Global Impact of Salmonella Pathogenicity Island 2-secreted Effectors on the Host Phosphoproteome

During the late stages of infection, Salmonella secretes numerous effectors through a type III secretion system that is encoded within Salmonella pathogenicity island 2 (SPI2). Despite the importance of SPI2 as a major virulence factor leading to the systemic spread of the bacteria and diseases, a global view of its effects on host responses is still lacking. Here, we measured global impacts of SPI2 effectors on the host phosphorylation and protein expression levels in RAW264.7 and in HeLa cells, as macrophage and nonphagocytic models of infection. We observe that SPI2 effectors differentially modulate the host phosphoproteome and cellular processes (e.g. protein trafficking, cytoskeletal regulation, and immune signaling) in a host cell-dependent manner. Our unbiased approach reveals the involvement of many previously unrecognized proteins, including E3 ligases (HERC4, RanBP2, and RAD18), kinases (CDK, SIK3, and WNK1), and histones (H2B1F, H4, and H15), in late stages of Salmonella infection. Furthermore, from this phosphoproteome analysis and other quantitative screens, we identified HSP27 as a direct in vitro and in vivo molecular target of the only type III secreted kinase, SteC. Using biochemical and cell biological assays, we demonstrate that SteC phosphorylates multiple sites in HSP27 and induces actin rearrangement through this protein. Together, these results provide a broader landscape of host players contributing to specific processes/pathways mediated by SPI2 effectors than was previously appreciated. During the late stages of infection, Salmonella secretes numerous effectors through a type III secretion system that is encoded within Salmonella pathogenicity island 2 (SPI2). Despite the importance of SPI2 as a major virulence factor leading to the systemic spread of the bacteria and diseases, a global view of its effects on host responses is still lacking. Here, we measured global impacts of SPI2 effectors on the host phosphorylation and protein expression levels in RAW264.7 and in HeLa cells, as macrophage and nonphagocytic models of infection. We observe that SPI2 effectors differentially modulate the host phosphoproteome and cellular processes (e.g. protein trafficking, cytoskeletal regulation, and immune signaling) in a host cell-dependent manner. Our unbiased approach reveals the involvement of many previously unrecognized proteins, including E3 ligases (HERC4, RanBP2, and RAD18), kinases (CDK, SIK3, and WNK1), and histones (H2B1F, H4, and H15), in late stages of Salmonella infection. Furthermore, from this phosphoproteome analysis and other quantitative screens, we identified HSP27 as a direct in vitro and in vivo molecular target of the only type III secreted kinase, SteC. Using biochemical and cell biological assays, we demonstrate that SteC phosphorylates multiple sites in HSP27 and induces actin rearrangement through this protein. Together, these results provide a broader landscape of host players contributing to specific processes/pathways mediated by SPI2 effectors than was previously appreciated. Type III secretion systems (T3SSs) 1The abbreviations used are:T3SStype III secretion systemSPISalmonella pathogenicity islandSCVSalmonella-containing vacuoleSILACstable isotope labeling by amino acids in cell cultureGOgene ontologyIP-MSimmunoprecipitation-mass spectrometryKEGGKyoto Encyclopedia of Genes and GenomesPTMPost-translational modification. 1The abbreviations used are:T3SStype III secretion systemSPISalmonella pathogenicity islandSCVSalmonella-containing vacuoleSILACstable isotope labeling by amino acids in cell cultureGOgene ontologyIP-MSimmunoprecipitation-mass spectrometryKEGGKyoto Encyclopedia of Genes and GenomesPTMPost-translational modification. are specialized virulence factors in Gram-negative pathogens that play an important role in delivering effector proteins to host cells. Salmonella enterica employs two distinct T3SSs encoded in Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2), with numerous effectors encoded around the genome, including a small number in SPI1 and SPI2 (1Haraga A. Ohlson M.B. Miller S.I. Salmonella interplay with host cells.Nat. Rev. Microbiol. 2008; 6: 53-66Crossref PubMed Scopus (597) Google Scholar). SPI1 T3SS effectors are required for the bacterial internalization by intestinal epithelial cells at early stages of infection after oral ingestion. Although Salmonella is subsequently taken up by intestinal macrophages via phagocytosis, SPI2 T3SS effectors function to promote intracellular replication. Part of the role of SPI2 effectors is to control the maturation of the membrane-enclosed, Salmonella-containing vacuole (SCV) where Salmonella survives and replicates, eventually leading to a systemic infection known as typhoid fever (2Abrahams G.L. Hensel M. Manipulating cellular transport and immune responses: Dynamic interactions between intracellular Salmonella enterica and its host cells.Cell. Microbiol. 2006; 8: 728-737Crossref PubMed Scopus (120) Google Scholar, 3Garai P. Marathe S.A. Chakravortty D. Effectors of Salmonella pathogenicity island 2: An island crucial to the life of Salmonella.Virulence. 2011; 2: 177-180Crossref PubMed Scopus (6) Google Scholar). type III secretion system Salmonella pathogenicity island Salmonella-containing vacuole stable isotope labeling by amino acids in cell culture gene ontology immunoprecipitation-mass spectrometry Kyoto Encyclopedia of Genes and Genomes Post-translational modification. type III secretion system Salmonella pathogenicity island Salmonella-containing vacuole stable isotope labeling by amino acids in cell culture gene ontology immunoprecipitation-mass spectrometry Kyoto Encyclopedia of Genes and Genomes Post-translational modification. Approximately 30 effectors are known to be translocated by the SPI2 T3SS but the actions and targets of most of these effectors are largely unknown (1Haraga A. Ohlson M.B. Miller S.I. Salmonella interplay with host cells.Nat. Rev. Microbiol. 2008; 6: 53-66Crossref PubMed Scopus (597) Google Scholar, 3Garai P. Marathe S.A. Chakravortty D. Effectors of Salmonella pathogenicity island 2: An island crucial to the life of Salmonella.Virulence. 2011; 2: 177-180Crossref PubMed Scopus (6) Google Scholar, 4Figueira R. Holden D.W. Functions of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system effectors.Microbiology. 2012; 158: 1147-1161Crossref PubMed Scopus (244) Google Scholar). A recent systematic study using a single mutant collection of SPI2 genes showed particular virulence factors (e.g. SpvB, SifA, and SteC) play a dominant role in replication within macrophages (5Buckner M.M. Croxen M.A. Arena E.T. Finlay B.B. A comprehensive study of the contribution of Salmonella enterica serovar typhimurium SPI2 effectors to bacterial colonization, survival, and replication in typhoid fever, macrophage, and epithelial cell infection models.Virulence. 2011; 2: 208-216Crossref PubMed Google Scholar). It is known that SpvB induces cytotoxicity through its ADP-ribosyltransferase activity (6Lesnick M.L. Reiner N.E. Fierer J. Guiney D.G. The Salmonella spvB virulence gene encodes an enzyme that ADP-ribosylates actin and destabilizes the cytoskeleton of eukaryotic cells.Mol. Microbiol. 2001; 39: 1464-1470Crossref PubMed Scopus (167) Google Scholar), and SifA is required for maturation of the SCV and the formation of Salmonella-induced filaments (7Beuzón C.R. Méresse S. Unsworth K.E. Ruíz-Albert J. Garvis S. Waterman S.R. Ryder T.A. Boucrot E. Holden D.W. Salmonella maintains the integrity of its intracellular vacuole through the action of SifA.EMBO J. 2000; 19: 3235-3249Crossref PubMed Scopus (458) Google Scholar). SteC has been identified as the sole serine/threonine protein encoded in the Salmonella J. A. M. P. Holden D.W. SteC is a Salmonella required for Microbiol. 2008; Google Scholar), but the target of this within the host are has been that SteC targets the A. J. M. Holden D.W. The Salmonella SteC targets the to the host actin 2012; PubMed Scopus Google Scholar). SteC is of of an around the this is on its activity but of through protein J. A. M. P. Holden D.W. SteC is a Salmonella required for Microbiol. 2008; Google Scholar), and K.E. M. M. Holden D.W. of the of Salmonella-induced actin of host cells and intracellular Microbiol. 6: PubMed Scopus Google Scholar). host proteins are the targets of effectors from many including the SPI1 system in Salmonella of the host actin cytoskeleton by in the of Microbiol. 8: PubMed Scopus Google and E. A. induces actin and formation epithelial J. PubMed Scopus Google Scholar). SteC is to actin in a through phosphorylation of host protein in to host gene expression S. M. Salmonella typhimurium type III secretion effectors immune responses in epithelial PubMed Scopus Google and host S. analysis of cells and host phosphorylation 2011; PubMed Scopus Google on the of SPI1 effectors in an comprehensive manner. is that SPI2 T3SS is a major virulence factor contributing to systemic infection, of its effects on host responses is this we used a spectrometry quantitative approach and measured global host phosphorylation as as by SPI2 Furthermore, we a molecular target of SPI2 effector SteC by the and other quantitative The was from Salmonella typhimurium to and sites for The was using the as in J. Finlay B.B. spectrometry Salmonella pathogenicity effectors and host 2011; PubMed Scopus Google and of as in J. Finlay B.B. spectrometry Salmonella pathogenicity effectors and host 2011; PubMed Scopus Google expression of in the from the was a expression using and RAW264.7 and HeLa cells Type and in and in the of and and Salmonella infection was at a of infection of for as previously (5Buckner M.M. Croxen M.A. Arena E.T. Finlay B.B. A comprehensive study of the contribution of Salmonella enterica serovar typhimurium SPI2 effectors to bacterial colonization, survival, and replication in typhoid fever, macrophage, and epithelial cell infection models.Virulence. 2011; 2: 208-216Crossref PubMed Google Scholar). the infection, cells with on and at the replication of the cells from a culture was and bacteria from host cells using and in bacteria in a and the number of was using cell to of HeLa cells in at a of in a and the with after HeLa cells with the cells and analysis as previously J. Finlay B.B. spectrometry Salmonella pathogenicity effectors and host 2011; PubMed Scopus Google that with was used for of and cells and cell was as An global for cell and analysis of 6: PubMed Scopus Google Scholar). the cells with and and was for protein with and with was at phosphoproteome using by by and M. and of by 2008; PubMed Scopus Google Scholar). to by J. M. for and of for using 2: PubMed Scopus Google Scholar). using J. M. for and of for using 2: PubMed Scopus Google to analysis was using an with an system as previously S. analysis of cells and host phosphorylation 2011; PubMed Scopus Google Scholar). An was with for analysis are as by using in the using 30 analysis using P. J. J. M. an for quantitative PubMed Scopus Google Scholar), by and SteC protein protein using two and including protein as as phosphorylation of and The was and the was and only with from the quantitative for of of SteC SteC using other and by J. M. and protein 2008; PubMed Scopus Google using the The was and the was was by of J. A. M. A the 2011; PubMed Scopus Google S. typhimurium protein S. typhimurium protein with and enzyme was to at and at protein as and III on P. M. in and phosphorylation in 2006; PubMed Scopus Google Scholar). protein and be in the analysis was with the and analysis of gene using PubMed Scopus Google Scholar). with D. An approach to the of protein phosphorylation from PubMed Scopus Google Scholar). analysis was using a A. A. PubMed Scopus Google Scholar). the in vitro of was with 1 of protein 1 of HSP27 in of a 1 and 1 that and of used for was for 30 at for and for at for the protein was to and to an spectrometry the protein was with as and the identified and on Using S. typhimurium as a SteC was by with that an a and at the and an at the The was and to the was in cells Type using HeLa cells with these and in culture for 2 The stable cell was as cells at in a a with for by the and HSP27 A and control used at of 1 was by using using of 30 and of in to a of 30 and with culture for 30 at cells on with up in with and for at in and and in for was on for 30 at at a of in the with and a was on for 30 at at a of in with the using on a replication of the SPI2 mutant Finlay B.B. SifA and replication of Salmonella typhimurium in Microbiol. 2001; PubMed Scopus Google was to in macrophages than with the (5Buckner M.M. Croxen M.A. Arena E.T. Finlay B.B. A comprehensive study of the contribution of Salmonella enterica serovar typhimurium SPI2 effectors to bacterial colonization, survival, and replication in typhoid fever, macrophage, and epithelial cell infection models.Virulence. 2011; 2: 208-216Crossref PubMed Google Scholar). a SPI2 system is important epithelial cells and (5Buckner M.M. Croxen M.A. Arena E.T. Finlay B.B. A comprehensive study of the contribution of Salmonella enterica serovar typhimurium SPI2 effectors to bacterial colonization, survival, and replication in typhoid fever, macrophage, and epithelial cell infection models.Virulence. 2011; 2: 208-216Crossref PubMed Google Scholar). Here, we used stable isotope labeling of amino acids in cell culture A. M. isotope labeling by amino acids in cell as a and approach to expression PubMed Scopus Google to spectrometry and A. M. by for in 6: PubMed Scopus Google to the of SPI2 T3SS on protein expression and levels in two cell models of Salmonella infection and Salmonella to the SPI2 T3SS and effectors within a after internalization macrophages J. S. Hensel M. of Salmonella pathogenicity island 2 gene Microbiol. PubMed Scopus Google Scholar, Méresse S. Salmonella-induced Microbiol. 2011; 19: PubMed Scopus Google Scholar), the bacteria to to after (5Buckner M.M. Croxen M.A. Arena E.T. Finlay B.B. A comprehensive study of the contribution of Salmonella enterica serovar typhimurium SPI2 effectors to bacterial colonization, survival, and replication in typhoid fever, macrophage, and epithelial cell infection models.Virulence. 2011; 2: 208-216Crossref PubMed Google Scholar). We to study this system are in intracellular bacteria between and at this (5Buckner M.M. Croxen M.A. Arena E.T. Finlay B.B. A comprehensive study of the contribution of Salmonella enterica serovar typhimurium SPI2 effectors to bacterial colonization, survival, and replication in typhoid fever, macrophage, and epithelial cell infection models.Virulence. 2011; 2: 208-216Crossref PubMed Google Scholar). from proteins in RAW264.7 cells biological and proteins in HeLa cells at the and at the protein only in at two biological used and and of the between for of these sites was than A SPI2 system infection a on the phosphoproteome of macrophages than that of epithelial cells to the of proteins most in the two we only phosphorylation that a of at up to the with in and and the proteins, of this in RAW264.7 and in HeLa cells of but a between the two the proteins in cell type distinct the that to most are as players in multiple processes (e.g. and of Salmonella SPI2 effectors on protein a of with and protein protein effector protein effector protein E3 protein only protein and protein factor factor in a an infection, in the phosphoproteome be to in the of a protein that is but to in protein expression R. of comprehensive phosphorylation by protein expression 2011; PubMed Scopus Google we measured the at after on in protein expression and The in expression as a of a SPI2 system the in cell type using the for a expression was only of the in cell type was proteins in RAW264.7 and proteins in the that in the levels of specific proteins are the for the in phosphorylation be as is between phosphoproteome and in The of the proteins in RAW264.7 and HeLa cells The of the SCV proteins after of infection, including proteins, and and D. The only to this was for an early SCV was in cells these results demonstrate that SPI2 effectors the two cell by Salmonella macrophages to be than epithelial cells, with a role for SPI2 T3SS as a for cells P. Marathe S.A. Chakravortty D. Effectors of Salmonella pathogenicity island 2: An island crucial to the life of Salmonella.Virulence. 2011; 2: 177-180Crossref PubMed Scopus (6) Google Scholar, 4Figueira R. Holden D.W. Functions of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system effectors.Microbiology. 2012; 158: 1147-1161Crossref PubMed Scopus (244) Google Scholar, M.M. Croxen M.A. Arena E.T. Finlay B.B. A comprehensive study of the contribution of Salmonella enterica serovar typhimurium SPI2 effectors to bacterial colonization, survival, and replication in typhoid fever, macrophage, and epithelial cell infection models.Virulence. 2011; 2: 208-216Crossref PubMed Google Scholar). an unbiased of the processes by the SPI2 system in epithelial cells and we cellular and the from cell as by and Kyoto Encyclopedia of Genes and Genomes and analysis of gene using PubMed Scopus Google Scholar). An of and with the host proteins phosphorylation is in of proteins in of and most by SPI2 in RAW264.7 and results with where the of SPI2 T3SS are to promote intracellular replication macrophages by host trafficking, and immune P. Marathe S.A. Chakravortty D. Effectors of Salmonella pathogenicity island 2: An island crucial to the life of Salmonella.Virulence. 2011; 2: 177-180Crossref PubMed Scopus (6) Google Scholar, 4Figueira R. Holden D.W. Functions of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system effectors.Microbiology. 2012; 158: 1147-1161Crossref PubMed Scopus (244) Google Scholar). Furthermore, these that these cellular processes/pathways in RAW264.7 cells are by SPI2 effectors at the phosphoproteome and of gene most in HeLa cells in with that Salmonella cell in epithelial cells D. A. M. of Salmonella-induced macrophage with expression of PubMed Google but using SPI2 effectors late of infection Guiney D.G. Genes in the Salmonella pathogenicity island 2 and the Salmonella virulence are for Salmonella-induced in intestinal epithelial cells.Cell. Microbiol. PubMed Scopus Google Scholar, of intestinal epithelial cells after bacterial PubMed Scopus Google Scholar). of the Salmonella infection is maturation of the SCV (7Beuzón C.R. Méresse S. Unsworth K.E. Ruíz-Albert J. Garvis S. Waterman S.R. Ryder T.A. Boucrot E. Holden D.W. Salmonella maintains the integrity of its intracellular vacuole through the action of SifA.EMBO J. 2000; 19: 3235-3249Crossref PubMed Scopus (458) Google Scholar), with the of host M.A. A Salmonella virulence protein that cellular J. PubMed Scopus Google Scholar). RAW264.7 proteins that play in these to be for phosphorylation by the SPI2 including protein protein and protein phosphorylation (e.g. and M. A. interactions of the proteins and and by PubMed Scopus Google the of phosphorylation at in RAW264.7 cells that SPI2 effectors control between and SCV through of the SPI2 effector SteC and other SPI2 effectors SpvB, and are to the interactions between host actin and the SCV D. Salmonella of the Microbiol. PubMed Scopus Google Scholar). 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The activity of a bacterial type III effector PubMed Scopus Google to subsequently Salmonella type III effector a to in intestinal of Microbiol. 2012; PubMed Scopus Google Scholar). protein kinases with but pathogens to T3SSs to virulence factors to of through phosphorylation of the host protein A. R. S. of a molecular target for the protein PubMed Scopus Google Scholar). but with a Salmonella SPI2 effector SteC is known to actin using its activity J. A. M. P. Holden D.W. SteC is a Salmonella required for Microbiol. 2008; Google Scholar). unbiased but of this in this for of actin and and of proteins to by SPI2 effectors host proteins be direct targets of we in a than that used and we to host of SteC by an immunoprecipitation-mass spectrometry approach as we previously for most effectors J. Finlay B.B. spectrometry Salmonella pathogenicity effectors and host 2011; PubMed Scopus Google Scholar, Finlay B.B. of host targets and specific sites on the Salmonella effector 2008; PubMed Scopus Google Scholar). HeLa with SteC and control HeLa and by after with the we identified proteins in in including and phosphorylation to be than SteC that of these proteins showed phosphorylation in the infection as and phosphorylation of of HSP27 in particular was in cells with Salmonella the we that Salmonella phosphorylation of Furthermore, the identified HSP27 as a of SteC the other proteins for SteC of to host HSP27 as a direct target of SteC. 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The Salmonella SteC targets the to the host actin 2012; PubMed Scopus Google that SteC targets the for actin cytoskeleton in cells. the of through HSP27 is of the we actin of the HeLa in the of a the we the cells for 30 with 30 was used in A. J. M. Holden D.W. The Salmonella SteC targets the to the host actin 2012; PubMed Scopus Google Scholar, of a of protein PubMed Scopus Google Scholar), is a and than but the actin A and and we observe the of the of the HSP27 with only HSP27 A and and that the of through HSP27 is of the and that SteC target at in the HeLa cells. results that HSP27 is of the molecular targets of SteC and that is of actin contributing to the formation of an in host cells. Here, we provide the analysis of the of SPI2 effectors on the host phosphoproteome and using two and nonphagocytic host models of Salmonella infection. Although the of the molecular interactions between SPI2 effectors and host targets has to be and be the of in this study we a landscape of host players and phosphorylation sites particular cellular processes/pathways by SPI2 effectors 2 and from study was the in phosphoproteome and cellular processes by SPI2 in RAW264.7 and HeLa cells 2 and SPI2 immune and protein transport in RAW264.7 cells but in HeLa cells. It is to that SPI2 a type of cell to macrophages epithelial cells, and this is an is that SPI2 a of effectors cell but the targets of a effector be in that specific be a where a SPI2 effector has targets in distinct host cell between type and the SPI2 single mutant (5Buckner M.M. Croxen M.A. Arena E.T. Finlay B.B. A comprehensive study of the contribution of Salmonella enterica serovar typhimurium SPI2 effectors to bacterial colonization, survival, and replication in typhoid fever, macrophage, and epithelial cell infection models.Virulence. 2011; 2: 208-216Crossref PubMed Google in these of global to between these the in the phosphoproteome mediated by the SPI2 T3SS as as we with SPI1 effectors at infection S. analysis of cells and host phosphorylation 2011; PubMed Scopus Google Scholar). are for this as the in the we to from the culture that was in this study to the the of the levels of phosphorylation phosphorylation within and SPI2 effectors to phosphorylation to effects to the that SPI1 effectors (1Haraga A. Ohlson M.B. Miller S.I. Salmonella interplay with host cells.Nat. Rev. Microbiol. 2008; 6: 53-66Crossref PubMed Scopus (597) Google Scholar). is by the that SPI2 effectors to be required for intracellular processes to a within host cells. The infection used than of for the cells to the effects of the infection the of cells. is to in phosphorylation a after in the only we are of in Salmonella infection models specific proteins, as S. M. infection in 6: PubMed Scopus Google of the D. A. A. by bacterial pathogens an host 2012; PubMed Scopus Google Scholar). are still a and the approach used to the in phosphorylation by SPI2 T3SS in an comprehensive manner. are kinases in and an number of are virulence The contribution of the has been the that a secreted bacterial host actin through the direct phosphorylation of a single host protein. this at the is still an in the HSP27 that phosphorylation of HSP27 induces its from actin in of from the actin J. by J. PubMed Scopus Google Scholar, E. D. A. M. protein is a of protein in actin by HSP27 2001; PubMed Scopus Google Scholar). of this be that of HSP27 actin We in vitro that SteC is of at sites in HSP27 and of kinases in only sites and D. D.G. P. M. of 2 as a major enzyme for the phosphorylation of the small PubMed Scopus Google Scholar). system from that used by the host to the phosphorylation of HSP27 to actin J. by J. PubMed Scopus Google Scholar). two showed that SteC targets in P. A. J. M. The Salmonella typhimurium effector SteC through to the factor in 2012; PubMed Google and in cells A. J. M. Holden D.W. The Salmonella SteC targets the to the host actin 2012; PubMed Scopus Google Scholar). We identified as a of SteC by the but in vitro in vivo phosphorylation of by SteC. SteC only be to using of the two proteins, that the of phosphorylation in vivo be Despite the SteC phosphorylation on to play a role in actin the of only by D. D.G. P. M. of 2 as a major enzyme for the phosphorylation of the small PubMed Scopus Google Scholar). that is to to actin in HeLa cells A for this is that be host targets of SteC in a cell is by where we the in phosphoproteome by SPI2 in the distinct cell as in the of these recent that HSP27 phosphorylation is a to host actin is cell be to the of the of actin through protein The provide the between SteC and actin effects be of the known to actin through and are targets of pathogens K.E. M. M. Holden D.W. of the of Salmonella-induced actin of host cells and intracellular Microbiol. 6: PubMed Scopus Google Scholar). the phosphorylation of HSP27 a for of the actin We and for and for with the in vitro and for with

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