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

Centre for Molecular Microbiology and Infection 1 and Centre for Structural Biology 3 , 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, UK 2 Author for corresponde...

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Veröffentlicht in:Microbiology (Society for General Microbiology) 2002-03, Vol.148 (3), p.657-665
Hauptverfasser: Reece, Stephen, Simmons, Cameron P, Fitzhenry, Robert J, Batchelor, Miranda, Hale, Christine, Matthews, Stephen, Phillips, Alan D, Dougan, Gordon, Frankel, Gad
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Sprache:eng
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Zusammenfassung:Centre for Molecular Microbiology and Infection 1 and Centre for Structural Biology 3 , 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, UK 2 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
ISSN:1350-0872
1465-2080
DOI:10.1099/00221287-148-3-657