title: Quantitative Mass Spectrometry Identifies Novel Host Binding Partners for Pathogenic Escherichia coli Type III Secretion System Effectors.
abstract: Enteropathogenic and enterohemorrhagic Escherichia coli cause enteric diseases resulting in significant morbidity and mortality worldwide. These pathogens remain extracellular and translocate a set of type III secreted effector proteins into host cells to promote bacterial virulence. Effectors manipulate host cell pathways to facilitate infection by interacting with a variety of host targets, yet the binding partners and mechanism of action of many effectors remain elusive. We performed a mass spectrometry screen to identify host targets for a library of effectors. We found five known effector targets and discovered four novel interactions. Interestingly, we identified multiple effectors that interacted with the microtubule associated protein, ensconsin. Using co-immunoprecipitations, we confirmed that NleB1 and EspL interacted with ensconsin in a region that corresponded to its microtubule binding domain. Ensconsin is an essential cofactor of kinesin-1 that is required for intracellular trafficking, and we demonstrated that intracellular trafficking was severely disrupted during wild type EPEC infections but not during infections with ΔnleB1 or ΔespL mutants. Our findings demonstrate the efficacy of quantitative proteomics for identifying effector-host protein interactions and suggest that vesicular trafficking is a crucial cellular process that may be targeted by NleB1 and EspL through their interaction with ensconsin.
authors.forename: Robyn J
authors.initials: RJ
authors.lastname: Law
authors.affiliation: Department of Biological Sciences, Simon Fraser University , Burnaby, British Columbia, Canada V5A 1S6.
authors.forename: Hong T
authors.initials: HT
authors.lastname: Law
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Scurll
authors.forename: Roland
authors.initials: R
authors.lastname: Scholz
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Santos
authors.forename: Stephanie R
authors.initials: SR
authors.lastname: Shames
authors.forename: Wanyin
authors.initials: W
authors.lastname: Deng
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Croxen
authors.forename: Yuling
authors.initials: Y
authors.lastname: Li
authors.forename: Carmen L
authors.initials: CL
authors.lastname: de Hoog
authors.forename: Joris
authors.initials: J
authors.lastname: van der Heijden
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Foster
authors.affiliation: Department of Biological Sciences, Simon Fraser University , Burnaby, British Columbia, Canada V5A 1S6.
authors.forename: Julian A
authors.initials: JA
authors.lastname: Guttman
authors.forename: B Brett
authors.initials: BB
authors.lastname: Finlay
mesh_terms: D002460:Cell Line; D004926:Escherichia coli / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D011485:Protein Binding; D000069296:Type III Secretion Systems / Q000737:chemistry / Q000378:metabolism*; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C000624428:EspL protein, E coli; D008869:Microtubule-Associated Proteins; C553814:NleB protein, E coli; D000069296:Type III Secretion Systems; D037521:Virulence Factors
keywords: MAP7; SILAC; effector proteins; ensconsin; enterohemorrhagic E. coli; enteropathogenic E. coli; quantitative mass spectrometry; type III secretion system