Characterization of Vibrio cholerae O1 El Tor galU and galE mutants: Influence on lipopolysaccharide structure, colonization, and biofilm formation

Recently we described the isolation of spontaneous bacteriophage K139-resis tant Vibrio cholerae O1 El Tor mutants. In this study, we identified phage- resistant isolates with intact O antigen but altered core oligosaccharide w hich were also affected in galactose catabolism; this strains have mutation s in the galU gene. We inactivated another gal gene, galE, and the mutant w as also found to be defective in the catabolism of exogenous galactose but synthesized an apparently normal lipopolysaccharide (LPS). Both gal mutants as well as a rough LPS (R-LPS) mutant were investigated for the ability to colonize the mouse small intestine. The galU and R-LPS mutants, but not th e galE mutant, were defective in colonization, a phenotype also associated with O-antigen-negative mutants. By investigating several parameters in vit ro, we could show that galU and R-LPS mutants were more sensitive to short- chain organic acids, cationic antimicrobial peptides, the complement system , and bile salts as well as other hydrophobic agents, indicating that their outer membrane no longer provides an effective barrier function. O-antigen -negative strains were found to be sensitive to complement and cationic pep tides, but they displayed significant resistance to bile salts and short-ch ain organic acids. Furthermore, we found that galU and galE are essential f or the formation of a biofilm in a spontaneous phage-resistant rugose varia nt, suggesting that the synthesis of UDP-galactose via UDP-glucose is neces sary for biosynthesis of the exopolysaccharide. In addition, we provide evi dence that the production of exopolysaccharide limits the access of phage K 139 to its receptor, the O antigen. In conclusion, our results indicate inv olvement of galU in V. cholerae virulence, correlated with the observed cha nge in LPS structure, and a role for galU and galE in environmental surviva l of V. cholerae.