Rapid Remodeling of the Host Epithelial Cell Proteome by the Listeriolysin O (LLO) Pore-forming Toxin

Bacterial pathogens use various strategies to interfere with host cell functions. Among these strategies, bacteria modulate host gene transcription, thereby modifying the set of proteins synthetized by the infected cell. Bacteria can also target pre-existing host proteins and modulate their post-translational modifications or trigger their degradation. Analysis of protein levels variations in host cells during infection allows to integrate both transcriptional and post-transcriptional regulations induced by pathogens. Here, we focused on host proteome alterations induced by the toxin Listeriolysin O (LLO), secreted by the bacterial pathogen Listeria monocytogenes. We showed that a short-term treatment with LLO remodels the host cell proteome by specifically decreasing the abundance of 149 proteins. The same decrease in host protein levels was observed in different epithelial cell lines but not in macrophages. We show in particular that this proteome remodeling affects several ubiquitin and ubiquitin-like ligases and that LLO leads to major changes in the host ubiquitylome. Strikingly, this toxin-induced proteome remodeling involves only post-transcriptional regulations, as no modification in the transcription levels of the corresponding genes was observed. In addition, we could show that Perfringolysin O, another bacterial pore-forming toxin similar to LLO, also induces host proteome changes. Taken together, our data reveal that different bacterial pore-forming toxins induce important host proteome remodeling, that may impair epithelial cell functions. Bacterial pathogens use various strategies to interfere with host cell functions. Among these strategies, bacteria modulate host gene transcription, thereby modifying the set of proteins synthetized by the infected cell. Bacteria can also target pre-existing host proteins and modulate their post-translational modifications or trigger their degradation. Analysis of protein levels variations in host cells during infection allows to integrate both transcriptional and post-transcriptional regulations induced by pathogens. Here, we focused on host proteome alterations induced by the toxin Listeriolysin O (LLO), secreted by the bacterial pathogen Listeria monocytogenes. We showed that a short-term treatment with LLO remodels the host cell proteome by specifically decreasing the abundance of 149 proteins. The same decrease in host protein levels was observed in different epithelial cell lines but not in macrophages. We show in particular that this proteome remodeling affects several ubiquitin and ubiquitin-like ligases and that LLO leads to major changes in the host ubiquitylome. Strikingly, this toxin-induced proteome remodeling involves only post-transcriptional regulations, as no modification in the transcription levels of the corresponding genes was observed. In addition, we could show that Perfringolysin O, another bacterial pore-forming toxin similar to LLO, also induces host proteome changes. Taken together, our data reveal that different bacterial pore-forming toxins induce important host proteome remodeling, that may impair epithelial cell functions. Bacterial pathogens have developed many strategies to exploit host functions for survival, replication and escape from immune responses. A first strategy consists in interfering with host cell protein activities (1Bhavsar A.P. Guttman J.A. Finlay B.B. Manipulation of host-cell pathways by bacterial pathogens.Nature. 2007; 449: 827-834Crossref PubMed Scopus (407) Google Scholar, 2Ribet D. Cossart P. Pathogen-mediated posttranslational modifications: A re-emerging field.Cell. 2010; 143: 694-702Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 3Ashida H. Sasakawa C. Bacterial E3 ligase effectors exploit host ubiquitin systems.Curr. Opin. Microbiol. 2016; 35: 16-22Crossref PubMed Scopus (33) Google Scholar). Another strategy is to remodel host cell composition, for example by modifying the abundance of specific proteins. This remodeling of host cell proteome may result from deregulation of gene transcription, which involves the targeting of transcription factors or chromatin remodelers (4Bierne H. Hamon M. Cossart P. Epigenetics and bacterial infections.Cold Spring Harb. Perspect. Med. 2012; 2: a010272Crossref PubMed Scopus (236) Google Scholar), or from protein degradation. Protein degradation can be achieved by targeting cellular factors to host degrading machineries such as the proteasome. Conversely, to respond to infection and to trigger anti-bacterial responses, host cells use similar processes, i.e. modulation of the activity of pre-existing components or remodeling of cell proteome. Characterization of the variations in host cell protein abundance in response to infection is thus critical to understand host-pathogen interactions (5Jean Beltran P.M. Federspiel J.D. Sheng X. Cristea I.M. Proteomics and integrative omic approaches for understanding host-pathogen interactions and infectious diseases.Mol. Syst. Biol. 2017; 13: 922Crossref PubMed Scopus (120) Google Scholar). Transcriptional profiling has been extensively used to study host cell responses to infections. mRNA concentrations are in this case used as proxies to evaluate the concentration of the corresponding proteins. In this context, it is assumed that transcript abundance correlates with protein abundance. However, it is now clear that protein abundance is strongly dependent on post-transcriptional mechanisms, which include stability of the RNA, its export rate to the cytosol, its translation efficiency by ribosomes, as well as the stability of the corresponding protein once synthetized (6Vogel C. Marcotte E.M. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.Nat. Rev. Genet. 2012; 13: 227-232Crossref PubMed Scopus (2499) Google Scholar). Proteomics approaches focusing on the direct quantification of proteins rather than RNA, are, in comparison, more informative as they integrate all these parameters (5Jean Beltran P.M. Federspiel J.D. Sheng X. Cristea I.M. Proteomics and integrative omic approaches for understanding host-pathogen interactions and infectious diseases.Mol. Syst. Biol. 2017; 13: 922Crossref PubMed Scopus (120) Google Scholar). Here, we monitored host proteome changes induced by the toxin Listeriolysin O (LLO) 1The abbreviations used are:LLOlisteriolysin OPFOperfringolysin OSILACstable isotope labeling by amino acids in cell cultureCHXcycloheximideUBC9SUMO-conjugating enzyme UBC9UBE2KUbiquitin-conjugating enzyme E2 KUBE2NUbiquitin-conjugating enzyme E2 N. 1The abbreviations used are:LLOlisteriolysin OPFOperfringolysin OSILACstable isotope labeling by amino acids in cell cultureCHXcycloheximideUBC9SUMO-conjugating enzyme UBC9UBE2KUbiquitin-conjugating enzyme E2 KUBE2NUbiquitin-conjugating enzyme E2 N. secreted by the bacterial pathogen Listeria monocytogenes. Listeria is a Gram-positive bacterium responsible for the foodborne disease listeriosis, a leading cause of death because of food-transmitted bacterial pathogens. Although most of human infections occur by ingestion of contaminated food, some unusual cases of nosocomial infections have been reported. Listeria is a facultative intracellular pathogen that can infect both phagocytic and nonphagocytic cells, such as epithelial cells. In contrast to the numerous reports of global transcriptional changes induced by Listeria in host cells during infection (7Hamon M.A. Batsche E. Regnault B. Tham T.N. Seveau S. Muchardt C. Cossart P. Histone modifications induced by a family of bacterial toxins.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 13467-13472Crossref PubMed Scopus (216) Google Scholar, 8Kutsch S. Degrandi D. Pfeffer K. Immediate lymphotoxin beta receptor-mediated transcriptional response in host defense against.L. monocytogenes. Immunobiology. 2008; 213: 353-366Crossref PubMed Scopus (16) Google Scholar, 9Lebreton A. Lakisic G. Job V. Fritsch L. Tham T.N. Camejo A. Mattei P.J. Regnault B. Nahori M.A. Cabanes D. Gautreau A. Ait-Si-Ali S. Dessen A. Cossart P. Bierne H. A bacterial protein targets the BAHD1 chromatin complex to stimulate type III interferon response.Science. 2011; 331: 1319-1321Crossref PubMed Scopus (141) Google Scholar, 10Wang C. Chou C.H. Tseng C. Ge X. Pinchuk L.M. Early gene response of human brain microvascular endothelial cells to Listeria monocytogenes infection.Can. J. Microbiol. 2011; 57: 441-446Crossref PubMed Scopus (4) Google Scholar, 11Archambaud C. Nahori M.A. Soubigou G. Becavin C. Laval L. Lechat P. Smokvina T. Langella P. Lecuit M. Cossart P. Impact of lactobacilli on orally acquired listeriosis.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 16684-16689Crossref PubMed Scopus (97) Google Scholar, 12Eskandarian H.A. Impens Nahori M.A. Soubigou G. Cossart P. Hamon M.A. A for in bacterial PubMed Scopus Google Scholar, S. K. L. A. E. V. J. D. A. Analysis of transcriptional in response to Listeria monocytogenes infection changes that result from type interferon 2016; PubMed Scopus Google Scholar, D. V. Nahori M.A. H. The H. Cossart P. protein Listeria monocytogenes 2017; Scopus Google Scholar), only post-transcriptional alterations of host protein abundance D. Hamon M. E. Nahori M.A. Impens H. K. J. A. Cossart P. Listeria monocytogenes for 2010; PubMed Scopus Google Scholar, A. Cossart P. for in Listeria monocytogenes 2012; PubMed Scopus Google Scholar, C. Cossart P. bacterial Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, A. Nahori M.A. V. L. Hamon M.A. Cossart P. Listeria monocytogenes the PubMed Scopus Google Scholar, B. S. Ge proteomic profiling for of the of in 2017; PubMed Scopus Google Scholar). focused on specific host proteins or and not global proteome alterations induced by the the decrease of some of these host such as or is by the pore-forming toxin LLO and was to be for Listeria infection D. Hamon M. E. Nahori M.A. Impens H. K. J. A. Cossart P. Listeria monocytogenes for 2010; PubMed Scopus Google Scholar, A. Nahori M.A. V. L. Hamon M.A. Cossart P. Listeria monocytogenes the PubMed Scopus Google Scholar). O O isotope labeling by amino acids in cell enzyme enzyme E2 enzyme E2 N. O O isotope labeling by amino acids in cell enzyme enzyme E2 enzyme E2 N. a of the LLO toxin may the host cell we to use a of transcriptomic and approaches to the and the of host proteins in cells to the We a decrease in the levels of 149 host proteins in response to a treatment with Strikingly, no in the transcription of the corresponding genes was that LLO induces remodeling of the host proteome post-transcriptional We several components of the host ubiquitin as by we observed a of the host in response to We show that the alterations of protein levels in epithelial cells not observed in but by another pore-forming toxin secreted by the bacterial pathogen and cells in a in for and cells with and for was with and isotope labeling by amino acids in cell B. H. A. M. isotope labeling by amino acids in cell as a and to Full Text Full Text PDF PubMed Scopus Google Scholar, M. of in isotope labeling by amino acids in cell 2: PubMed Scopus Google Scholar), cells in or and with and and or and was its concentration in but the concentration of was to of the concentration in to of to for to of the and Listeria in brain or used in this study L. monocytogenes and the corresponding Cossart P. D. of epithelial cell induced by bacterial Microbiol. 2017; PubMed Scopus Google Scholar). in cells a of cells the Bacteria in of and in cells for bacteria to cells of infection of and on cells for of cells and or for with with and to and LLO and toxins from with of LLO and as in D. Hamon M. E. Nahori M.A. Impens H. K. J. A. Cossart P. Listeria monocytogenes for 2010; PubMed Scopus Google Scholar, Cossart P. D. of epithelial cell induced by bacterial Microbiol. 2017; PubMed Scopus Google Scholar, and J.A. The Listeria monocytogenes has to activity and to infected host Biol. PubMed Scopus Google Scholar). from E. bacterial The of for toxin was by by toxins in of cells for as in the cell only was proteomic proteomic transcriptomic from cell from the proteomic cells in or cells for and with of toxin for or the first cells in cells in with was for the cells in with LLO, and cells in cells by on in and from and cells as to and with of for components by for and was to the to a concentration of and by with and for on for on for and with of The was in a in and for to in a and in for into by focusing in to the of the on and in in of which was for on to was for in on a and from the the was on a by a from to in a rate of The was in and for the most acquired a of in the to a target of The most a of for by a of in the the a target of for proteomic cells for in for and with of LLO for or cells for with and and or not with cells by on in The protein concentration in the was and of protein from was to the by of and for and by of for in the with to a concentration of and proteins with for to and with The was by of and on for to on and in in and of was for on to a with a was for in on a and the was on a by a from to in a rate of by a and with A in The was in a The was in and for the most acquired a of in the to a target of The most a of of for a of the a target of for acquired a of in the The was set and we with and from was with for the for the J. M. and protein 2008; PubMed Scopus Google the J. N. A. M. a into the 2011; PubMed Scopus Google with a rate set on both the and protein the human proteins in the of human protein for the of human protein for the with the of O with a for and of and during the was set as to and also and a of of protein was set as of was set as of was set as a modification in the and as a modification in the the of the was set to with or in the for a of amino acids In both and only proteins with or and a of or was for the for protein the by is in the was with a of and of and proteins by the in the J. N. M. quantification by and 13: Full Text Full Text PDF PubMed Scopus Google Scholar). data of the was with the the from only by and and protein proteins with than in and from a the leading to a of host proteins in the with LLO treatment and host proteins in the with LLO treatment a was and to reveal proteins of which the was by LLO treatment and to the in for and the of gene PubMed Scopus Google Scholar). from cells or not with LLO for of was monitored on on of and in transcription of the in of by a to on The cell with was by to was by the N. J. T. K. M. for genes with a of PubMed Scopus Google Scholar). The that the in a gene observed could occur by by the used for are in and used as cells with for and protein was by on and with specific host proteome alterations induced by LLO, we used on cells or not with the LLO We a first isotope labeling by amino acids in cell B. H. A. M. isotope labeling by amino acids in cell as a and to Full Text Full Text PDF PubMed Scopus Google Scholar). The is on isotope labeling of proteins during cell by of amino acids and that or and cell proteins are to The is and by are by the protein and quantification is by and for In our set we the protein from cell and with a of LLO (7Hamon M.A. Batsche E. Regnault B. Tham T.N. Seveau S. Muchardt C. Cossart P. Histone modifications induced by a family of bacterial toxins.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 13467-13472Crossref PubMed Scopus (216) Google Scholar). to LLO during only to protein changes from transcriptional We with labeling to Among the proteins that in both we a of proteins for which protein levels in cells with LLO with a in both and that LLO a remodeling of the host proteome by decreasing the protein of many host In to this we a to protein abundance in cells or not with in this to a of proteins. of a protein to be LLO treatment D. Hamon M. E. Nahori M.A. Impens H. K. J. A. Cossart P. Listeria monocytogenes for 2010; PubMed Scopus Google Scholar), was monitored in to LLO treatment efficiency Among the proteins that in all we a of 149 proteins for which protein levels in cells with LLO and In contrast to these only proteins showed levels in cells. This result that LLO remodels the cell proteome by decreasing the of host We proteins levels in response to LLO by gene cellular we not in proteins from specific cell in the of proteins to the of proteins. This that affects different cellular and both and proteins. protein we that a specific of is in the of proteins in response to LLO This that LLO not only with host as D. Hamon M. E. Nahori M.A. Impens H. K. J. A. Cossart P. Listeria monocytogenes for 2010; PubMed Scopus Google Scholar, L. Cossart P. D. of and of changes induced by Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar), but also targets ubiquitin and ubiquitin-like focusing on proteins in ubiquitin we observed that only some components a in host proteins. we could several E2 such as or that are in response to LLO such as or We observed that components of the ubiquitin such as the and E3 ligases and and and several are by LLO that affects specific components of the and that LLO may thus only a of the host In to and we that the levels of ubiquitin-like and with their E2 and in response to LLO This that both and of ubiquitin-like modification by Listeria during the observed decrease in protein levels was to transcriptional we used data from a transcriptomic of cells or not with LLO for (7Hamon M.A. Batsche E. Regnault B. Tham T.N. Seveau S. Muchardt C. Cossart P. Histone modifications induced by a family of bacterial toxins.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 13467-13472Crossref PubMed Scopus (216) Google Scholar). these transcriptomic data with our we could on levels corresponding to of the 149 proteins by Strikingly, the transcription levels of all these targets not in response to LLO Although several genes as of LLO of for the proteins levels in LLO cells. This that the of host protein This is with our that used a treatment with LLO and that variations in protein levels because of host transcription the host was in the levels of these 149 we our proteomic on cells with to The efficiency of was by the in in cells with both and we observed that the of host proteins as strongly in response to LLO also levels in the of and and This result that the of observed decrease in host protein levels induced by LLO is not because of degradation. our we on cells with LLO for or and the protein of several host targets in our different proteomic We that LLO a decrease in the of protein A the E2 ubiquitin and and the E2 This decrease LLO than In addition, we that this is as with not the decrease in the of these host proteins LLO was to induce a of translation in host cells M. B. S. toxins induce cellular responses Microbiol. 2011; 13: PubMed Scopus Google Scholar), we the observed in host protein levels a of proteins of this we translation LLO treatment by cells with for treatment not to a decrease in the levels of host i.e. and that these proteins have a than We observed a decrease in the levels of and in cells with and with LLO, that the decrease in the levels of these proteins our to cell we a human epithelial cell and a cell with LLO for or of cell showed a decrease in response to LLO in the of targets in cells and that LLO induces proteome alterations in different epithelial cell In no changes in or protein levels observed in strongly that are some proteome alterations host proteome alterations observed in response to LLO are also induced in the of bacterial we infected cells with L. monocytogenes or Listeria or of infection and by We that infection with Listeria induces a decrease in the of host proteins in our This decrease was not observed during infection with a the of LLO in this our data show that several E2 ubiquitin ligases are in response to LLO, we this bacterial toxin host protein cells with LLO for or we with specific for or that this toxin a decrease in the of these of cells with not this that these in and are We infected cells with L. monocytogenes or a We observed a decrease in and proteins in cells infected with Listeria of but not with the Listeria LLO thus strongly with host which the observed decrease in several E2 ligases We showed that the pore-forming toxin Perfringolysin O secreted by the pathogen induces a decrease in the of host to LLO D. Hamon M. E. Nahori M.A. Impens H. K. J. A. Cossart P. Listeria monocytogenes for 2010; PubMed Scopus Google Scholar). We monitored the of host proteins such as or We cells with and observed that this toxin induces a decrease in both and levels The decrease was not observed cells with of LLO or toxins D. Hamon M. E. Nahori M.A. Impens H. K. J. A. Cossart P. Listeria monocytogenes for 2010; PubMed Scopus Google Scholar, Cossart P. D. of epithelial cell induced by bacterial Microbiol. 2017; PubMed Scopus Google Scholar), that host protein by these toxins that host proteome remodeling may occur in response to infection by different bacterial pathogens that pore-forming toxins in the cells, the protein abundance is strongly dependent on post-transcriptional (6Vogel C. Marcotte E.M. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.Nat. Rev. Genet. 2012; 13: 227-232Crossref PubMed Scopus (2499) Google Scholar). Proteomics the abundance of of proteins now to integrate transcription translation efficiency and protein such a we host proteins and showed that the pore-forming toxin LLO remodels the host of protein Strikingly, most of protein are not because of transcriptional that proteome remodeling by LLO the post-transcriptional The observed decrease in the abundance of 149 host proteins by concentration of LLO is a that in than in this LLO also affects the transcription of a of but of for the 149 targets in this study (7Hamon M.A. Batsche E. Regnault B. Tham T.N. Seveau S. Muchardt C. Cossart P. Histone modifications induced by a family of bacterial toxins.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 13467-13472Crossref PubMed Scopus (216) Google Scholar). to LLO several of proteome by targeting pre-existing proteins or by In the of Listeria proteome remodeling only of the different transcriptional and post-transcriptional changes that are by this show that host proteome remodeling is observed during infection and for several infections proteome alterations be and leading to a different proteomic of the host cells. A showed that LLO induces in protein M. B. S. toxins induce cellular responses Microbiol. 2011; 13: PubMed Scopus Google Scholar). This allows cells to a in which is is M. B. S. toxins induce cellular responses Microbiol. 2011; 13: PubMed Scopus Google Scholar). A direct of this pore-forming toxin-induced of protein is the of proteins M. B. S. toxins induce cellular responses Microbiol. 2011; 13: PubMed Scopus Google Scholar). Here, we show that the observed decrease in the abundance of and is not because of such a translation This that these proteins are in response to in a we that LLO in epithelial cells, a of host and a of such as in the host Cossart P. D. of epithelial cell induced by bacterial Microbiol. 2017; PubMed Scopus Google Scholar). these are not in degradation Cossart P. D. of epithelial cell induced by bacterial Microbiol. 2017; PubMed Scopus Google Scholar). these are responsible for the degradation of some of the proteins to be In to a of from several host to be in response to In LLO induces cell which is by the of and J.A. B. Listeriolysin O from Listeria monocytogenes is a PubMed Scopus Google Scholar). In this toxin induces a that a to the and the of the T. H. K. T. S. T. H. S. M. Listeriolysin bacterial into the is for and in infected with Listeria 2010; PubMed Scopus Google Scholar). LLO was also to in and cells, the of a and the which in and K. J. B. S. H. P. M. T. N. B. Listeria human cells on O and 2010; PubMed Scopus Google Scholar, M.A. Cossart P. is for by Listeria monocytogenes O and pore-forming 2011; PubMed Scopus Google Scholar). LLO was to in which to the of the cell the induced by the toxin J.A. L. G. during Listeria monocytogenes infection a dependent 2012; PubMed Scopus Google Scholar). these different host in response to LLO, are in the of the proteins to be Strikingly, we could not degradation of the different LLO This may that protein the of these targets are by host This also that the levels of these targets decrease to mechanisms, such as host cells the by LLO M. M. M. A. M. L. M. G. of O and its regulation by and PubMed Scopus Google Scholar). in this study as in response to LLO are in various cellular We several E2 ubiquitin ligases that by the This result correlates with our of a of the host more of proteins by and ubiquitin This result the of LLO on another ubiquitin-like the E2 enzyme is in response to the toxin D. Hamon M. E. Nahori M.A. Impens H. K. J. A. Cossart P. Listeria monocytogenes for 2010; PubMed Scopus Google Scholar). for is a modification D. 2016; PubMed Scopus Google Scholar). The observed decrease in proteins from the of E2 ubiquitin that cellular and to a in the the of the proteins. targeting of E2 ubiquitin ligases such as has also been in the case of the pathogen this bacterium that the of in cells. This modification activity and response in the of infection T. M. H. M. M. A. H. T. T. S. J. J. T. Sasakawa C. The to the 2012; PubMed Scopus Google Scholar). be a host by different pathogens during by different In to E2 ubiquitin our data a of host proteins in response to LLO that may during of some of these may may infection their decrease is as a by the host cell that trigger responses. we showed that another pore-forming toxin secreted by also host proteome In to LLO or that both to the family of it has been that the pore-forming toxin secreted by some also induces degradation of specific host proteins in cell or cell M.A. The pore-forming toxin of host proteins to cell and 2012; Full Text Full Text PDF PubMed Scopus Google Scholar). This that host proteome remodeling by pore-forming toxins is a strategy used by different of pathogens. intracellular was also to remodel the host from changes in transcription L.M. alterations in the host cell proteome are for intracellular Full Text Full Text PDF PubMed Scopus Google Scholar). and the of approaches that on protein abundance rather than mRNA levels to host-pathogen The data have been to the the J.A. A. N. J.A. J. G. T. H. of the and its 2016; PubMed Scopus Google with the of the to and are LLO with

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