Kim Lam Chiok, Narayan C. Paul, Ezekiel O. Adekanmbi, Soumya K. Srivastava, Devendra H. Shah

We previously reported that inactivation of a universally conserved dimethyl adenosine transferase (KsgA) attenuates virulence and increases sensitivity to oxidative and osmotic stress in Salmonella Enteritidis. Here, we show a role of KsgA in cell-envelope fitness as a potential mechanism underlying these phenotypes in Salmonella. We assessed structural integrity of the cell-envelope by transmission electron microscopy, permeability barrier function by determining intracellular accumulation of ethidium bromide and electrophysical properties by dielectrophoresis, an electrokinetic tool, in wild-type and ksgA knock-out mutants of S. Enteritidis. Deletion of ksgA resulted in disruption of the structural integrity, permeability barrier and distorted electrophysical properties of the cell-envelope. The cell-envelope fitness defects were alleviated by expression of wild-type KsgA (WT-ksgA) but not by its catalytically inactive form (ksgAE66A), suggesting that the dimethyl transferase activity of KsgA is important for cell-envelope fitness in S. Enteritidis. Upon expression of WT-ksgA and ksgAE66A in inherently permeable E. coli cells, the former strengthened and the latter weakened the permeability barrier, suggesting that KsgA also contributes to the cell-envelope fitness in E. coli. Lastly, expression of ksgAE66A exacerbated the cell-envelope fitness defects, resulting in impaired S. Enteritidis interactions with human intestinal epithelial cells, and human and avian phagocytes. This study shows that KsgA contributes to cell-envelope fitness and opens new avenues to modulate cell-envelopes via use of KsgA-antagonists.