Silencing of oxidative stress response in Mycobacterium tuberculosis: expression patterns of ahpC in virulent and avirulent strains and effect of ahpC inactivation

Abstract

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.

FIG. 1

(A) Genetic organization of the oxyR-ahpC and furA-katG regions in the M. tuberculosis complex. The M. tuberculosis oxyR pseudogene (inactivated by multiple mutations; vertical bars) and ahpC are tightly linked and divergently transcribed. The furA and katG genes are linked in all mycobacteria. The M. bovis BCG furA carries a missense mutation (producing A43V). GFP box preceded by thick line, ahpC-gfp promoter fusion used in this work. (B) Low or undetectable ahpC expression in virulent M. tuberculosis and M. bovis and high level of AhpC production in M. bovis BCG. Shown is a Western blot with AhpC antibodies using extracts from various strains of M. tuberculosis (Mt), M. bovis (Mb), and M. bovis BCG (BCG) grown under standard aerated conditions in roller bottles. Equal amounts of protein were loaded in each lane. The same extracts tested for KatG showed no differences in all lanes (as in panel D). (C) Multicopy titration of ahpC repression in M. tuberculosis H37Rv. Plasmid pP/OahpC′-oxyR′-gfp, carrying the promoter region of ahpC (A), was introduced into M. tuberculosis H37Rv and M. bovis BCG by electroporation, with selection on media supplemented with antibiotics. Strains were grown under standard, aerated conditions, and cell extracts were examined by Western blotting using an antibody against M. tuberculosis AhpC (32). (D) Induction of AhpC production in M. tuberculosis H37Rv under static growth conditions. AhpC levels (Western blot) in M. bovis BCG and M. tuberculosis H37Rv grown under oxygenated (O) and static (S) growth conditions as previously described (45) are shown. Extracts were prepared, and Western blot analysis was carried out with KatG or AhpC antibodies.

FIG. 2

Expression analyses of ahpC in M. tuberculosis H37Rv and M. bovis BCG in infected macrophages. Expression of ahpC-gfp was monitored by flow cytometry and compared with that in bacteria carrying no gfp (control) and the hsp60-gfp fusion. Macrophages (J774 cells) were infected with mycobacteria containing the gfp fusions indicated, and after 3 days samples were prepared and flow cytometry was carried out as described previously (16). (A and B) Fluorescence intensity distribution in macrophage-grown H37Rv and BCG. (C to E) Dot plots corresponding to panel A. (F to H) Dot plots corresponding to panel B.

FIG. 3

Inactivation of ahpC in M. tuberculosis H37Rv. (A) Southern blot using ahpC as a probe. Two independent isolates in each case (lanes 2 and 3 and lanes 4 and 5) are shown. (B and C) Western blots with KatG and AhpC antibodies, respectively. Mycobacteria were grown under static conditions for Western blot analysis. Equal amounts of protein were loaded per lane. wt, wild type; sxo, single-crossover strain; dxo, double-crossover strain.

FIG. 4

Effects of ahpC inactivation on M. tuberculosis virulence in mice. BALB/c mice were infected intravenously with 5 × 10⁴ M. tuberculosis H37Rv CFU. The numbers of CFU per tissue were determined for spleens (A) and lungs (B). Shown are results from one of the two separate experiments with similar outcomes (n = 6 per time point). Each point represents the mean ± standard errors. Circles, H37Rv Str^r ahpC⁺; triangles, H37Rv Str^r ahpC::Km^r mutant; squares, H37Rv Str^r ahpC ahpC::Km^r merodiploid single-crossover strain (ahpC⁺ ahpC::Km^r).

FIG. 5

Survival curves of H37Rv and H37Rv ahpC::Km^r treated with cumene hydroperoxide (CHP) (A) or hydrogen peroxide (H₂O₂) (B). Percent starting OD₆₀₀ was determined from the OD₆₀₀ (see Materials and Methods) normalized to the OD at the beginning of the experiment. Solid symbols, ahpC⁺ strain; open symbols, ahpC::Km^r mutant. wt, wild type.