Regulated delayed synthesis of lipopolysaccharide and enterobacterial common antigen of Salmonella Typhimurium enhances immunogenicity and cross-protective efficacy against heterologous Salmonella challenge

Lipopolysaccharide (LPS) O-antigen and enterobacterial common antigen (ECA) are two major polysaccharide structures on the surface of Salmonella enterica serovar Typhimurium. Previous studies have demonstrated that regulated truncation of LPS enhances the cross-reaction against conserved outer membrane proteins (OMPs) from enteric bacteria. We speculate that the regulation of both O-antigen and ECA may enhance the induction of immune responses against conserved OMPs from enteric bacteria. In this work we targeted rfbB and rffG genes which encode dTDP-glucose 4,6-dehydratases and share the same function in regulating O-antigen and ECA synthesis. We constructed a mutant, S496 (ΔrfbB6 ΔrffG7 ΔpagL73::TT araC PBADrfbB-3), in which rfbB gene expression was dependent on exogenously supplied arabinose during in vitro growth and achieved the simultaneous tight regulation of both LPS and ECA synthesis, as demonstrated by the LPS profile and Western blotting using antisera against LPS and ECA. When administered orally, S. Typhimurium S496 was completely attenuated for virulence but still retained the capacity to colonize and disseminate in mice. In addition, we found that oral immunization with S496 resulted in increased immune responses against OMPs from enteric bacteria and enhanced survival compared with immunization with S492 possessing ΔrfbB6 ΔrffG8 mutations when challenged with lethal doses of Salmonella Choleraesuis or Salmonella Enteritidis. These results indicate that S. Typhimurium arabinose-regulated rfbB strain S496 is a good vaccine candidate, conferring cross-protection against lethal challenge with heterologous Salmonella.