Construction and Characterization of PIMT Gene Deletion Mutant of Salmonella typhimurium

Among all enteric bacterial pathogens Salmonella alone is the major cause of food borne infections. Salmonella enterica serovar Typhimurium (ST) and Salmonella enterica serovar Enteritidis (SE) are the most important serovars associated with gastroenteritis in human. While in European countries SE is most prevalent serovar, in India ST has been most frequently isolated from poultry and poultry products. ST and SE colonize poultry asymptomatically to mild enteritis, these strains cause serious infections in human if ingested with contaminated poultry products. Elder and immuno-compromised people are at high risk where infection can be fatal. However Salmonella infections can be treated with antibiotics but growing resistance to current drugs is a serious concern. To identify newer therapeutic targets and live attenuated vaccines in depth-mechanism of Salmonella-host interaction is required. Salmonella not only survives but also replicates inside the phagocytic cells. Phagocytes kill invading microorganisms by deploying a battery of oxidants. Almost all macromolecules, including DNA, RNA, lipids and proteins are susceptible to oxidant mediated inactivation. Due to abundance and reactivity proteins (amino acids) are the primary targets of phagocyte generated oxidants. Although many types of amino acid modifications are known (like formation of methionine sulfoxide, isoaspartate etc), only few amino acids repair systems have been identified till date which can repair modified amino acids to normal residues. One of the repairable modifications is the repair of isoaspartate to aspartate by protein isoaspartyl methyl transferase (PIMT). PIMT repairs protein bound isoaspartate to aspartate thus maintain protein function under adverse conditions i. e. at the time of infection. To ascertain the involvement of PIMT in ST virulence, we generated a PIMT gene deletion mutant strain. The PIMT gene was deleted from the ST genome by allelic replacement technique using lambda Red recombinase. The deletion was confirmed by PCR using primers located flanking to the PIMT gene sequence. Stability of deletion was confirmed by growing mutants for many generations or by co-culturing with ST wild type bacteria. The growth of mutant strain was evaluated in vitro in Luria Bertani media, where mutant grew comparable to wild type suggesting that PIMT gene deletion doesn't affect the growth of ST. Bacterial counts (cfu/ml) were similar in both wild type as well as in mutant strain cultures. Interestingly, PIMT mutant strain was two folds more susceptible to H₂O₂ mediated killing in comparison with wild type. Our preliminary data suggests that caecum of ST infected chicks showed severe inflammation while caecum of mutant infected was normal. Studies regarding attenuation and vaccine potential of mutant in poultry are underway.