Salmonella enterica Serovar Typhi Uses Type IVB Pili To Enter Human Intestinal Epithelial Cells

Abstract

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,...

Salmonella Typhi: from a Human Pathogen to a Vaccine Vector

“…Another important island is the SPI-7 which is responsible for the production of the Vi polysaccharide capsule (20). This island is 134 kb in length and encodes a variety of putative virulence-associated gene clusters, including the Vi locus, a phage encoding the SopE effector protein of PSI-1 (21-23), a type IV pilus, and a putative type IV secretion system (24)(25)(26)(27). The type IV pilus is involved in aiding attachment to eukaryotic cells (24)(25)(26)(27) and the SopE prophage harbors a gene encoding an effector protein secreted through the TTSS (14).…”

Aptasensors for biosecurity applications

“…It was observed that when mutations are introduced to the gene that encodes the pili (pilS) in S. typhi, their ability to adhere and invade human gastrointestinal epithelial cells in vitro was drastically reduced. [26] This suggests that the structural protein PilS may have important roles in the pathogenesis of S. Typhi in humans. Pan and coworkers have recently reported the generation of RNA aptamers against the type IVB pili of S. Typhi, which is composed largely of polymerized PilS protein.…”

SPI-7: Salmonella’s Vi-Encoding Pathogenicity Island

“…The pili have a diameter of 6nm, as observed from an overexpressing construct under electron microscopy [43], and are expressed primarily during stationary phase in liquid culture [44].…”

“…SPI-7 carries an operon responsible for the production of type IVB pili (pilL-pilV; STY4539-STY4550) [43]. The pili have a diameter of 6nm, as observed from an overexpressing construct under electron microscopy [43], and are expressed primarily during stationary phase in liquid culture [44].…”

“…The pili were initially characterised as enhancing the adhesion to and/or invasion of human epithelial cells: in both S. Typhi and S. Dublin, knocking out the major structural prepilin subunit, PilS, was observed to reduce bacterial uptake by target cells [4,43,45]. This protein, PilS, also appears to mediate the interaction with the chloride channel CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) [46], which is involved in the uptake of S. Typhi into epithelial cells [47].…”