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

Abstract

Summary 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 M. 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 (H2O2), 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 (H2O2) and organic (CHP and t-BHP) peroxides. Further, an M. smegmatis mutant (MSΔosmC) 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 MSΔosmC 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.

Functional Characterization of Osmotically Inducible Protein C (MG_427) from Mycoplasma genitalium

“…The reduction of organic hydroperoxides t-BHP and CHP by rMG_427 is consistent with the known activities of Ohr/ OsmC proteins, which preferentially detoxify OHPs. In addition, the observation that MG_427 was able to reduce H 2 O 2 is consistent with the reported property of OsmC from several bacteria (25,35,36).…”

“…We showed that purified rMG_427 protein responded to DTT and oxidant exposures and reduced OHPs and H 2 O 2 in a concentration-dependent manner. These results are consistent with the reported hydroperoxide peroxidase properties of OsmC proteins in E. coli (17,34), Thermus thermophilus (36), Thermococcus kodakarensis KOD1 (35), as well as M. tuberculosis and M. smegmatis (25). Further, we demonstrated that the ⌬MG_427 strain of M. genitalium exhibited hypersensitivity to t-BHP and increased sensitivity to H 2 O 2 and this sensitivity could be reversed in the mutant strain complemented with an intact MG_427 gene, similar to osmC mutants of E. coli (24) and M. smegmatis (25).…”

“…These results are consistent with the reported hydroperoxide peroxidase properties of OsmC proteins in E. coli (17,34), Thermus thermophilus (36), Thermococcus kodakarensis KOD1 (35), as well as M. tuberculosis and M. smegmatis (25). Further, we demonstrated that the ⌬MG_427 strain of M. genitalium exhibited hypersensitivity to t-BHP and increased sensitivity to H 2 O 2 and this sensitivity could be reversed in the mutant strain complemented with an intact MG_427 gene, similar to osmC mutants of E. coli (24) and M. smegmatis (25). We also observed a slower growth of the ⌬MG_427 mutant than the wild type in SP-4 medium containing glycerol as the carbon source, a growth condition known to generate H 2 O 2 by mycoplasmas (37,38).…”

“…Compared to their parental strains, ohr deletion mutants exhibit hypersensitivity to killing by OHPs, such as tertbutyl hydroperoxide (t-BHP) and cumene hydroperoxide (CHP). Similar findings were also reported for osmC deletion mutants of Escherichia coli (24) and Mycobacterium smegmatis (25).…”

Antioxidants: Friend or foe for tuberculosis patients

“…In case of atypical Mycobacterium species bacterial antioxidants are thought to modulate host derived oxidative killing mechanisms [19]. In Mycobacterium species new antioxidant mechanisms that protect the bacterium from the phagosomal respiratory burst is an established phenomenon [20,21]. Even recombinant BCG over-expressed with superoxide dismutase A confers less protection for development of tuberculosis [22].…”