Detection and proteomic identification of in vivo S-nitrosylated proteins in Vibrio cholerae: A novel evidence

Abstract

Conserved across the phylogeny, S-nitrosylation and S-denitrosylation of biological thiols is a reversible protein post-translational modification of cysteine thiol residues involving nitric oxide (NO) and NO^•-derived metabolites. S-nitrosylation of proteins is observed to transduce signalling pathways with significant pathological and physiological relevance. Although endogenous S-nitrosylation is also an obligate non-classical cellular signalling pathway of NO^• in single-celled organisms, but very little information is available in prokaryotes. Hitherto unknown, we present experimental evidence for the first time in vivo S-nitrosylation (without using any NO^•/RNS donor) of proteins of the enteropathogenic, Gram-negative bacteria O1 El Tor strains of Vibrio cholerae N16961 and C6706. In the present study, PSNO was quantified by 2, 3-diaminonaphthalene (DAN) using a spectrofluorometer, which was further supported by confocal microscopy. Western blot and mass spectrometry-based proteomic analyses identified ten S-nitrosylated proteins via DMPO-nitrone adduct formation. V. cholerae contained high amounts of the in vivo pool of S-nitrosylated proteome in different respiratory conditions. Experimental evidence shows that physiological levels of glutathione (GSH) can efficiently S-denitrosylate Vibrio cholerae PSNO in a concentration-dependent manner, suggesting that the intracellular GSH tends to reset the redox state of these protein thiols. Our data suggests that V. cholerae possesses more amount of in vivo PSNO during semi-anaerobic respiration than aerobic respiration and irrespective of media and strain used; stationary phase cells are relatively more stable to GSH-catalyzed S-denitrosylation than their log-phase counterparts. Additionally, the in vivo PSNO accumulation was found to be elevated in the nitrate reductase deletion mutant (ΔnapA), indicating the role of napA in the nitroso-oxidative stress response mechanism of V. cholerae. This could aid in its remarkable adaptability and survivability in the hostile conditions of the human intestine, thereby paving the way for cholera, a highly contagious diarrheal disease.