Site-directed mutagenesis of intimin α modulates intimin-mediated tissue tropism and host specificity

被引:31
作者
Reece, S
Simmons, CP
Fitzhenry, RJ
Matthews, S
Phillips, AD
Dougan, G
Frankel, G [1 ]
机构
[1] Univ London Imperial Coll Sci Technol & Med, Ctr Mol Microbiol & Infect, Dept Biochem, London SW7 2AZ, England
[2] Royal Free Hosp, Ctr Paediat Gastroenterol, London NW3 2QG, England
[3] Univ London Imperial Coll Sci Technol & Med, Ctr Struct Biol, Dept Biochem, London SW7 2AZ, England
关键词
D O I
10.1046/j.1365-2958.2001.02371.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The hallmark of enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherchia coli adhesion to host cells is intimate attachment leading to the formation of distinctive 'attaching and effacing' lesions. This event is mediated, in part, by binding of the bacterial adhesion molecule intimin to a second bacterial protein, Tir, delivered by a type III secretion system into the host cell plasma membrane. The receptor-binding activity of intimin is localized to the C-terminal 280 amino acids (Int280) and at least five distinct intimin types (alpha, beta, gamma, delta and epsilon) have been identified thus far. In addition to binding to Tir, intimin can also bind to a component encoded by the host. The consequence of latter intimin-binding activity may determine tissue tropism and host specificity. In this study we selected three amino acids in intimin, which are implicated in Tir binding, far site-directed mutagenesis. We used the yeast two-hybrid system and gel overlays to study intimin-Tir protein interaction. In addition, the biological consequences of the mutagenesis was tested using a number of infection models (cultured epithelial cells, human intestinal explants and a mouse model). We report that while an 1237/897A substitution (positions numbered according to Int280 alpha /whole intimin alpha) in intimin or did not have any affect on its biological activity, a T255/914A substitution attenuated intimin activity in vivo. In contrast, the mutation V252/911A affected tissue targeting in the human intestinal explant model and attenuated the biological activity of intimin in the mouse model. This study provides the first clues of the molecular basis of how intimin mediates tissue tropism and host specificity.
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页码:86 / 98
页数:13
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