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J. Biol. Chem., Vol. 279, Issue 19, 20127-20136, May 7, 2004

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Proteomic Analysis of the Intestinal Epithelial Cell Response to Enteropathogenic Escherichia coli^*

Philip R. Hardwidge, Isabel Rodriguez-Escudero, David Goode, Sam Donohoe¶, Jimmy Eng¶, David R. Goodlett¶, Reudi Aebersold¶, and B. Brett Finlay||

From the Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada, the Departamento de Microbiologia II, Universidad Complutense de Madrid, 28040 Madrid, Spain, and the ^¶Institute for Systems Biology, Seattle, Washington 98103

We present the first large scale proteomic analysis of a human cellular response to a pathogen. Enteropathogenic Escherichia coli (EPEC) is an enteric human pathogen responsible for much childhood morbidity and mortality worldwide. EPEC uses a type III secretion system (TTSS) to inject bacterial proteins into the cytosol of intestinal epithelial cells, resulting in diarrhea. We analyzed the host response to TTSS-delivered EPEC effector proteins by infecting polarized intestinal epithelial monolayers with either wild-type or TTSS-deficient EPEC. Host proteins were isolated and subjected to quantitative profiling using isotope-coded affinity tagging (ICAT) combined with electrospray ionization tandem mass spectrometry. We identified over 2000 unique proteins from infected Caco-2 monolayers, of which 13% are expressed differentially in the presence of TTSS-delivered EPEC effector proteins. We validated these data in silico and through immunoblotting and immunofluorescence microscopy. The identified changes extend cytoskeletal observations made in less relevant cell types and generate testable hypotheses with regard to host proteins potentially involved in EPEC-induced diarrhea. These data provide a framework for future biochemical analyses of host-pathogen interactions.

Received for publication, February 4, 2004 , and in revised form, February 23, 2004.

^* This work was supported in part by fellowships from the National Institutes of Health and the Michael Smith Foundation for Health Research (to P. R. H.) and the Canadian Institutes for Health Research, Howard Hughes Medical Institute, and Genome Canada (to B. B. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. S1–S3.

^|| Canadian Institutes of Health Research Distinguished Investigator and the Peter Wall Distinguished Professor. To whom correspondence should be addressed: Biotechnology Laboratory, University of British Columbia, Rm. 237 Wesbrook Bldg., 6174 University Blvd., Vancouver, British Columbia V6T 1Z3, Canada. Tel.: 604-822-2210; Fax: 604-822-9830; E-mail: bfinlay{at}interchange.ubc.ca.

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