Abstract
Salmonella typhimurium exhibits a distinct tropism for mouse enterocytes that is linked to their expression of type 1 fimbriae. The distinct binding traits of Salmonella type 1 fimbriae is also reflected in their binding to selected mannosylated proteins and in their ability to promote secondary bacterial aggregation on enterocyte surfaces. The determinant of binding in Salmonella type 1 fimbriae is a 35-kDa structurally distinct fimbrial subunit, FimHS, because inactivation of fimHS abolished binding activity in the resulting mutant without any apparent effect on fimbrial expression. Surprisingly, when expressed in the absence of other fimbrial components and as a translational fusion protein with MalE, FimHS failed to demonstrate any specific binding tropism and bound equally to all cells and mannosylated proteins tested. To determine if the binding specificity of Salmonella type 1 fimbriae was determined by the fimbrial shaft that is intimately associated with FimHS, we replaced the amino-terminal half of FimHS with the corresponding sequence from Escherichia coli FimH (FimHE) that contains the receptor binding domain of FimHE. The resulting hybrid fimbriae bearing FimHES on a Salmonella fimbrial shaft exhibited binding traits that resembled that of Salmonella rather than E. coli fimbriae. Apparently, the quaternary constraints imposed by the fimbrial shaft on the adhesin determine the distinct binding traits of S. typhimurium type 1 fimbriae.
Highlights
Salmonella typhimurium is a highly evolved pathogen that has adapted to invading and surviving for long periods in its human and mouse hosts [1]
In view of the tropism exhibited by Salmonella for gut tissue, the association of type 1 fimbrial expression with virulent strains of Salmonella, and the heterogeneity in binding specificity among type 1 fimbriae of Salmonella and other enterobacteria, we hypothesized that the type 1 fimbriae of S. typhimurium may play a critical role in modulating bacterial tropism to the gut of the host
Type 1 Fimbriae of S. typhimurium Promote Bacterial Binding to Murine Enterocytes but Not to Bladder Epithelial Cells—We examined the adherence of two pairs of strains, the clinical S. typhimurium strain X4252 and its fim minus derivative, S. typhimurium X4253, and the fim minus laboratory E. coli strain, ORN103, and its recombinant derivative, ORN103(pISF101) harboring the recombinant plasmid pISF101 encoding S. typhimurium type 1 fimbriae
Summary
The immortalized mouse proximal small intestinal epithelial cell line SI-H10 was established from LFABPϪ596 to ϩ21/tsA58 transgenic mice [16]. The crystals of formazan that are produced could be observed microscopically in individual bacterial cells and could be quantitated in a multiwell scanning spectrophotometer (enzyme-linked immunosorbent assay reader) This MTT bacterial adherence assay is a new, simple, inexpensive, and rapid colorimetric assay that we have developed in our laboratory to study the adherence of live, metabolically active bacteria. Enterocytes (SI-H10 cells) or bladder epithelial cells (MM45T.BL cells) at a concentration of 0.15 ϫ 106/ml were seeded in a 96-well tissue culture plate and allowed to grow in enriched medium (at 39 °C in the case of SI-H10 cells or 37 °C in the case of MM45T.BL cells) with 5% CO2 Under these conditions, most of the cells attached to the plastic surface of the dish and formed a monolayer. Fifty l of 2 mg/ml MTT was added to all wells, and the plates were incubated for 15 min at 37 °C to allow reduction of MTT to formazan by live bacteria.
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