Identification of oxidant susceptible proteins in Salmonella Typhimurium

The human gut pathogen, Salmonella Typhimurium (S. Typhimurium) not only survives but also replicates inside the phagocytic cells. Bacterial proteins are the primary targets of phagocyte generated oxidants. Because of the different amino acid composition, some proteins are more prone to oxidation than others. Many oxidant induced modifications to amino acids have been described. Introduction of carbonyl group is one of such modifications, which takes place quite early following exposure of proteins to oxidants and is quite stable. Therefore, carbonyl groups can be exploited to identify oxidant susceptible proteins. Hypochlorous acid (HOCl) is one of the most potent oxidants produced by phagocytes. Incubation of S. Typhimurium with 3 mM HOCl resulted in more than 150 folds loss of bacterial viability. Proteins extracted from HOCl exposed S. Typhimurium cells showed about 60 folds (p < 0.001) more carbonyl levels as compared to unexposed cells. Similarly, 2, 4-Dinitrophenylhydrazine (2, 4-DNPH) derivatized proteins of HOCl treated S. Typhimurium cultures reacted strongly with anti-DNP antibodies as compared to buffer treated counterpart. Next, we have derivatized carbonyl groups on the proteins with biotin hydrazide. The derivatized proteins were then isolated by avidin affinity chromatography. Mass spectrometry based analysis revealed the presence of 204 proteins.