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The Tol–Pal system of Gram-negative bacteria is necessary for maintaining outer membrane integrity. It is a multiprotein complex of five envelope proteins, TolQ, TolR, TolA, TolB, and Pal. These proteins were first investigated in  E. coli , and subsequently been identified in many other bacterial genera. However, the function of the Tol–Pal system in  Salmonella  Choleraesuis pathogenesis is still unclear. Here, we reported the role of three of these proteins in the phenotype and biology of  S.  Choleraesuis. We found that mutations in  tolA ,  tolB , and  tolR  caused severe damage to the cell wall, which was supported by observing the microstructure of spherical forms, long chains, flagella defects, and membrane blebbing. We confirmed that all the mutants significantly decreased  S.  Choleraesuis survival when exposed to sodium deoxycholate and exhibited a high sensitivity to vancomycin, which may be explained by the disruption of envelope integrity. In addition,  tolA ,  tolB , and  tolR  mutants displayed attenuated virulence in a mouse infection model. This could be interpreted as a series of defective phenotypes in the mutants, such as severe defects in envelope integrity, growth, and motility. Further investigation showed that all the genes participate in outer membrane vesicles (OMVs) biogenesis. Interestingly, immunization with OMVs from Δ tolB  efficiently enhanced murine viability in contrast to OMVs from the wild-type  S.  Choleraesuis, suggesting its potential use in vaccination strategies. Collectively, this study provides an insight into the biological role of the  S.  Choleraesuis Tol–Pal system.

The role of TolA, TolB, and TolR in cell morphology, OMVs production, and virulence of Salmonella Choleraesuis