Identification of a new alanine racemase in Salmonella Enteritidis and its contribution to pathogenesis

Non-typhoidal Salmonella (NTS) infections caused primarily by S. Enteritidis and S. Typhimurium particularly in immunocompromised hosts have accounted for a large percentage of fatalities in developed nations. Antibiotics have revolutionized the cure of enteric infections but have also led to the rapid emergence of pathogen resistance. New powerful therapeutics involving metabolic enzymes are expected to be potential targets for combating microbial infections and ensuring effective health management. Therefore, the need for new antimicrobials to fight such health emergencies is paramount. Enteric bacteria successfully evade the gut and colonize their hosts through specialized virulence strategies. An important player, alanine racemase is a key enzyme facilitating bacterial survival.

This study aims at understanding the contribution of alanine racemase genes alr, dadX and SEN3897 to Salmonella survival in vitro and in vivo. We have shown SEN3897 to function as a unique alanine racemase in S. Enteritidis which displayed essential alanine racemase activity. Interestingly, the sole presence of this gene in alr dadX double mutant showed a strict dependence on d-alanine supplementation both in vitro and in vivo. However, Alr complementation in d-alanine auxotrophic strain restored the alanine racemase deficiency. The K_m and V_max of SEN3897 was 89.15 ± 10.2 mM, 400 ± 25.6 µmol/(min mg) for l-alanine and 35 ± 6 mM, 132.5 ± 11.3 µmol/(min mg) for d-alanine, respectively. In vitro assays for invasion and survival as well as in vivo virulence assays involving SEN3897 mutant showed attenuated phenotypes. Further, this study also showed attenuation of d-alanine auxotrophic strain in vivo for the development of potential targets against Salmonella that can be investigated further.

This study identified a third alanine racemase gene unique in S. Enteritidis which had a potential effect on survival and pathogenesis in vitro and in vivo. Our results also confirmed that SEN3897 by itself wasn't able to rescue d-alanine auxotrophy in S. Enteritidis which further contributed to its virulence properties.