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Pathogenicity and Medical Microbiology |
University Department of Paediatric Gastroenterology, Royal Free Hospital, London NW3 2QG, UK1
Centro de Investigaciones Microbiológicas, Benemérita Universidad Autónoma de Puebla, Puebla, México2
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK3
Author for correspondence: Alan D. Phillips. Tel: +44 207 830 2783. Fax: +44 207 830 2146. e-mail: adphill{at}rfhsm.ac.uk
Adhesion to cultured epithelial cells by enteropathogenic Escherichia coli (EPEC) is associated with extensive rearrangement of the host cell cytoskeleton. Evidence has been presented that EPEC adhesion is associated with activation of signal transduction pathways leading to production of a characteristic histopathological feature known as the attaching and effacing (A/E) lesion. A/E lesion formation requires intimin, an EPEC adhesion molecule and several EPEC secreted proteins (EspA, B, D and Tir) involved in cell signalling and protein translocation. In this study it is shown that HEp-2 cells respond during the early stages of infection with two wild-type EPEC strains (B171 and E2348/69) by producing microvillus-like processes (MLP) at the site of initial bacterial adherence. Intimin appears to play a key role in MLP elongation. At later stages of infection with these wild-type EPEC strains, when A/E lesions have formed, the MLP were reduced in number and length to appear as at time zero, and the cell surface in the vicinity of bacterial clusters appeared unaffected. In contrast, infection with EspA- or EspB-negative, but intimin-positive, EPEC strains (UMD872 and UMD864, respectively) resulted in enhanced MLP proliferation and formation of ‘cage-like’ structures engulfing the bacteria. Inoculating HEp-2 cells with intimin-coated latex spheres induced similar ‘cage-like’ structures. Caco-2 cells did not show intimin-induced microvillus elongation in response to EPEC infection, although microvillus effacement and reduction in number occurred. Similar phenomena appeared on B171 and E2348/69 infection of paediatric intestine using in vitro organ culture, i.e. elongated microvilli were seen in association with small colonies and at the periphery of large localized colonies, along with evidence of microvillus breakdown and debris in the colony centre. These results show that intimin activates signal transduction pathways involved in the remodelling of the eukaryotic cell surface, probably via binding to a receptor encoded by the host cell.
Keywords: enteropathogenic Escherichia coli, intimin, cell culture, in vitro organ culture, microvilli
Abbreviations: A/E, attaching and effacing; Bfp, bundle-forming pilus; EAF, EPEC adherence factor; EPEC, enteropathogenic Escherichia coli; Esp, EPEC secreted protein; IVOC, in vitro organ culture; LA, localized adherence; LEE, locus of enterocyte effacement; MLP, microvillus-like processes; SEM, scanning electron microscopy; Tir, translocated intimin receptor
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