DNA sequencing upstream of the Salmonella typhi pilV and rci genes previously identified in the ca. 118 Kb major "pathogenicity island" 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 raised in mice. Mutants of S. typhi lacking either the whole pil operon, or with an insertion mutation in the pilS gene, were constructed, as was a strain in which the pilN-V genes were driven by the tac promoter. The pil⁺ strains synthesized Type IVB pili, as judged by (a) visualization in the electron microscope of thin pili in culture supernatants of one such strain, and (b) the presence of PilS protein (smaller than the prePilS protein by removal of the l...[ Read more ]

DNA sequencing upstream of the Salmonella typhi pilV and rci genes previously identified in the ca. 118 Kb major "pathogenicity island" 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 raised in mice. Mutants of S. typhi lacking either the whole pil operon, or with an insertion mutation in the pilS gene, were constructed, as was a strain in which the pilN-V genes were driven by the tac promoter. The pil⁺ strains synthesized Type IVB pili, as judged by (a) visualization in the electron microscope of thin pili in culture supernatants of one such strain, and (b) the presence of PilS protein (smaller than the prePilS protein by removal of the leader peptide) on immunoblotting of material pelleted by high-speed centrifugation of either the culture supernatant or sonicates of pil⁺ strains. Control pil^- mutants did not express the PilS protein.

Next, a pilS mutant of S. typhi entered human intestinal INT407 cells, in tissue culture, to levels only 5-25% those of the wild-type strain, and S. typhi entry was strongly inhibited by soluble prePilS protein (50% inhibition of entry at 1.42 μM prePilS) while the entry of S. typhimurium was not. This evidence, at the tissue culture level, suggests that Type IVB pili are important to facilitate the entry by S. typhi, of human gastrointestinal cells.

Then, it was shown that S. typhi prePilS protein (the signal sequence-bearing precursor of the PilS pilin) of the Type IVB pili interacts with a 15-mer peptide representing the first extracellular domain of the cystic fibrosis transmembrane conductance regulator (CFTR). Inhibition, by prePilS protein, of S. typhi entry into human epithelial gastrointestinal cells in vitro was abrogated by the 15-mer peptide. This suggested that S. typhi may use Type IVB pili to interact with CFTR during infection.

Finally, a δpilV mutant invaded INT407 cells at a level ca. 65% that of the wild-type S. typhi strain. Human INT407 cell entry levels by pilV1^cand pilV2^c mutants, however, were ca. 247% and 217%, respectively, when compared to wild type (100%). With all 3 pilV mutants, prePilS protein inhibited INT407 cell entry in a concentration-dependent manner similar to that seen with the wild type strain. The entry of S. typhimurium into INT407 cells was again not affected by prePilS protein in the bacteria-containing medium overlaying the human intestinal cells. These results indicated that the PilV proteins are not required for human epithelial cell invasion, and the proteins may serve as initiators for pilus synthesis. The functions of the PilV proteins of S. typhi remain to be ascertained.