Salmonella enterica serovar Typhi uses type IVB pili to enter human intestinal epithelial cells

DNA sequencing upstream of the Salmonella enterica serovar Typhi pilV and r ci genes previously identified in the ca, 118-kb major pathogenicity island (X.-L. Zhang, C. Morris, and J. Hackett, Gene 202:139-146, 1997) identifie d a further 10 pil genes apparently forming a pil operon, The product of th e pilS gene, prePilS protein (a putative type IVB structural prepilin) was purified, and an anti-prePilS antiserum was raised in mice. Mutants of sero var 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 pi lV genes were driven by the tac promoter. The pil(+) strains synthesized ty pe IVB pill, as judged by (i) visualization in the electron microscope of t hin pill in culture supernatants of one such strain and (ii) the presence o f PilS protein (smaller than the prePilS protein by removal of the leader p eptide) 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. A pilS mutant of serovar Typh i entered human intestinal INT407 cells in culture to levels only 5 to 25% of those of the wild-type strain, and serovar Typhi entry was strongly inhi bited by soluble prePilS protein (50% inhibition of entry at 1.4 mu M prePi lS).