Salmonella enterica serovar Typhi can establish a chronic, asymptomatic infection of the human gallbladder, suggesting that this bacterium utilizes novel mechanisms to mediate enhanced colonization and persistence in a bile-rich environment. Gallstones are one of the most important risk factors for developing carriage, and we have previously demonstrated that salmonellae form biofilms on human gallstones in vitro. Thus, we hypothesize that bile-induced biofilms on gallstone surfaces promote gallbladder colonization and maintenance of the carrier state. A colanic acid/cellulose S. enterica serovar Typhimurium double mutant formed a mature biofilm on gallstones in a test tube assay and in a new, gallstone-independent assay using cholesterol-coated Eppendorf tubes. These data suggest the presence of an unidentified exopolysaccharide necessary for mature biofilm development and demonstrate specific binding affinity between salmonellae and cholesterol. Our experiments indicate that the Salmonella O-antigen capsule (yihU-yshA and yihV-yihW) is a crucial determinant in gallstone and cholesterol biofilms but that expression of this exopolysaccharide is not necessary for binding to glass or plastic. Real-time PCR revealed that growth in bile resulted in upregulation of the O-antigen capsule-encoding operon in an agfD-independent manner. Thus, the O-antigen capsule genes are bile induced, and the capsule produced by the enzymes of this operon is specifically required for biofilm formation on cholesterol gallstones. These studies provide new therapeutic targets for preventing asymptomatic serovar Typhi gallbladder carriage.
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