Reactive nitrogen species induced catalases promote a novel nitrosative stress tolerance mechanism in Vibrio cholerae

Abstract

Vibrio cholerae faces nitrosative stress during successful colonization in intestine. Very little information is available on the nitrosative stress protective mechanisms of V. cholerae. Reports show that NorR regulon control two genes hmpA and nnrS responsible for nitric oxide (NO) detoxification in V. cholerae. In the present study we first time report a novel role of V. cholerae catalases under nitrosative stress. Using zymogram analysis of catalase we showed that KatB and KatG activity were induced within 30 min in V. cholerae in the presence of sodium nitroprusside (SNP), a NO donor compound. Surprisingly, V. cholerae cell survival was found to be decreased under nitrosative stress if catalase activities were blocked by ATz, a catalase inhibitor. Flow cytometry study was conducted to detect reactive oxygen species (ROS) and reactive nitrogen species (RNS) using DHE and DHR123, fluorescent probes respectively. Short exposure of SNP to V. cholerae did not generate ROS but RNS was detectable within 30 min. Total glutathione content was increased in V. cholerae cells under nitrosative stress. Furthermore, Superoxide dismutase (SOD) and Glutathione reductase (GR) activities remained unchanged under nitrosative stress in V. cholerae indicated antioxidant role of NO which could produce peroxynitrite. To investigate the role of catalase induction under nitrosative stress in V. cholerae, we conducted peroxynitrite reductase assay using cell lysates. Interestingly, SNP treated V. cholerae cell lysates showed lowest DHR123 oxidation compared to the control set. The extent of DHR123 oxidation was more in V. cholerae cell lysate when catalases were blocked by ATz.