Proteomic analysis of the binding partners to enteropathogenic Escherichia coli virulence proteins expressed in Saccharomyces cerevisiae.
Enteropathogenic Escherichia coli (EPEC) is an enteric human pathogen responsible for much worldwide morbidity and mortality. EPEC uses a type III secretion system to inject bacterial proteins into the cytosol of intestinal epithelial cells to cause diarrheal disease. We are interested in determining the host proteins to which EPEC translocator and effector proteins bind during infection. To facilitate protein enrichment, we created fusions between GST and EPEC virulence proteins, and expressed these fusions individually in Saccharomyces cerevisiae. The biology of S. cerevisiae is well understood and often employed as a model eukaryote to study the function of bacterial virulence factors. We isolated the yeast proteins that interact with individual EPEC proteins by affinity purifying against the GST tag. These complexes were subjected to ICAT combined with ESI-MS/MS. Database searching of sequenced peptides provided a list of proteins that bound specifically to each EPEC virulence protein. The dataset suggests several potential mammalian targets of these proteins that may guide future experimentation.
Veterinary Science Department, South Dakota State University, Brookings, SD, USA. philip.hardwidge@sdstate.edu
Philip R
PR
Hardwidge
Sam
S
Donohoe
Ruedi
R
Aebersold
B Brett
BB
Finlay
D000345:Affinity Labels; D001665:Binding Sites / Q000235:genetics; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000378:metabolism / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D013058:Mass Spectrometry; D011485:Protein Binding / Q000235:genetics; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D037521:Virulence Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D000345:Affinity Labels; D029968:Escherichia coli Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors