Functional loss ofrffGandrfbB,encoding dTDP-glucose 4,6-dehydratase, changes colony morphology, cell shape, motility and virulence inSalmonellaTyphimurium

Abstract

AbstractLipopolysaccharide (LPS) O-antigen and enterobacterial common antigen (ECA) play crucial roles in maintaining the structural integrity of the outer membrane in Gram-negative bacteria. Previous studies conducted with either LPS or ECA mutants have highlighted the importance of these cell surface polysaccharides in the physiology ofSalmonella entericaserovar Typhimurium (S. Typhimurium). However, the functional consequences resulting from the abrogation of both O-antigen and ECA synthesis inS. Typhimurium are not well studied. In the present study, we generated single and double gene-deleted mutants ofrffGandrfbB, which are paralogs, encoding dTDP-glucose 4,6-dehydratase that catalyze steps in the synthesis of both O-antigen and ECA. The functional loss of bothrffGandrfbB(ΔrffGΔrfbB), but not in single gene-deleted strains, results in a round cell morphology, smaller colony formation and altered LPS profile. In addition, the ΔrffGΔrfbBstrain displays defects in outer membrane permeability, causing hypersensitivity to bile and cell wall targeting antibiotics, e.g., meropenem and polymyxin B. Transcriptomic analysis identified flagellar and SPI-1 pathway to be highly down-regulated in the ΔrffGΔrfbBstrain which leads to impaired swimming and swarming motility and lower adhesion and invasion of HeLa cells. Importantly, the ΔrffGΔrfbBstrain is less proficient in colonizing Peyer’s patches, spleen and liver, is unable to induce pro-inflammatory cytokines and is attenuated in both the oral and intra-peritoneal models ofS. Typhimurium infection in mice. Overall, this study highlights the importance ofrffGandrfbBin maintaining cell wall integrity, colony and cellular morphology, motility and virulence inS. Typhimurium.