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.
Reece
Stephen
S
Centre for Molecular Microbiology and Infection and Centre for Structural Biology, Department of Biological Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK.
Simmons
Cameron P
CP
Fitzhenry
Robert J
RJ
Batchelor
Miranda
M
Hale
Christine
C
Matthews
Stephen
S
Phillips
Alan D
AD
Dougan
Gordon
G
Frankel
Gad
G
D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001422:Bacterial Adhesion; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D016971:Citrobacter freundii / Q000378:metabolism* / Q000472:pathogenicity*; D003106:Colon / Q000382:microbiology; D004195:Disease Models, Animal; D004756:Enterobacteriaceae Infections / Q000382:microbiology / Q000503:physiopathology; D029968:Escherichia coli Proteins; D005260:Female; D006801:Humans; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011956:Receptors, Cell Surface / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics; D014774:Virulence
D018829:Adhesins, Bacterial; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; C109287:Tir protein, E coli; C073819:eaeA protein, E coli; D014364:Tryptophan