The Type IVB Pili of Salmonella enterica Serovar Typhi Bind to the Cystic Fibrosis Transmembrane Conductance Regulator

Abstract

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...

Type IV Pilin Proteins: Versatile Molecular Modules

“…Not all pilins are lectin-like, however; PilS from S. Typhi was reported to bind to the first extracellular loop of the cystic fibrosis transmembrane conductance regulator (CFTR) protein on intestinal epithelial cells (or peptide mimetics thereof), while its equivalent from S. enterica serovar Typhimurium does not (394). This specific interaction was thought to explain why only S. Typhi is capable of causing human epidemics.…”

Temporal upregulation of host surface receptors provides a window of opportunity for bacterial adhesion and disease

“…Salmonella enterica has been identified to utilize the adhesin PilS, interacting with the epithelial receptor, cystic fibrosis transmembrane conductance regulator (CFTR), allowing entry to the intestinal epithelial cells [85]. Interestingly, CFTR gene expression has been shown to be induced in ulcerative colitis, which might predispose an individual with this condition to subsequent Salmonella infection [86].…”

Host Specificity of Bacterial Pathogens

“…The effects of InvH on Salmonella interactions with host-intestinal mucosa appear to be host specific, as an invH mutation reduces the severity of Salmonella enteritis in calves but does not affect intestinal colonization or virulence in chicks (Watson et al 1995(Watson et al , 1998Porter and Curtiss 1997). The hostspecific pathogen S. Typhi also requires InvH for efficient interaction with epithelial cells (Altmeyer et al 1993) but enters human cells via an interaction between its type-IVB pili and the CFTR (cystic fibrosis transmembrane conductance regulator) protein (Pier et al 1998;Tsui et al 2003). Resistance to typhoid has there-fore been proposed as a selection pressure that may have maintained the cystic fibrosis mutation at high frequencies in the population (van de Vosse et al 2005).…”