Methionine sulfoxide reductase B (MsrB) of Mycobacterium smegmatis plays a limited role in resisting oxidative stress

Abstract

Pathogenic mycobacteria including Mycobacterium tuberculosis resists phagocyte generated reactive oxygen intermediates (ROI) and this constitutes an important virulence mechanism. We have previously reported, using Mycobacterium smegmatis as a model to identify the bacterial components that resist intracellular ROI, that an antioxidant methionine sulfoxide reductase A (MsrA) plays a critical role in this process. In this study, we report the role of methionine sulfoxide reductase B (MsrB) in resistance to ROI by constructing a msrB mutant (MSDeltamsrB) and MsrA/B double mutant (MSDeltamsrA/B) strains of M. smegmatis and testing their survival in unactivated and interferon gamma activated mouse macrophages. WhilemsrB mutant exhibited significantly lower intracellular survival than its wild type counterpart, the survival rate seemed to be much higher than msrA mutant (MSDeltamsrA) strain. Further, the msrB mutant showed no sensitivity to oxidants in vitro. The msrA/B double mutant (MSDeltamsrA/B), on the other hand, exhibited a phenotype similar to that of msrA mutant in terms of both intracellular survival and sensitivity to oxidants. We conclude, therefore, that MsrB of M. smegmatis plays only a limited role in resisting intracellular and in vitro ROI.

Fig. 1

SDS-PAGE protein profile showing overexpression and purification of M. tuberculosis MsrA and MsrB. Lane 1, Molecular marker. Lanes 2 and 3, extracts of E. coli strain BL21 carrying overexpression plasmid pTBMSRAEX in the absence (lane 2) and presence (lane 3) of 0.1 mM IPTG. Lanes 5 and 6, extracts of E. coli strain BL21 carrying overexpression plasmid pTBMSRBEX in the absence (lane 5) and presence (lane 6) of 0.1 mM IPTG. Lanes 4 and 7, His₁₀-MsrA and His₁₀-MsrB, respectively after Ni-NTA chromatography.

Fig. 2

Immunoblot analysis of MsrA and MsrB from different mycobacteria. Whole extracts were prepared from mycobacteria grown in 7H9-ADC-TW medium. Each lane contained about 150 μg of protein. Anti-MsrA antiserum and anti-MsrB antiserum were used at 1:500 dilutions. Peroxidase conjugated anti-rabbit IgG was used as secondary antibody (1:10,000 dilution; Sigma). Bands were visualized by chemiluminescence method (Amersham).

Fig. 3

(A) Organization of msrA and msrB in the genome of M. smegmatis. Black arrows in the upper and lower panels represent the genes in the flanking regions of msrA and msrB, respectively. The genes are named according to JVCI (TIGR) annotations. Open arrows represent msrA (MSMEG6477) and msrB (MSMEG2784). (B) Immunoblot showing the expression of MsrA and MsrB in M. smegmatis. W, whole extract; S, soluble fraction; M, membrane fraction. Anti-MsrA antiserum and anti-MsrB antiserum were used at 1:500 dilutions. Peroxidase conjugated anti rabbit IgG was used as secondary antibody (1:10,000 dilution; Sigma). Bands were visualized by chemiluminescence method (Amersham).

Fig. 4

(A) Southern analysis of M. smegmatis strains. Genomic DNA of M. smegmatis wild-type strain (MSWt) and msrB mutant (MS20; MSΔmsrB) strains were cut with SalI and probed with 1.75 kb hygromycin-resistance gene (hyg) and 1 kb M. smegmatis msrB gene (msrB). (B) Schematic explaining msrB locus in the wild-type (WT) and MSΔmsrB (MS20) strain. White boxes represent the flanking region of msrB, stippled boxes represent the msrB gene, black box represents the hygromycin resistance gene. Arrows indicate the direction of transcription of genes. The sites for restriction enzymes in and around msrB gene are indicated. The lines below the boxes indicate the expected size of fragments from SalI-cut genomic DNA of WT and MS20 when probed with 1 kb msrB gene containing fragment.

Fig. 5

(A) Immunoblot analysis of M. smegmatis strains. MSWt, wild-type M. smegmatis; MS97, msrA mutant (MSΔmsrA); MS20, msrB mutant (MSΔmsrB); MS441, msrA/B double mutant (MSΔmsrA/B). Note the absence of MsrA, MsrB and both MsrA and MsrB in MSΔmsrA,MSΔmsrB and MSΔmsrA/B strains, respectively. (B) Graph showing the growth of M. smegmatis strains in broth culture. MSWt, wild-type M. smegmatis; MS97, msrA mutant (MSΔmsrA); MS20, msrB mutant (MSΔmsrB); MS441, msrA/B double mutant (MSΔmsrA/B). Cultures were grown in 7H9-ADC-TW broth for forty hours and their growth was assessed at different time points.

Fig. 6

Survival of M. smegmatis strains in murine macrophage-like J774A.1 cells. Naive (unactivated) IFN-γ-activated macrophages were infected at an MOI of 1:1 for 4 h, washed, lysed at intervals and plated onto 7H10-ADC-TW plates for CFUs. Solid squares, wild-type (MSWt); open squares, msrA mutant (MS97; MSΔmsrA); solid circles, msrB mutant (MS20; MSΔmsrB); open circles msrA/B double mutant (MS441; MSΔmsrA/B). Error bars are not seen for some time points because of the small SE.

Fig. 7

Survival of M. smegmatis after exposure to hydroperoxides. MSWt, wild-type M. smegmatis; MS20, msrB mutant (MSΔmsrB); MS441, msrA/B double mutant (MSΔmsrA/B). Aliquots (1 ml) of culture were exposed to 5 mM H₂O₂,5mM t-butyl hydroperoxide or 5 mM cumene hydroperoxide for 1 h at 37°C. Cultures were serially diluted and plated on 7H10-ADC-TW plates.

Fig. 8

Oxyblot of M. smegmatis strains. MSWt, wild-type M. smegmatis; MS97, msrA mutant (MSΔmsrA); MS20, msrB mutant (MSΔmsrB); MS441, msrA/B double mutant (MSΔmsrA/B). 7 μg of protein extract was loaded in each lane. Protein extracts were treated with DNPH (dinitrophenyl hydrazine) and then with DTT (dithiothreitol) and mercaptoethanol to avoid further oxidation of proteins. Proteins separated on SDS-PAGE and transferred to nitrocellulose were detected with oxyblot protein oxidation detection kit (Chemicon).