title: GELFrEE fractionation combined with mass spectrometry for proteome analysis of secreted toxins from Enteropathogenic Escherichia coli (EPEC).
abstract: Enteropathogenic Escherichia coli, or EPEC, is a human pathogen associated with gastroenteritis and diarrheal disease whose pathogenicity is related to the secretion of effector proteins (exotoxins). Determining exotoxin expression level is of considerable interest to those studying toxin function and pathological phenotypes. Mass spectrometry (MS) provides an ideal platform for detection and quantification of proteins from complex mixtures. Here, we apply a solution-phase electrophoretic platform (GELFrEE) followed by MS to characterize the secreted proteome of a wild type and mutant strain of EPEC (ΔsepD), exhibiting enhanced secretion of effector proteins. Through peptide-level analysis, a total of 363 and 155 proteins were identified from the wild type and mutant strains, respectively. Semi-quantitative analysis of the MS data reveals the effector proteins EspB, EspC, and EspD appear in a relatively greater abundance from wild type EPEC, while two major virulence factors in EPEC, Tir and NleA appear in greater abundance from the secreted proteome of the mutant strain. Additionally, intact proteins may further be characterized following GELFrEE with MS to improve throughput of analysis. This study demonstrates the application of GELFrEE-MS to differentiate wild type and mutant strains of EPEC. This platform is therefore a powerful tool to study exotoxin secretion from pathogenic bacteria.
authors.affiliation: Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Orton
authors.forename: Dan J
authors.initials: DJ
authors.lastname: Arsenault
authors.forename: Nikhil A
authors.initials: NA
authors.lastname: Thomas
authors.forename: Alan A
authors.initials: AA
authors.lastname: Doucette
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000302:isolation & purification; D001427:Bacterial Toxins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D054308:Enteropathogenic Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D020543:Proteome / Q000032:analysis*; D011956:Receptors, Cell Surface / Q000032:analysis / Q000302:isolation & purification; D037521:Virulence Factors / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001427:Bacterial Toxins; C087863:EaeB protein, E coli; D029968:Escherichia coli Proteins; C102541:EspC protein, E coli; C474481:EspD protein, E coli; C488216:NleA protein, E coli; D020543:Proteome; D011956:Receptors, Cell Surface; C109287:Tir protein, E coli; D037521:Virulence Factors
keywords: Enteropathogenic Escherichia coli; GELFrEE; Mass spectrometry; Proteomics; Secreted proteins; Toxins