A role for <em>Salmonella</em> fimbriae in intraperitoneal infections

Abstract

Fimbriae play a critical role in virulence by allowing bacteria to interact with host cells and other solid substrates (1, 2). The distribution of fimbrial operons among enteric bacteria suggests a role for fimbriae in pathogenesis; broadly distributed fimbrial operons may provide general adhesive functions, but fimbriae whose distribution is limited may provide specific functions required in virulence. For example, the common type I fimbriae found in many Gram-negative bacteria mediates adherence to the pharynx, intestinal epithelium, and bladder, whereas plasmid encoded fimbriae found only in Salmonella bind specifically to M cells in the intestine (1, 3).

Most fimbriae (with the notable exception of type IV pili) have a conserved mechanism of translocation to the bacterial surface. Fimbrial proteins are secreted into the periplasm by means of the general secretory system. Two accessory proteins assist in construction of the fimbrial shaft. First, the fimbrial subunits are bound by a chaperone in the periplasm to prevent premature aggregation and then the subunits are translocated across the outer membrane by an usher protein. Recently, the crystal structures of FimC–FimH and PapD–PapK complexes have been solved, demonstrating an elegantly simple mechanism for the interactions between the subunit and chaperone and between pairs of subunits (4, 5). In all fimbrial systems that have been studied, the genes encoding the chaperone and usher are located in the same operon as the major subunit. Cursory examination of the Escherichia coli, Yersinia pestis, Salmonella enterica serovar Typhimurium (S. typhimurium), and Salmonella Typhi (S. typhi) genomes identified about one dozen chaperone–usher-dependent fimbrial operons per genome, although only a handful of these have currently been characterized (R.A.E., B. M. Matlock, and S.R.M., unpublished observations). It is expected that Salmonella enteritidis contains a similar number of fimbrial operons to these other enteric bacteria. The S. enteritidis fimbriae (SEF14) is restricted to S. enteritidis and other closely related group D Salmonella (7). Therefore, an analysis of SEF14 fimbrial function may provide insight into the unique aspects of virulence that distinguish this group of Salmonella.

The sef operon is located on a small pathogenicity island. The operon contains four structural genes (sefABCD) required for the translocation and biogenesis of SEF14 fimbriae: sefA encodes the major subunit, sefB and sefC encode the chaperone and usher, respectively, and sefD encodes the putative adhesin. Adjacent to sefD, there is an AraC-like regulatory protein (encoded by sefR) that activates transcription of the sef genes (6) (Fig. ​(Fig.1).1). Some evidence suggests that SEF14 fimbriae may play a role in pathogenesis. For example, immunization of mice with purified SEF14 subunits induces a strong T lymphocyte response, and the mice present a delayed-type hypersensitive response to whole S. enteritidis, demonstrating that these fimbriae are expressed in vivo and stimulate cell-mediated immunity (8). Furthermore, pretreatment of mice with anti-SEF14 antibodies protects mice from S. enteritidis infection (9). However, other studies on the effect of sef mutants on virulence showed conflicting results (10, 11).

Figure 1

The sef pathogenicity island. Organization of the sef genes, direction of transcription (arrows), and predicted function of the ORFs.

Because discrepancies in earlier studies may be the result of genetic differences in the mutants used, we constructed several defined sef mutants (R.A.E., B. M. Matlock, and S.R.M., unpublished observations; L. H. Keller, R. C. Boston, and D.M.S., unpublished observations; ref. 13). Like many other fimbrial operons, the sefABCD genes are cotranscribed so that insertion of a kanamycin cassette into sefA eliminates transcription of sefD, and translocation of SefD across the outer membrane is a prerequisite for translocation of SefA (6). Based on these studies, we predict that the SEF14 fimbrial proteins displayed on the surface of the sef mutants is as shown in Fig. ​Fig.2.2. In this work, we assay the role of the various sef mutants in virulence and show that SEF14 fimbriae mediate interactions with phagocytes in the peritoneal cavity, which allows S. enteritidis to survive the macrophage assault. This role for fimbriae in vivo is in contrast to the routine modus operandi of previously characterized fimbriae involved in binding to host epithelial surfaces.

Figure 2

Predicted export of SEF14 fimbrial subunits from the periplasm in the mutants used in this study. Note that all subunits contain signal sequences, suggesting that they are exported across the cytoplasmic membrane in a secretory-dependent manner. Wild-type SEF14 fimbriae (A) consist of the major subunit, SefA. Based on sequence homology, SefB is the chaperone that binds subunits in the periplasm and prevents premature aggregation. SefC is the membrane-located usher through which the subunits are translocated. SefD is probably a tip-located adhesin. Export of SefD occurs in the absence of SefA (ΔsefA; B) and may form a fibrillar structure on the cell surface. The polar sefA∷Kan strain (C) does not produce any subunits. Export of SefA does not occur in the absence of SefD (ΔsefD; D).

*Only the sef genotype is shown.

^{To reduce the number of mice required, we only tested two mice for each dilution of} S. enteritidis; therefore, all LD₅₀ values are approximate.

Acknowledgments

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