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

The hallmark of enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Esche rchia coli adhesion to host cells is intimate attachment leading to the for mation of distinctive 'attaching and effacing' lesions. This event is media ted, in part, by binding of the bacterial adhesion molecule intimin to a se cond 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 dist inct intimin types (alpha, beta, gamma, delta and epsilon) have been identi fied thus far. In addition to binding to Tir, intimin can also bind to a co mponent encoded by the host. The consequence of latter intimin-binding acti vity may determine tissue tropism and host specificity. In this study we se lected three amino acids in intimin, which are implicated in Tir binding, f ar site-directed mutagenesis. We used the yeast two-hybrid system and gel o verlays to study intimin-Tir protein interaction. In addition, the biologic al consequences of the mutagenesis was tested using a number of infection m odels (cultured epithelial cells, human intestinal explants and a mouse mod el). We report that while an 1237/897A substitution (positions numbered acc ording to Int280 alpha /whole intimin alpha) in intimin or did not have any affect on its biological activity, a T255/914A substitution attenuated int imin activity in vivo. In contrast, the mutation V252/911A affected tissue targeting in the human intestinal explant model and attenuated the biologic al activity of intimin in the mouse model. This study provides the first cl ues of the molecular basis of how intimin mediates tissue tropism and host specificity.