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

BACKGROUND

Non-typhoidal (NTS) infections caused primarily by Enteritidis and 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.

RESULTS

This study aims at understanding the contribution of alanine racemase genes , and to survival in vitro and in vivo. We have shown to function as a unique alanine racemase in . Enteritidis which displayed essential alanine racemase activity. Interestingly, the sole presence of this gene in 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 and V 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 SEN mutant showed attenuated phenotypes. Further, this study also showed attenuation of d-alanine auxotrophic strain in vivo for the development of potential targets against that can be investigated further.

CONCLUSION

This study identified a third alanine racemase gene unique in . 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 . Enteritidis which further contributed to its virulence properties.