Mutagenesis of conserved tryptophan residues within the receptor-binding domain of intimin: influence on binding activity and virulence

Intimate bacterial adhesion to intestinal epithelium is a pathogenic mechanism shared by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli and Citrobacter rodentium. The proteins directly involved in this process are the outer-membrane adhesion molecule intimin and the translocated intimin receptor, Tir. The receptor-binding activity of intimin resides within the carboxy terminus 280 aa (Int280) of the polypeptide. Four tryptophan residues, W117/776, W136/795, W222/881 and W240/899, are conserved within different Int280 molecules that otherwise show considerable sequence variation. In this study the influence of site-directed mutagenesis of each of the four tryptophan residues on intimin-Tir interactions and on intimin-mediated intimate attachment was determined. The mutant intimins were also studied using a variety of in vitro and in vivo infection models. The results show that all the substitutions modulated intimin activity, although some mutations had more profound effects than others. </p

Mutagenesis of conserved tryptophan residues within the receptor-binding domain of intimin: influence on binding activity and virulence.

Intimate bacterial adhesion to intestinal epithelium is a pathogenic mechanism shared by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli and Citrobacter rodentium. The proteins directly involved in this process are the outer-membrane adhesion molecule intimin and the translocated intimin receptor, Tir. The receptor-binding activity of intimin resides within the carboxy terminus 280 aa (Int280) of the polypeptide. Four tryptophan residues, W117/776, W136/795, W222/881 and W240/899, are conserved within different Int280 molecules that otherwise show considerable sequence variation. In this study the influence of site-directed mutagenesis of each of the four tryptophan residues on intimin-Tir interactions and on intimin-mediated intimate attachment was determined. The mutant intimins were also studied using a variety of in vitro and in vivo infection models. The results show that all the substitutions modulated intimin activity, although some mutations had more profound effects than others

Tyrosine residues at the immunoglobulin-C-type lectin inter-domain boundary of intimin are not involved in Tir-binding but implicated in colonisation of the host.

Intimin is an outer membrane adhesion molecule involved in bacterial adhesion to intestinal epithelium by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli and Citrobacter rodentium. Intimin binds to the translocated intimin receptor, Tir, which is delivered to the plasma membrane of the host cell by a type III protein translocation system. Intimin is also implicated in binding to a host cell-encoded intimin receptor (Hir). The receptor-binding activity of intimin resides within the carboxy terminus 280 amino acids (Int280) of the polypeptide. Structural analysis of this region revealed two immunoglobulin-like domains, the second of which forms a number of contacts with the distal C-type lectin-like module. Specific orientation differences at this inter-domain boundary, which consists of several tyrosine residues, were detected between the crystal and solution structures. In this study, we determined the influence of site-directed mutagenesis of each of four tyrosine residues on intimin-Tir interactions and on intimin-mediated intimate attachment. The mutant intimins were also studied using a variety of in vitro and in vivo infection models. The results show that three of the four Tyr, although not essential for A/E lesion formation in vitro, are required for efficient colonisation of the mouse host following oral challenge

Early interactions of Salmonella enterica serovar typhimurium with human small intestinal epithelial explants.

BACKGROUND: Salmonella enterica serovar typhimurium (S typhimurium) causes invasive gastroenteritis in humans, a disease involving significant penetration of the intestinal mucosa. However, few studies have been undertaken to investigate this interaction directly using differentiated human gut tissue. AIMS: To investigate the early interactions of an enteropathogenic strain of S typhimurium with human intestinal mucosa using human intestinal in vitro organ culture (IVOC). METHODS: Wild-type and mutant derivatives of S typhimurium TML were used to compare interactions with cultured human epithelial cells, bovine ligated loops, and human intestinal IVOC. RESULTS: S typhimurium TML was shown to attach to cultured Caco-2 brush border expressing cells and cause tissue damage and fluid accumulation in a ligated bovine loop model.S typhimurium TML bound predominantly to the mucus layer of human IVOC explants during the first four hours of IVOC incubation. From four to eight hours of IVOC incubation, small but characteristic foci of attaching and invading S typhimurium TML were detected as clusters of bacteria interacting with enterocytes, although there was no evidence for large scale invasion of explant tissues. Ruffling of enterocyte membranes associated with adherent Salmonella was visualised using electron microscopy. CONCLUSIONS: Human IVOC can be used as an alternative model for monitoring the interactions between S typhimurium and human intestinal epithelium, thus potentially offering insight into the early stages of human Salmonella induced gastroenteritis