Flagellated but not hyperfimbriated Salmonella enterica serovar Typhimurium attaches to and forms biofilms on cholesterol-coated surfaces

Abstract

The asymptomatic, chronic carrier state of Salmonella enterica serovar Typhi occurs in the bile-rich gallbladder and is frequently associated with the presence of cholesterol gallstones. We have previously demonstrated that salmonellae form biofilms on human gallstones and cholesterol-coated surfaces in vitro and that bile-induced biofilm formation on cholesterol gallstones promotes gallbladder colonization and maintenance of the carrier state. Random transposon mutants of S. enterica serovar Typhimurium were screened for impaired adherence to and biofilm formation on cholesterol-coated Eppendorf tubes but not on glass and plastic surfaces. We identified 49 mutants with this phenotype. The results indicate that genes involved in flagellum biosynthesis and structure primarily mediated attachment to cholesterol. Subsequent analysis suggested that the presence of the flagellar filament enhanced binding and biofilm formation in the presence of bile, while flagellar motility and expression of type 1 fimbriae were unimportant. Purified Salmonella flagellar proteins used in a modified enzyme-linked immunosorbent assay (ELISA) showed that FliC was the critical subunit mediating binding to cholesterol. These studies provide a better understanding of early events during biofilm development, specifically how salmonellae bind to cholesterol, and suggest a target for therapies that may alleviate biofilm formation on cholesterol gallstones and the chronic carrier state.

FIG. 1.

Schematic diagram of Tn10d transposon insertion sites in serovar Typhimurium cholesterol biofilm mutants with mutations affecting type 1 fimbria and flagellum structure and biogenesis. The arrows indicate the sizes and orientations of neighboring genes, and the triangles indicate the locations of transposon insertions.

FIG. 2.

Overexpression of type 1 fimbriae inhibits biofilm formation on cholesterol-coated surfaces, while normal or decreased expression of type 1 fimbriae has no effect. Biofilm formation by serovar Typhimurium strains grown with 3% crude ox bile extract on cholesterol-coated Eppendorf tubes was examined. Crystal violet-stained TBA biofilms were extracted with acetic acid, and absorbance at 570 nm was measured. *, statistically significantly different (P < 0.005) based on a two-tailed Student t test. OD570, optical density at 570 nm.

FIG. 3.

Flagella are necessary for biofilm formation on cholesterol-coated surfaces, whereas motility is dispensable. Serovar Typhimurium strains were grown with and without 3% crude ox bile extract and added to cholesterol-coated Eppendorf tubes. Biofilms were stained with crystal violet and extracted with acetic acid, and the absorbance at 570 nm was measured. *, statistically significantly different (P < 0.005) based on a two-tailed Student t test. OD570, optical density at 570 nm.

FIG. 4.

Expression of purified flagellum proteins from serovar Typhimurium wild-type, fliC mutant, fljB mutant, and fliC fljB mutant strains adhering to cholesterol-coated wells. An ELISA was performed using antiflagellum or anti-FliC monoclonal antibodies, and binding to cholesterol was quantified by measuring the bound secondary HRP-conjugated substrate using the optical density at 415 nm (OD415). Asterisks indicate statistically significant differences based on a two-tailed Student t test (*, P < 0.05; **, P < 0.005). Ab, antibody.