Contribution of protein isoaspartate methyl transferase (PIMT) in the survival of Salmonella Typhimurium under oxidative stress and virulence

The enteric pathogen Salmonella Typhimurium (ST) survives inside the oxidative environment of phagocytic cells. Phagocyte generated oxidants primarily target proteins and modify amino acids in them. These modifications render the targeted proteins functionally inactive. Conversion of Asp to iso-Asp is one of the several known oxidant mediated amino acids modifications. By repairing iso-Asp to Asp, protein-isoaspartyl methyltransferase (PIMT) maintains the activities of proteins and thus helps in cellular survival under oxidative stress. To elucidate the role of PIMT in ST survival under oxidative stress, we have constructed a pimt gene deletion strain (Δpimt strain) of ST. The Δpimt strain grows normally in various culture media in vitro. However, in comparison to wild type ST, the Δpimt strain is found significantly (p < 0.001) more susceptible to H2O2 and hypochlorite (HOCl). Further, the Δpimt mutant strain shows hypersusceptibility (p < 0.001) to INF-γ stimulated macrophages. This susceptibility is reversed by pharmacological inhibition of reactive oxygen species (ROS) but not reactive nitrogen species (RNS) production. Further, plasmid based complementation enhances the survival of Δpimt mutant strain against oxidants in vitro and also inside the macrophages. In mice model, the LD50 for wild type ST and mutant Δpimt has been 1.73 × 104 and 1.38 × 105, respectively. Further, the mutant strain shows reduced dissemination to spleen and liver in mice. Following infection with a mixture of wild type ST and the Δpimt mutant (co-infection experiment), we recover significantly (p < 0.001) less numbers of mutant bacteria from the spleen and liver of mice.