DNA sequencing upstream of the Salmonella enterica serovar Typhi pilV and rci genes previously identified in the ca. 118-kb major pathogenicity island (X.-L. Zhang, C. Morris, and J. Hackett, Gene 202:139-146, 1997) identified a further 10 pil genes apparently forming a pil operon. The product of the pilS gene, prePilS protein (a putative type IVB structural prepilin) was purified, and an anti-prePilS antiserum was raised in mice. Mutants of serovar Typhi either lacking the whole pil operon or with an insertion mutation in the pilS gene were constructed, as was a strain in which the pilN to pilV genes were driven by the tac promoter. The pil Earlier, it was reported that the major pathogenicity island of Salmonella enterica serovar Typhi, which is ca. 118 kb in size (11), contained pilV and rci genes, which were cloned and sequenced (22). The Rci gene product was shown to be a site-specific recombinase, active to invert DNA in the C-terminal region of the pilV gene, so that two PilV proteins could be synthesized. Comparisons with database sequences indicated that the two possible pilV genes might code for pilus-tip adhesins, as the serovar Typhi PilV sequence was similar to that of PilV proteins encoded by the Escherichia coli R64 plasmid. In R64-bearing strains, different PilV proteins, borne on type IV pili, select various recipients in liquid mating (the R64-bearing cell is the donor) (10). Both serovar Typhi PilV proteins were seen when the two pilV genes were transcribed from the T7 promoter. The discovery of the serovar Typhi pilV and rci genes in the ca. 118-kb pathogenicity island (henceforth in this work termed the large pathogenicity island) suggested that serovar Typhi might synthesize thin pili belonging to the type IV pilin family (9). As type IV pili, encoded in a Vibrio cholerae pathogenicity island (7, 8) are used by V. cholerae as mediators of adhesion to human cells (13, 18), it was of interest to ask (i) if serovar Typhi also synthesizes type IV pili and (ii) if such pili are important in adherence to or invasion of human intestinal cells. These topics are the subject of this paper. MATERIALS AND METHODSMaterials. All reagents were of molecular biology grade. Enzymes active on DNA were obtained from either GibcoBRL or Boehringer Mannheim and were used as directed by the suppliers. 5-Bromo-4-chloro-3-indolyl-␤-D-galactopyranoside and isopropyl-␤-D-thiogalactopyranoside were purchased from Amersham. Anti-mouse immunoglobulin G (from sheep), conjugated with horseradish peroxidase, was from Amersham. Phosphatase-labeled goat anti-mouse immunoglobulin G (heavy and light chains) was purchased from KPL Laboratories. p-Nitrophenyl phosphate tablets were from Sigma. Bio-Rad was the supplier of polyvinylidene difluoride membrane. Freund's adjuvant was from GibcoBRL.Strains and vectors. Serovar Typhi J341 (Ty2 Vi Ϫ ) (22) was the source of DNA for a cosmid bank (partially Sau3AI-cut DNA in BamHI-cut pHC79), which was probed with 32 P-labeled total DNA (including the virulence plasmid pSLT) of (wild-type,...

Previously, it was shown that type IVB pili encoded by the Salmonella enterica serovar Typhi pil operon are used to facilitate bacterial entry into human intestinal epithelial cells in vitro and that such entry is inhibited by purified prepilin (pre-PilS) protein (X. . The pil operon concludes with a simple shufflon, and a recombinase gene product (Rci) inverts DNA in the C-terminal region of the pilV gene to allow synthesis of two distinct PilV proteins, PilV1 and PilV2, which are presumptive minor pilus proteins. We show here that the type IVB pili mediate bacterial self-association, but only when the PilV1 and PilV2 proteins are not expressed. This may be achieved in wild-type serovar Typhi by rapid DNA inversion activity of the shufflon. We show that the inversion activity inhibits the expression of genes inserted between the 19-bp inverted repeats used for Rci-mediated recombination and that the activity of Rci increases when DNA is supercoiled. The data suggest that serovar Typhi self-associates under conditions (such as low oxygen tension in the gut) that favor DNA supercoiling. These results explain (i) the function of the serovar Typhi shufflon and (ii) why there are only two possible shufflon states, in contrast to the many possible states of other shufflon systems. The data further indicate that a very early step in serovar Typhi pathogenesis may be type IVB pilus-mediated self-association of bacteria in the anaerobic human small intestine prior to invasion of the human gut epithelium. The suggested type IVB pilus-dependent step in typhoid fever pathogenesis may partially explain the enhanced invasiveness of serovar Typhi for humans.-The type IVB pilus operon of Salmonella enterica serovar Typhi contains a pilS gene encoding the structural pilin (3, 15). A pilS mutant of serovar Typhi exhibited much reduced adhesion to and invasion of human epithelial gastrointestinal cells in vitro, and soluble purified pre-PilS protein (retaining the signal sequence normally cleaved when the protein is excreted to form insoluble pili based on polymerized PilS) inhibited bacterial invasion (15). These data indicated that the type IVB pili might have an important function in the pathogenesis of serovar Typhi in humans.The serovar Typhi pil operon concludes with a simple shufflon, and the Rci gene product, encoded by the rci gene adjacent to the pil operon, acts to invert DNA between a pair of 19-bp inverted repeats to place one of two possible C termini on the pilV gene (14). No function in pathogenesis or otherwise has yet been assigned to the serovar Typhi shufflon. It is notable that the serovar Typhi shufflon is simple compared to the shufflons which terminate the pil operons of plasmids R64 and R721. The latter shufflons have seven and six 19-bp repeats, respectively, to form seven or six different pilV genes by Rcimediated recombination. In these plasmids, the various PilV proteins selectively recognize determinants on the exterior of enterobacterial species, facilitating transfer of the conjugative plasmids in ...

The type IVB pilus operon of Salmonella enterica serovar Typhi contains a pilS gene encoding the structural pilin (1, 5). A pilS mutant of serovar Typhi was much reduced in adhesion to and invasion of human epithelial gastrointestinal cells in vitro, and soluble purified prePilS protein (retaining the signal sequence normally cleaved when the protein is excreted to form insoluble pili based on polymerized PilS) inhibited bacterial invasion (5). While the pili mediate bacterial self-association (3), these data did not explain why purified prepilin should inhibit serovar Typhi entry to human intestinal epithelial cells. This rather suggested that the prepilin might interact with an epithelial cell receptor.It is known that the first extracellular domain of the cystic fibrosis transmembrane conductance regulator (CFTR) is a serovar Typhi receptor domain (4). To determine if soluble prePilS protein could interact with this domain of CFTR, bacteria of serovar Typhi strain J341 (Ty2 Vi Ϫ ) (5), grown in Luria broth for 14 to 16 h at 30°C to reach optical densities at 600 nm of 0.5 to 0.7, were resuspended in Eagle's basal medium, which also contained CFTR peptides, prePilS protein, or both. Then the bacteria were centrifuged for 10 min at 3,500 ϫ g onto washed cells (bacterium/cell ratio of 1:1) of the human embryonic intestine cell line INT407 (4). Bacterium-cell interaction proceeded for 2 h at 37°C in a 5% (vol/vol) CO 2 atmosphere. After incubation, cells were washed, treated with gentamicin, washed once more, and lysed for enumeration of internalized bacteria (3 to 5% of added bacteria, in the absence of an inhibitor of INT407 cell entry by serovar Typhi) (5).The 15-mer peptides representing the first extracellular human CFTR domain (GRIIASYDPDNKEER) and a scrambled version of this domain (GKDPNYRDEAIRSIE) (4) were synthesized by Research Genetics, Huntsville, Ala.. The prePilS protein was purified from a glutathione S-transferase (GST) fusion protein, expressed in Escherichia coli K-12 as previously described (5). Affinity beads with bound fusion protein were washed with 100 volumes of phosphate-buffered saline (PBS) before thrombin cleavage of prePilS, and the prePilS preparation was confirmed to be free of bacterial lipopolysaccharide in assays detecting periodate-cleavable carbohydrate. Also, heat (boiling for 5 min) destroyed the ability of prePilS to inhibit eukaryotic cell entry by serovar Typhi, in that boiled prePilS at 4 M did not detectably inhibit such entry, while ca. 75% cell entry inhibition was afforded by the same concentration of native prePilS (described below).As demonstrated before (5), prePilS protein inhibited serovar Typhi entry into INT407 cells, with 50% inhibition noted at ca. 2 M prePilS (Fig. 1A). Addition of the scrambled version of the CFTR 15-mer to assays including prePilS protein did not affect the inhibition noted. When the authentic CFTR 15-mer was present (at 2 M) in assays in which the prePilS protein was included at 1, 2, 3, or 4 M, the CFTR 15-mer effectively neutralized th...