Type IVb Pili Research Articles

Salmonella enterica serovar Paratyphi C carries an inactive shufflon.

Salmonella enterica serovar Typhi uses type IVB pili to facilitate bacterial self-association, but only when the PilV proteins (potential minor pilus proteins) are not synthesized. This pilus-mediated event may be important in typhoid fever pathogenesis. We initially show that S. enterica serovar Paratyphi C strains harbor a pil operon very similar to that of serovar Typhi. An important difference, however, is located in the shufflon which concludes the pil operon. In serovar Typhi, the Rci recombinase acts upon two 19-bp inverted repeats to invert the terminal region of the pilV gene, thereby disrupting PilV synthesis and permitting bacterial self-association. In serovar Paratyphi C, however, the shufflon is essentially inactive because each of the Rci 19-bp substrates has acquired a single base pair insertion. A PilV protein is thus synthesized whenever the pil operon is active, and bacterial self-association therefore does not occur in serovar Paratyphi C. The data thus suggest that serovar Typhi bacterial self-association using type IVB pili may be important in the pathogenesis of epidemic enteric fever.

Salmonella enterica serovar Dublin strains which are Vi antigen-positive use type IVB pili for bacterial self-association and human intestinal cell entry

Some strains of Salmonella enterica serovar Dublin are Vi antigen-positive. S. enterica serovar Typhi uses Type IVB pili, encoded adjacent to the viaB locus required for Vi antigen synthesis, to facilitate both eukaryotic cell attachment and bacterial self-association under conditions that favour DNA supercoiling. These pilus-mediated events may be important in typhoid fever pathogenesis. A survey of 17 isolates of S. enterica serovar Dublin showed that all strains which carried the viaB region also carried a serovar Typhi-like Type IVB pil operon, and all serovar Dublin Vi antigen-negative isolates lacked the pil operon. The pil operon was completely sequenced from one of the Vi + serovar Dublin strains, and was almost identical (4 nt changes; 3 aa changes, in over 10 kb) to that of serovar Typhi. A pilS mutant of one serovar Dublin strain was constructed, and shown to invade cultured human intestinal INT407 cells to an extent only 20% that of the wild-type parent. Purified prePilS protein inhibited INT407 cell entry by serovar Dublin. The wild-type serovar Dublin strain, but not the pilS mutant, self-associated. The data suggest that the serovar Dublin Type IVB pil operon may increase the human-invasiveness of serovar Dublin, compared to pil-free strains.

The type IVB pili of Salmonella enterica serovar Typhi bind to the cystic fibrosis transmembrane conductance regulator.

Salmonella enterica serovar Typhi expresses type IVB pili. We show that the prePilS protein (the soluble precursor form of the structural pilin) interacts with a 15-mer peptide representing the first extracellular domain of the cystic fibrosis transmembrane conductance regulator (CFTR), a recognized human epithelial cell receptor for serovar Typhi (G. B. Pier et al., Nature 393:79-82, 1998). This indicates that after mediating bacterial self-association (C. Morris et al., Infect. Immun. 71:1141-1146, 2003), the pili then act to attach the bacterial clumps to CFTR in the membrane of gut epithelial cells. These sequential type IVB pilus-mediated events cannot be performed by (for example) S. enterica serovar Typhimurium, which may explain why only serovar Typhi causes epidemics of enteric fever in humans.

The shufflon of Salmonella enterica serovar Typhi regulates type IVB pilus-mediated bacterial self-association.

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.-L. Zhang, I. S. M. Tsui, C. M. C. Yip, A. W. Y. Fung, D. K.-H. Wong, X. Dai, Y. Yang, J. Hackett, and C. Morris, Infect. Immun. 68:3067-3073, 2000). 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.

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 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(+) strains synthesized type IVB pili, as judged by (i) visualization in the electron microscope of thin pili in culture supernatants of one such strain and (ii) 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. A pilS mutant of serovar Typhi 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 inhibited by soluble prePilS protein (50% inhibition of entry at 1.4 microM prePilS).