Proteomic analysis of the intestinal epithelial cell response to enteropathogenic Escherichia coli.
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 approximately 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.
Biotechnology Laboratory, University of British Columbia, Wesbrook Building, 6174 University Boulevard, Vancouver, British Columbia V6T 1Z3, Canada.
Philip R
PR
Hardwidge
Isabel
I
Rodriguez-Escudero
David
D
Goode
Sam
S
Donohoe
Jimmy
J
Eng
David R
DR
Goodlett
Reudi
R
Aebersold
B Brett
BB
Finlay
D001692:Biological Transport; D015153:Blotting, Western; D018938:Caco-2 Cells; D003599:Cytoskeleton / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D015536:Down-Regulation; D004847:Epithelial Cells / Q000382:microbiology*; D004926:Escherichia coli / Q000378:metabolism*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007422:Intestines / Q000382:microbiology*; D007477:Ions; D008856:Microscopy, Fluorescence; D020543:Proteome; D021241:Spectrometry, Mass, Electrospray Ionization; D015854:Up-Regulation
D007477:Ions; D020543:Proteome