In-vivo protein nitration facilitates Vibrio cholerae cell survival under anaerobic, nutrient deprived conditions

Abstract

Protein tyrosine nitration (PTN), a highly selective post translational modification, occurs in both prokaryotic and eukaryotic cells under nitrosative stress. However, its physiological function is not yet clear. Like many gut pathogens, Vibrio cholerae also faces nitrosative stress, which makes its proteome more vulnerable to PTN. Here, we report for the first time in-vivo PTN in V. cholerae by immunoblotting and LC-ESI-MS/MS proteomic analysis. Our results indicated that in-vivo PTN in V. cholerae was culture media independent. Surprisingly, in-vivo PTN was reduced in V. cholerae proteome under anaerobic or hypoxic condition in a nutrient deprived state. Interestingly, intracellular nitrate content was more than the nitrite content in V. cholerae under anaerobic conditions. Additionally, biochemical measurement of GSH/GSSG ratio, activities of catalase and SOD, ROS and RNS imaging by confocal microscopy confirmed a relative intracellular oxidizing environment in V. cholerae under anaerobic conditions. This altered redox environment favors the oxidation of nitrite which may be generated from protein denitration enriching the intracellular nitrate pool. The cell survival of V. cholerae can finally be facilitated by nitrate reductase (NapA) utilizing that nitrate pool. Our cell viability study using wild type and ΔnapA strain of V. cholerae also supported the role of NapA mediated cell survival under nutrient deprived anaerobic conditions. In spite of having nitrate reductase (NapA), V. cholerae lacks any nitrite reductase (NiR). Hence, in-vivo nitration may provide an avenue for toxic nitrite storage and also may help in nitrosative stress tolerance mechanism preventing further unnecessary protein nitration in V. cholerae proteome.