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bioRxiv · 10.1101/2023.06.27.546680

Functional loss of rffG and rfbB, encoding dTDP-glucose 4,6-dehydratase, changes colony morphology, cell shape, motility and virulence in Salmonella Typhimurium

Chakravorty, S.·Banerjee, P.·Joseph, J. P.·Pathak, S.·Verma, T.·Das, M.·Nandi, D.
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Abstract

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 ({Delta}rffG{Delta}rfbB), but not in single gene-deleted strains, results in a round cell morphology, smaller colony formation and altered LPS profile. In addition, the {Delta}rffG{Delta}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 {Delta}rffG{Delta}rfbB strain which leads to impaired swimming and swarming motility and lower adhesion and invasion of HeLa cells. Importantly, the {Delta}rffG{Delta}rfbB strain is less proficient in colonizing Peyers 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.

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Chakravorty, S., Banerjee, P., Joseph, J. P., Pathak, S., Verma, T., Das, M., Nandi, D.. 2023-06-27. Functional loss of rffG and rfbB, encoding dTDP-glucose 4,6-dehydratase, changes colony morphology, cell shape, motility and virulence in Salmonella Typhimurium. https://doi.org/10.1101/2023.06.27.546680

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