Novel Host Proteins and Signaling Pathways in Enteropathogenic E. coli Pathogenesis Identified by Global Phosphoproteome Analysis *

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. 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. Diarrheagenic E. coli are a major global health burden and cause much morbidity and mortality worldwide. Enteropathogenic E. coli (EPEC) 1 is the causative agent of potentially fatal infantile diarrhea and remains an endemic health threat for children in developing countries. EPEC and the closely related enterohemorrhagic E. coli (EHEC) belong to the group of attaching and effacing (A/E) pathogens that form distinct A/E lesions on the surface of intestinal epithelial cells causing the loss of the characteristic intestinal brush border architecture (1.Croxen M.A. Law R.J. Scholz R. Keeney K.M. Wlodarska M. Finlay B.B. Recent advances in understanding enteric Escherichia to intestinal EPEC uses a the type III secretion system to at bacterial effector proteins into the host cell Finlay B.B. analysis of type III of Escherichia coli an effector repertoire for bacterial M. M.A. and analysis of Escherichia coli type III secretion into these a of host signaling subverting host cell and Enteropathogenic and Escherichia bacterial is of the first and into the host of EPEC to the surface its with the bacterial surface R. M. Finlay B.B. Enteropathogenic E. coli (EPEC) its for adherence into E. analysis of effector the type III secretion system of Escherichia In with also an cytoskeletal to the of the of bacterial effector proteins diarrheagenic Escherichia coli the host cell EPEC also cellular host immune and and to cause Enteropathogenic and Escherichia in has to the identification of key in the host and the the EPEC and the epithelial host and the are is that host such as protein phosphorylation is a key for bacterial pathogens to host cell in host cells during bacterial and are that this the for is into the host cell and this a role in the of the R. Finlay B.B. Enteropathogenic E. coli to in host a the of directly host cell E. Finlay B.B. effector to protein the phosphorylation of a few host proteins such as of and protein 1 as as alterations in phosphorylation of host proteins, are in an EPEC E. phosphorylation and its to E. E. M. in Escherichia coli (EPEC) to Enteropathogenic Escherichia coli of the of host Finlay B.B. E. coli type III effector with host and host cell M. Finlay B.B. Enteropathogenic Escherichia coli infection of the protein in epithelial Finlay B.B. of Escherichia coli to host cells novel signaling including phosphorylation of Finlay B.B. Enteropathogenic Escherichia coli are a analysis is needed to how and to EPEC host cell phosphorylation to cause disease. Recent advances in quantitative to the changes in host protein phosphorylation infection the diarrheagenic bacterial pathogens and E. of host protein phosphorylation during infection analysis of cells key and host phosphorylation Finlay B.B. of on the host our such analysis has been for a diarrheagenic bacterial such as In this we a in cell M. for in cell quantitative approach to the of EPEC infection on the host cell of time and the of an EPEC mutant in type III secretion insights into the as as the effector of these This experimental approach enabled identification of and EPEC bacterial and host infection on a identified host the biological of the of host protein phosphorylation in EPEC of septin-9 a SEPT9 was and into the and of to a was to of with and are in the cells in with and at and of cells was in and with as as and and EPEC and EPEC and the EPEC in this study Escherichia coli that cause and are Finlay B.B. of the Escherichia coli type III secretion system of the type III for and to infection, EPEC on to bacterial was at in a bacterial at for at bacterial was in and cell to a of and in a with for cells first and with and cell to infection with EPEC at a of infection of of and cell during infection to EPEC and infection cells with and cell time points cells with and on was to has been previously Finlay B.B. of on the host cells in and and for was to a of and was at for the of and cells proteins with at for and with at for in the was at a of at for with M. for and of for proteomics and and as previously M. for in proteomics M. and of with M. for and of for proteomics to analysis an with a a for in at a of to was a in and of at a of Both and the the to over directly into an and was in a the in the signal of 1 and with of and for and of for analysis on the to a and as with the in and was for the the with to in to in to in and for an and was in a approach of and events of 1 with a time of an of and an of with and M. identification and protein and including protein as as phosphorylation of and was to with of the the of for and for Both the and the of was M. a into the EPEC of with and and a of at and protein and as the to phosphorylation as and III M. in and phosphorylation in signaling was and in the of the for phosphorylation of and protein a of was In the EPEC proteins that of as and the analysis with a protein and of as into and for changes the protein and observed in a of of biological for a of regulated phosphorylation and protein during EPEC infection, phosphorylation and proteins that showed a compared with the at at time and an a of in at at that time to EPEC a determined in of the that protein was at analysis was this of regulated and for regulated and proteins was determined with at time on a of of biological and proteins as when the of EPEC and EPEC on the host phosphoproteome in at time when a and was observed of compared with was T3SS-dependent a was as T3SS-dependent for time as In a of and proteins was determined the extended of regulated and proteins This regulated and proteins with that a with an of in the of the was with the M. 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This the the mutant and the role of SEPT9 phosphorylation in EPEC with host cells. in EPEC cells. 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Finlay B.B. of cytoskeletal and signaling proteins to and enterohemorrhagic Escherichia coli phosphorylation has been to the of a of this during infection of phosphorylation of An that the the in that form and of infection M.A. Escherichia coli to M.A. of distinct host cell signaling Escherichia coli effector We observed an of to an role and to of showed phosphorylation at in a T3SS-dependent during EPEC infection. of of M. of as a major for the phosphorylation of the E. of and phosphorylation of protein This in to phosphorylation also as a phosphorylation that during EPEC infection. 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M.A. M. E. R. M. M. of SEPT9 phosphorylation was also in of and epithelial cells E. of host protein phosphorylation during infection analysis of cells key and host phosphorylation a SEPT9 phosphorylation and the of SEPT9 to the of and also of bacterial such as and these a of EPEC-mediated alterations the host We identified SEPT9 as a novel host in EPEC pathogenesis, established the biological relevance of SEPT9 and mechanistic insights into to host We for with the and and for of the is the in in this was the for the and the with attaching and effacing for and Escherichia coli Escherichia coli enterohemorrhagic Escherichia coli analysis of and of infection for in cell and for the of type III secretion type III secretion system

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