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

Lipopolysaccharide (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 of Salmonella enterica serovar Typhimurium ( S . Typhimurium). However, the functional consequences resulting from the abrogation of both O-antigen and ECA synthesis in S . Typhimurium are not well studied. In the present study, we generated single and double gene-deleted mutants of rffG and rfbB , 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 both rffG and rfbB (Δ rffG Δ rfbB ), but not in single gene-deleted strains, results in a round cell morphology, smaller colony formation and altered LPS profile. In addition, the Δ rffG Δ rfbB strain 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 Δ rffG Δ rfbB strain which leads to impaired swimming and swarming motility and lower adhesion and invasion of HeLa cells. Importantly, the Δ rffG Δ rfbB strain 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 of S . Typhimurium infection in mice. Overall, this study highlights the importance of rffG and rfbB in maintaining cell wall integrity, colony and cellular morphology, motility and virulence in S . Typhimurium.