title: Mass Spectrometry-Based Proteomics Identification of Enteropathogenic Escherichia coli Pedestal Constituents.
abstract: Enteropathogenic Escherichia coli (EPEC) co-opt host signaling pathways and recruit numerous host proteins to motile morphological structures, called pedestals, at sites of bacterial attachment. These pedestals are hallmarks of EPEC-based disease, and the identification and characterization of the functions of pedestal proteins continue to steadily increase. To identify additional constituents in an unbiased manner, we developed a strategy where EPEC pedestals were elongated artificially, severed, and then concentrated prior to their analysis by mass spectrometry (MS)-based proteomics. We identified >90 unique mammalian proteins over multiple experimental trials from our preparations. Seventeen predicted molecules were significantly higher in abundance (p < 0.05) when compared to both the negative controls and sample means. Validation of two identified proteins (cyclophilin A [nonactin-associated] and transgelin [actin-associated]) by immunolocalization was used to confirm our analysis, and both showed enrichment at EPEC pedestals. The EPEC pedestal concentration technique developed here together with the identification of novel pedestal proteins not only provides a resource for the further characterization of molecular components within these structures but also demonstrates that EPEC pedestals can be used as a model system for the identification of novel functions of proteins not normally thought to be at actin-based structures.
authors.forename: Hong T
authors.initials: HT
authors.lastname: Law
authors.forename: Michael
authors.initials: M
authors.lastname: Chua
authors.forename: Kyung-Mee
authors.initials: KM
authors.lastname: Moon
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Foster
authors.forename: Julian A
authors.initials: JA
authors.lastname: Guttman
mesh_terms: D054308:Enteropathogenic Escherichia coli / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D040901:Proteomics
keywords: BDM; Transgelin; cyclophilin A; epithelial cells; stable isotope labeling