Abstract
Objective To analyze the enzymatic properties of alkyl hydroperoxide reductase subunit C (AhpC) from Leptospira interrogans (L.interrogans) and to elucidate its physiological roles in host-pathogen interactions in macrophages during Leptospira infection. Methods A prokaryotic expression system for ahpC gene of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was established to express the recombinant AhpC (rAhpC). After purified by Ni-NTA affinity chromatography, the enzymatic activity of the rAhpC and its role in protecting DNA from oxidation were analyzed. The importance of each cysteine in its molecule was evaluated through site-directed mutation. L. interrogans strains were pretreated with or without Conoidin A, a covalent inhibitor of peroxiredoxin, and then were used to infect macrophages. Changes in oxidative status in leptospires and survival rates of L. interrogans strains were analyzed by fluorescence-activated cell sorting and colony counting method. Results The rAhpC was successfully expressed in the established prokaryotic expression system. It had peroxiredoxin activity that was able to catalyze the reduction of hydrogen peroxide. Its ability of reducing hydrogen peroxide depended on the thioredoxin/thioredoxin reductase system. Cys47 (a peroxidatic cysteine) and Cys167 (a resolving cysteine) were critical to maintaining the enzymatic activity of AhpC. AhpC could protect DNA from hydrogen peroxide induced-oxidative damage. When L. interrogans strains were pretreated with Conoidin A, the oxidative status in leptospires was elevated and the survival of L. interrogans in macrophages was significantly reduced in a dose-dependent manner. Conclusion The AhpC of L. interrogans is a thioredoxin-dependent peroxiredoxin that plays an important role in protecting L. interrogans against oxidative stress in macrophages. Key words: Leptospira interrogans; ahpC gene; Oxidative stress; Survival
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