title: Novel Host Proteins and Signaling Pathways in Enteropathogenic E. coli Pathogenesis Identified by Global Phosphoproteome Analysis.
abstract: Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to directly translocate effector proteins into host cells where they play a pivotal role in subverting host cell signaling needed for disease. However, our knowledge of how EPEC affects host protein phosphorylation is limited to a few individual protein studies. We employed a quantitative proteomics approach to globally map alterations in the host phosphoproteome during EPEC infection. By characterizing host phosphorylation events at various time points throughout infection, we examined how EPEC dynamically impacts the host phosphoproteome over time. This experimental setup also enabled identification of T3SS-dependent and -independent changes in host phosphorylation. Specifically, T3SS-regulated events affected various cellular processes that are known EPEC targets, including cytoskeletal organization, immune signaling, and intracellular trafficking. However, the involvement of phosphorylation in these events has thus far been poorly studied. We confirmed the MAPK family as an established key host player, showed its central role in signal transduction during EPEC infection, and extended the repertoire of known signaling hubs with previously unrecognized proteins, including TPD52, CIN85, EPHA2, and HSP27. We identified altered phosphorylation of known EPEC targets, such as cofilin, where the involvement of phosphorylation has so far been undefined, thus providing novel mechanistic insights into the roles of these proteins in EPEC infection. An overlap of regulated proteins, especially those that are cytoskeleton-associated, was observed when compared with the phosphoproteome of Shigella-infected cells. We determined the biological relevance of the phosphorylation of a novel protein in EPEC pathogenesis, septin-9 (SEPT9). Both siRNA knockdown and a phosphorylation-impaired SEPT9 mutant decreased bacterial adherence and EPEC-mediated cell death. In contrast, a phosphorylation-mimicking SEPT9 mutant rescued these effects. Collectively, this study provides the first global analysis of phosphorylation-mediated processes during infection with an extracellular, diarrheagenic bacterial pathogen.
authors.affiliation: From the ‡Michael Smith Laboratories and.
authors.forename: Roland
authors.initials: R
authors.lastname: Scholz
authors.affiliation: §Centre for High-Throughput Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.
authors.forename: Koshi
authors.initials: K
authors.lastname: Imami
authors.affiliation: §Centre for High-Throughput Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.
authors.forename: Nichollas E
authors.initials: NE
authors.lastname: Scott
authors.affiliation: ¶Cell Biology Program, Hospital for Sick Children and ‖Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1X8, Canada.
authors.forename: William S
authors.initials: WS
authors.lastname: Trimble
authors.affiliation: §Centre for High-Throughput Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada, **Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada, and bfinlay@msl.ubc.ca foster@chibi.ubc.ca.
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Foster
authors.affiliation: From the ‡Michael Smith Laboratories and **Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada, and ¶¶Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada bfinlay@msl.ubc.ca foster@chibi.ubc.ca.
authors.forename: B Brett
authors.initials: BB
authors.lastname: Finlay
mesh_terms: D000595:Amino Acid Sequence; D058947:Bacterial Secretion Systems; D054308:Enteropathogenic Escherichia coli / Q000472:pathogenicity*; D004847:Epithelial Cells / Q000378:metabolism / Q000382:microbiology; D004927:Escherichia coli Infections / Q000378:metabolism / Q000382:microbiology; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D058112:Septins / Q000378:metabolism; D012760:Shigella / Q000378:metabolism; D015398:Signal Transduction; D014774:Virulence
chemical_list: D058947:Bacterial Secretion Systems; D010750:Phosphoproteins; D020543:Proteome; C474315:SEPTIN9 protein, human; D058112:Septins