria suchas Caulobacter crescentus (22), S. enterica serovar Typhimurium(7, 9), N. meningitidis (27), and Haemophilus ducreyi (21). Theincreased sensitivity to superoxide-generating agents ofCuZnSOD-deficient M. tuberculosis and the location of theenzyme at the periphery of bacilli (28) suggest the potential fora similar protective role against exogenous oxidative stress.

The increased sensitivity to exogenous hydrogen peroxide ofCuZnSOD-deficient M. tuberculosis, like S. enterica serovarTyphimurium and Escherichia coli sodC mutants (11), could bedue to the Haber-Weiss reaction, in which iron reduced bysuperoxide reacts with peroxides to generate hydroxyl radicals(23, 25).

Survival of CuZnSOD-deficient M. bovis BCG in mousebone marrow macrophages. To assess the role of SodC inintracellular growth and protection against killing by macro-phages, the survival rates of the BCGsodC mutant strain andits parental strain were compared during infection of unstimu-lated and activated mouse bone marrow-derived macrophages.Bone marrow-derived macrophages were obtained from fem-

FIG. 2. Sensitivity of the M. tuberculosis sodC-defective mutant toROI. Mycobacteria were diluted in 7H9 medium at 108 CFU/ml andincubated at 37°C for 5 h with various concentrations of menadione(A), plumbagin (B), or hydrogen peroxide (C). The inhibitory effect ofthese reagents on M. tuberculosis H37Rv (solid circles) and M. tuber-culosis MTsodC (open circles) was measured as percentage of [3H]u-racil incorporation in wells without reagent. The P values (paired ttest) were considered significant (0.02 for panel B) to extremely sig-nificant (,0.0001 for panel A and 0.0007 for panel C).

FIG. 3. Sensitivity of the M. bovis sodC-defective mutant to ROI.Mycobacteria were diluted in 7H9 medium at 108 CFU/ml and incu-bated at 37°C for 5 h with various concentrations of menadione (A),plumbagin (B), and hydrogen peroxide (C). The inhibitory effect ofthese reagents on M. bovis BCG (solid circles) and M. bovis BCGsodC(open circles) was measured as a percentage of [3H]uracil incorpora-tion 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).

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oral bones of 6- to 8-week-old female C57BL/6 mice and cul-tivated for 8 to 10 days in Dulbecco modified Eagle medium(Gibco BRL, Glasgow, Scotland) supplemented with 2 mML-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 3105 cells/well 24 h before infection. For experiments with ac-tivated macrophages, 100 U of gamma interferon per ml and10 ng of E. coli lipopolysaccharide per ml were added 24 hbefore infection. The macrophage activation status was con-firmed before each experiment by measurement of CD54(ICAM-1) up-regulation and induction of inducible nitric ox-ide synthase. Macrophages were infected with the mutant andwild-type BCG strains at a multiplicity of infection of 0.5 to 1bacterium/cell. After 2 to 3 h at 37°C, the cells were washedtwice in phosphate-buffered saline, and fresh medium wasadded. The number of mycobacteria associated with the mono-layers was assessed at 0, 1, 2, and 3 days postinfection foractivated macrophages and 0, 1, 3, 5, 7, and 9 days for unstimu-

lated macrophages. The cell monolayer was washed once withphosphate-buffered saline, and then 1 ml of 0.1% Triton X-100was added to lyse the macrophages. Lysates were serially di-luted and plated onto 7H11 medium, and CFU were countedafter 17 to 21 days. The experiments were performed twice,with three determinations per time point. The BCGsodC strainand its parental strain showed similar kinetics of intracellulargrowth in nonstimulated bone marrow macrophages (Fig. 4A).In activated macrophages, approximately 90% killing was ob-served at 3 days for both the BCGsodC mutant and parentalstrains (Fig. 4B). These data suggest that SodC does not pro-tect M. bovis BCG against killing by macrophages.

Survival of CuZnSOD-deficient M. tuberculosis in guineapigs. It has been reported that Brucella abortus and S. entericaserovars Typhimurium, Choleraesuis, and Dublin sodC mu-tants behaved similarly to their respective parental strainswithin macrophages, although their CuZnSOD was shown tocontribute to pathogenicity in vivo (9, 24). Therefore, it was ofinterest to test the effect of M. tuberculosis sodC disruption onpathogenicity. Outbred female Hartley guinea pigs were in-jected subcutaneously with 104 viable units of parental andmutant M. tuberculosis strains in 0.2 ml of saline solution (fivereplicates/strain). Animals were sacrificed 5 weeks after infec-tion, and there were no visible differences in tuberculosis le-sions in the spleen, lungs, lymph nodes, or liver or at the site ofinjection. The lymph nodes draining the site of injection andspleen were homogenized, and serial dilutions were platedonto 7H11 medium. There was no difference between strains inthe number of CFU recovered from spleens or lymph nodes(Fig. 5). Thus, SodC does not make an obvious contribution tothe pathogenicity of M. tuberculosis. However, its in vitro sen-sitivity to ROI suggests that it could protect the periphery ofthe bacilli against ROI at some stage of its life cycle. IfCuZnSOD does not form a major component of defenseagainst ROI, FeSOD may be important. Using an identicalstrategy to that used to interrupt sodC, we have been unable todisrupt sodA under aerobic or microaerophilic conditions. It isnot known whether this is due to technical problems, to the factthat FeSOD is essential for the viability of M. tuberculosis, or to

FIG. 4. Survival of sodC-defective mutants in macrophages. Un-stimulated mouse bone marrow-derived macrophages (A) and macro-phages activated by 100 U of gamma interferon per ml and 10 ng of E.coli lipopolysaccharide per ml (B) were infected with the M. bovis BCGsodC mutant strain (open circles) and the parental strain (solid circles)at a multiplicity of infection of 0.5 to 1 mycobacterium per cell. Mac-rophages were lysed, and the number of mycobacteria associated withmacrophages was assessed by plating on 7H11. p.i., postinfection.

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

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detrimental polar effects on the expression of downstreamgenes. The role of FeSOD in mycobacterial pathogenicity re-mains an open question.

We thank Fang-Jen Lee for providing antiserum and Simon Krollfor helpful discussion.

This research was supported by the Institut Pasteur (O.D., P.P., andG.M.) and by the Wellcome Trust (G.S. and O.N.).

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Editor: S. H. E. Kaufmann

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