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

Centre for Molecular Microbiology and Infection¹ and Centre for Structural Biology³, Department of Biological Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK
Centre for Paediatric Gastroenterology, Royal Free Hospital, London NW3 2QG, UK²

Author for correspondence: Gad Frankel. Tel: +44 20 7594 5253. Fax: +44 20 7594 5255. e-mail: g.frankel{at}ic.ac.uk

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.

Keywords: Citrobacter rodentium, EPEC, EHEC, Tir

Abbreviations: A/E lesion, ‘attaching and effacing’ lesion; EHEC, enterohaemorrhagic Escherichia coli; EPEC, enteropathogenic Escherichia coli; FAS, fluorescent actin stain; LEE, locus of enterocyte effacement; MBP, maltose-binding protein; Tir, translocated intimin receptor

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