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.
Highlights
From the ‡Michael Smith Laboratories and §Centre for HighThroughput Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada, ¶Cell Biology Program, Hospital for Sick Children and ʈDepartment of Biochemistry, University of Toronto, Toronto, Ontario M5G 1X8, Canada, **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
To determine an appropriate late stage time point when the first cytotoxic effects occur, cytotoxicity-associated release of lactate dehydrogenase (LDH) from human epithelial (HeLa) cells was assessed at 0.5 h, 1.5 h, and 3.0 h postinoculation with the wild-type Enteropathogenic E. coli (EPEC) O127:H6 strain E2348/69 (EPEC WT) and the T3SS-deficient strain EPEC ⌬escN
The intermediate stage of EPEC infection was defined as 1.5 hpi, a time point at which no significant LDH release was observed but when the majority of effectors are already translocated into the host cytosol (Fig. 1A) [42]
Summary
A/E, attaching and effacing; cfu, colony-forming unit; DAVID, Database for Annotation Visualization and Integrated Discovery; DMEM, Dulbecco’s modified Eagle medium; E. coli, Escherichia coli; EPEC, enteropathogenic Escherichia coli; FTMS, Fourier transform mass spectrometry; EHEC, enterohemorrhagic Escherichia coli; GOTERM_BP_FAT, Gene Ontology Biological Process Functional Annotation Tool; GOTERM_CC_FAT, Gene Ontology Cellular Component Functional Annotation Tool; hpi, hours postinoculation; HPLC, high-performance liquid chromatography; ITMS, ion trap mass spectrometer; KEA, kinase enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; LC-MS/MS, liquid-chromatography tandem mass spectrometry; LDH, lactate dehydrogenase; MOI, multiplicity of infection; MS, mass spectrometry; NCBI, National Center for Biotechnology Information; S. typhimurium, Salmonella enterica serovar typhimurium; S. flexneri, Shigella flexneri; SILAC, stable isotope labeling by amino acids in cell culture; SP_PIR, Swiss-Prot and Protein Information Resource; STRING, Search Tool for the Retrieval of Interacting Genes/Proteins; T3S, type III secretion; T3SS, type III secretion system; WT, wild-type. Tir is phosphorylated upon insertion into the host cell membrane and this event plays a role in the rearrangement of the actin cytoskeleton [10] Another EPEC-encoded effector, NleH, contains a functional kinase domain suggesting the potential of directly phosphorylating host cell targets [11]. The integration of time course experiments and the use of an EPEC mutant deficient in type III secretion (T3S) provided further insights into the dynamics as well as the effector dependence of these processes This experimental approach enabled identification of both stable and transient interactions between EPEC bacterial effectors and host proteins. Additional infection studies focusing on a newly identified host target, septin-9, further emphasizes the biological significance of the manipulation of host protein phosphorylation in EPEC pathogenesis
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