OsmC proteins of Mycobacterium tuberculosis and Mycobacterium smegmatis protect against organic hydroperoxide stress

Abstract

Bacterial antioxidants play a critical role in the detoxification of endogenously and host derived oxidative radicals during host-pathogen interactions. Recently, the osmotically induced bacterial protein C (OsmC) is included in the antioxidant category of enzymes as it shows structural and functional relationships with organic hydroperoxide reductase (Ohr) enzyme. A copy of the gene encoding OsmC is conserved across mycobacterial species, including Mycobacterium tuberculosis (Rv2923c) and Mycobacterium smegmatis (MSMEG2421), but its role in protecting these species against oxidative stress is unknown. To determine the role of OsmC in mycobacterial oxidative stress, we overexpressed and purified OsmCs of M. tuberculosis and M. smegmatis and assessed their ability to reduce peroxide substrates like hydrogen peroxide (H(2)O(2)), cumene hydroperoxide (CHP) and t-butyl hydroperoxide (t-BHP) in Ferrous Ion Oxidation in Xylenol (FOX) assay. This revealed that OsmCs from both species were capable of reducing both inorganic (H(2)O(2)) and organic (CHP and t-BHP) peroxides. Further, an M. smegmatis mutant (MS∆osmC) deficient in OsmC exhibited reduced reduction of CHP and t-BHP than the parental wild type strain, indicating that OsmC protein contributes significantly for the total peroxide reductase activity of mycobacteria. The MS∆osmC strain was also sensitive to organic hydroperoxides, which could be reversed by complementing with a plasmid borne osmC. Plasmid borne osmC also increased the resistance of M. smegmatis wild type strain to isoniazid (INH) but at a relatively lower level than ahpC, an organic hydroperoxide reductase. These results suggest that OsmC plays an important role in peroxide metabolism and protecting mycobacteria against oxidative stress.

Keywords: INH; Ohr; OsmC; antioxidants; evasion; macrophage; mycobacteria; organic hydroperoxides; oxidative stress.

Fig. 1

osmC gene organization and its overexpression. A. Schematic showing the organization of osmC gene in the genomes of M. tuberculosis and M. smegmatis. I, M. tuberculosis; II, M. smegmatis. Arrows represent genes and their orientations. Numbers above the arrows indicate the assigned number for each gene. B. SDS-PAGE protein profile showing overexpression and purification of M. tuberculosis and M. smegmatis OsmC. E. coli was grown in LB broth at 37°C, and lysates were prepared by sonication. Lane M, EZ-Run Rec Protein Ladder (Fisher Scientific); lanes 1 and 4, extracts of E. coli strain BL21 carrying the overexpression plasmids p16MTBOSMCEX and p16MSOSMCEX before the addition of 1 mM IPTG; lanes 2 and 5, extracts of E. coli strain BL21 carrying the overexpression plasmids p16MTBOSMCEX and p16MSOSMCEX after the addition of 1 mM IPTG; lanes 3 and 6, His₁₀TBOsmC and His₁₀MSOsmC after Ni-NTA affinity chromatography. The arrow points to the His₁₀OsmC bands.

Fig. 2

Reduction of peroxides by His₁₀OsmC proteins in FOX assay. Reduction of peroxides was assayed by FOX assay. A. Reduction of peroxides by His₁₀TBOsmC. B. Reduction of peroxides by His₁₀MSOsmC. Each reaction contained 200 μM of substrates cumene hydroperoxide (CHP) or t-Butyl hydroperoxide (t-BHP) or hydrogen peroxide (H₂O₂) and 2 μg of the overexpressed His₁₀OsmC protein.

Fig. 3

Southern and Real time RT-PCR analyzes of M. smegmatis strains. A. Southern analysis of genomic DNA. Genomic DNA was digested with BamHI and PstI, separated on 1% agarose gels and transferred to nitrocellulose membranes. Membranes were hybridized with [³²P] dCTP labeled 824 bp DNA fragment containing osmC region and signals captured by autoradiography. Arrows indicate the sizes of the signals MSWt, M. smegmatis wild type; ΔosmC, M. smegmatis osmC mutant (MSΔosmC); SCO, M. smegmatis osmC single crossover (MSosmC/SCO). B. Expression of osmC by M. smegmatis strains. Determination of transcripts by real time RT-PCR and normalization are detailed in material and methods section. MSWt, M. smegmatis wild type; SCO, M. smegmatis osmC single crossover (MSosmC/SCO); ΔosmC, M. smegmatis osmC mutant (MSΔosmC).

Fig. 4

Reduction of peroxides by crude cell lysates of M. smegmatis strains. Reduction of peroxides was assayed by FOX assay. Each reaction contained 200 μM of the substrates cumene hydroperoxide (CHP) or t-Butyl hydroperoxide (t-BHP) or hydrogen peroxide (H₂O₂) and 2 μg of the protein extract. EC, E. coli DH5α strain; MSWt, M. smegmatis wild-type strain; ΔosmC, M. smegmatis mutant (MSΔosmC), ΔahpC, M. smegmatis ahpC mutant (MSΔahpC). Asterisk indicates that the values are significantly (P≤0.01) lower than the values of MSWt strain.

Fig. 5

Survival of M. smegmatis after exposure to hydroperoxides. Aliquots (1 ml) of cultures at OD₆₀₀ of 0.6 were exposed to 5 mM hydrogen peroxide (H₂O₂) or 5 mM cumene hydroperoxide (CHP) or 5 mM t-butyl hydroperoxide (t-BHP) for 1 h at 37°C. The cultures were serially diluted and plated onto 7H10-TW-ADC plates and the colonies counted after 3–4 days of incubation at 37°C. Solid grey bars, untreated cultures; Solid black bars, treated cultures. MSWt, M. smegmatis wild-type strain; ΔosmC, M. smegmatis mutant (MSΔosmC); SCO, M. smegmatis osmC single crossover (MSosmC/SCO); Δos/c, M. smegmatis osmC mutant (MSΔosmC) harboring plasmid pMRV2923 (MSΔosmC2923). Asterisk indicates that the values are significantly (P≤0.01) lower than untreated cultures.

Fig. 6

Stress response of osmC of M. tuberculosis and M. smegmatis. A. Stress of response osmC of M. tuberculosis; B. Stress of response of osmC of M. smegmatis. Total RNA was isolated after exposure to stress conditions and real time RT-PCR was performed. M. smegmatis and M. tuberculosis osmC genes transcripts were normalized with their sigA genes transcripts. H₂O₂, 5 mM hydrogen peroxide; CHP, 5 mM cumene hydroperoxide; BHP, 5 mM t-butyl hydroperoxide; NaCl, 400 mM Sodium chloride; EtOH, 5% Ethanol; heat, 52°C; cold, 4°C.