Glyceraldehyde-3-phosphate dehydrogenase present in extracellular vesicles from Leishmania major suppresses host TNF-alpha expression

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills various physiological roles that are unrelated to its glycolytic function. However, to date, the nonglycolytic function of GAPDH in trypanosomal parasites is absent from the literature. Exosomes secreted from Leishmania, like entire parasites, were found to have a significant impact on macrophage cell signaling and function, indicating cross talk with the host immune system. In this study, we demonstrate that the Leishmania GAPDH (LmGAPDH) protein is highly enriched within the extracellular vesicles (EVs) secreted during infection. To understand the function of LmGAPDH in EVs, we generated control, overexpressed, half-knockout (HKO), and complement cell lines. HKO cells displayed lower virulence compared with control cells when macrophages and BALB/c mice were infected with them, implying a crucial role for LmGAPDH in Leishmania infection and disease progression. Furthermore, upon infection of macrophages with HKO mutant Leishmania and its EVs, despite no differences in TNFA mRNA expression, there was a considerable increase in TNF-α protein expression compared with control, overexpressed, and complement parasites as determined by ELISA, RT-PCR, and immunoblot data. In vitro protein translation studies suggest that LmGAPDH-mediated TNF-α suppression occurs in a concentration-dependent manner. Moreover, mRNA binding assays also verified that LmGAPDH binds to the AU-rich 3′-UTR region of TNFA mRNA, limiting its production. Together, these findings confirmed that the LmGAPDH contained in EVs inhibits TNF-α expression in macrophages during infection via posttranscriptional repression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills various physiological roles that are unrelated to its glycolytic function. However, to date, the nonglycolytic function of GAPDH in trypanosomal parasites is absent from the literature. Exosomes secreted from Leishmania, like entire parasites, were found to have a significant impact on macrophage cell signaling and function, indicating cross talk with the host immune system. In this study, we demonstrate that the Leishmania GAPDH (LmGAPDH) protein is highly enriched within the extracellular vesicles (EVs) secreted during infection. To understand the function of LmGAPDH in EVs, we generated control, overexpressed, half-knockout (HKO), and complement cell lines. HKO cells displayed lower virulence compared with control cells when macrophages and BALB/c mice were infected with them, implying a crucial role for LmGAPDH in Leishmania infection and disease progression. Furthermore, upon infection of macrophages with HKO mutant Leishmania and its EVs, despite no differences in TNFA mRNA expression, there was a considerable increase in TNF-α protein expression compared with control, overexpressed, and complement parasites as determined by ELISA, RT-PCR, and immunoblot data. In vitro protein translation studies suggest that LmGAPDH-mediated TNF-α suppression occurs in a concentration-dependent manner. Moreover, mRNA binding assays also verified that LmGAPDH binds to the AU-rich 3′-UTR region of TNFA mRNA, limiting its production. Together, these findings confirmed that the LmGAPDH contained in EVs inhibits TNF-α expression in macrophages during infection via posttranscriptional repression. Leishmania spp., the organism causing leishmaniasis in humans, divides its life cycle between the sand fly vector and the mammalian host. Earlier researchers revealed that extracellular vesicles (EVs)-based secretion by Leishmania is involved in the delivery of proteins into host cells (1Silverman J.M. Clos J. de’Oliveira C.C. Shirvani O. Fang Y. Wang C. Foster L.J. Reiner N.E. An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages.J. Cell Sci. 2010; 123: 842-852Crossref PubMed Scopus (304) Google Scholar, 2Silverman J.M. Clos J. Horakova E. Wang A.Y. Wiesgigl M. Kelly I. Lynn M.A. McMaster W.R. Foster L.J. Levings M.K. Reiner N.E. Leishmania exosomes modulate innate and adaptive immune responses through effects on monocytes and dendritic cells.J. Immunol. 2010; 185: 5011-5022Crossref PubMed Scopus (200) Google Scholar). Comparative quantitative proteomics studies unambiguously identified numerous proteins in Leishmania EVs (1Silverman J.M. Clos J. de’Oliveira C.C. Shirvani O. Fang Y. Wang C. Foster L.J. Reiner N.E. An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages.J. Cell Sci. 2010; 123: 842-852Crossref PubMed Scopus (304) Google Scholar, 3Silverman J.M. Chan S.K. Robinson D.P. Dwyer D.M. Nandan D. Foster L.J. Reiner N.E. Proteomic analysis of the secretome of Leishmania donovani.Genome Biol. 2008; 9: R35Crossref PubMed Scopus (216) Google Scholar). Leishmania EVs and EV mediate proteins were detected in the cytosolic compartment of infected macrophages, and the incubation of macrophages with EVs selectively regulated the secretion of different cytokines (2Silverman J.M. Clos J. Horakova E. Wang A.Y. Wiesgigl M. Kelly I. Lynn M.A. McMaster W.R. Foster L.J. Levings M.K. Reiner N.E. Leishmania exosomes modulate innate and adaptive immune responses through effects on monocytes and dendritic cells.J. Immunol. 2010; 185: 5011-5022Crossref PubMed Scopus (200) Google Scholar, 4Silverman J.M. Reiner N.E. Leishmania exosomes deliver preemptive strikes to create an environment permissive for early infection.Front. Cell. Infect. Microbiol. 2011; 1: 26PubMed Google Scholar). However, the exact functions of parasite-derived EV mediate proteins in host parasite interaction are far from being elucidated. Earlier, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been considered simply as a housekeeping glycolytic enzyme. Recent studies indicate GAPDH as a multifunctional protein displaying numerous physiological roles that are unrelated to its glycolytic function. For example, GAPDH shows phosphotransferase/kinase activity, autophosphorylation, or phosphorylation of other proteins, thus acting as a cellular kinase (5Kawamoto R.M. Caswell A.H. Autophosphorylation of glyceraldehydephosphate dehydrogenase and phosphorylation of protein from skeletal muscle microsomes.Biochemistry. 1986; 25: 657-661Crossref PubMed Scopus (103) Google Scholar). It can interact with tubulin and catalyzes tubulin polymerization into microtubules (6Durrieu C. Bernier-Valentin F. Rousset B. Binding of glyceraldehyde 3-phosphate dehydrogenase to microtubules.Mol. Cell. 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In GAPDH has been as a protein virulence M. M. A. A. D. M. GAPDH is a Immunol. PubMed Scopus Google Scholar, R.A. glyceraldehyde-3-phosphate dehydrogenase of binds Microbiol. PubMed Scopus Google Scholar) to its to interact with host proteins A protein on A is a with binding PubMed Scopus Google Scholar). of GAPDH in and of the cytosolic GAPDH have been identified in Leishmania Leishmania and Leishmania activity suggest that GAPDH in and has also been detected in the M. S. analysis of the cytosolic and glyceraldehyde-3-phosphate dehydrogenase in Leishmania Biochem. PubMed Scopus Google Scholar, cytosolic and glyceraldehyde-3-phosphate dehydrogenase from and with J. Biochem. 1991; PubMed Scopus Google Scholar, O. J. R. dehydrogenase from of the and cytosolic J. Biochem. 1987; PubMed Scopus Google Scholar). However, Leishmania and Leishmania have of the of GAPDH and the cytosolic GAPDH K. D. M.A. E. A. M. A. A. A. analysis of Leishmania that human PubMed Scopus Google Scholar, J. D.P. D. Y. M. K. M. and between and of 2011; PubMed Scopus Google Scholar). A of that the cytosolic GAPDH enzyme in a role in the to in G. of cytosolic glyceraldehyde-3-phosphate dehydrogenase in infection by Leishmania Cell. PubMed Scopus Google Scholar). from and have been and of glyceraldehyde-3-phosphate dehydrogenase from Leishmania for and a for the binding 1995; 34: PubMed Scopus Google Scholar, G. glyceraldehyde-3-phosphate dehydrogenase: and 1998; PubMed Scopus Google Scholar, J. A of glyceraldehyde dehydrogenase from Biol. 1995; PubMed Google Scholar), nonglycolytic role of GAPDH is in the parasites, and In this we have a possible nonglycolytic role of GAPDH from is in the as as in the To understand the nonglycolytic function of this we have different of Leishmania cell control and complement for various in vitro and in with these cell we are to between LmGAPDH secretion during Leishmania infection and the of the of host It is that the cytosolic human GAPDH are to the membrane R.A. glyceraldehyde-3-phosphate dehydrogenase of binds Microbiol. PubMed Scopus Google Scholar, E. R. M. J. J. of the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase by the secretion in J. Biochem. Cell Biol. PubMed Scopus Google Scholar) and the role of nuclear of glyceraldehyde-3-phosphate dehydrogenase in and Cell Sci. PubMed Google Scholar) for LmGAPDH the the enzyme in other for displaying nonglycolytic To the LmGAPDH protein was in other the as a of cells were by were from C. F. C. and of from Leishmania and PubMed Google Scholar) and were the EVs from to with incubation of the for we verified the and of the EVs via A and and and confirmed the and of the vesicles to in were to C. E. R. A. F. D. for studies of extracellular vesicles A of the for and of the PubMed Scopus Google Scholar). To these analysis was from confirmed that the of the EVs from Leishmania was with that the LmGAPDH protein was from and EVs the kinase and the EVs protein a were indicating that the enzyme is in the and the EV of the other the LmGAPDH protein was absent in the and the cytosolic and nuclear a were the GAPDH activity in the was from the and the EVs that the enzyme is in the and the EV of Leishmania that the LmGAPDH has its in To the nonglycolytic role of LmGAPDH in a was the LmGAPDH is an for the of Leishmania of the we of the of the as generated LmGAPDH HKO by of and of and with and the of a of LmGAPDH in cells been To the HKO we a analysis on DNA with generated from the and analysis with confirmed the of LmGAPDH in the HKO cell and that of a of LmGAPDH been in the cell that is to and the other the of of LmGAPDH protein expression in cells compared with the of LmGAPDH in and is and we the of LmGAPDH was in the EVs from HKO and cells compared with and confirmed that the EVs from HKO cells of LmGAPDH protein and the EVs from the cell an of LmGAPDH compared with or To the of HKO is to or cell analysis was that the HKO no significant in the compared with or cells a of GAPDH in HKO of proteins that glycolytic function of for can host macrophages, we the interaction of with the to and cells were by the of were or of incubation HKO on the other a lower or cells and of infected macrophages parasites and the of parasites in HKO infected macrophages indicate that the HKO parasites were the In the of macrophages infected with compared with infection. mice infection that HKO cells a with an of compared with or were by the virulence in in mice of parasite during infection that HKO parasites, compared with or parasite in of findings that the GAPDH in parasites an role in macrophage infection and disease in a to have a crucial role in the of infection. GAPDH has been found to TNF-α and responses by macrophage activity B. GAPDH binding to TNF-α mRNA to posttranscriptional in A of communication between and Immunol. PubMed Scopus Google Scholar). TNF-α expression in and HKO infected macrophages was determined by quantitative and analysis and that the TNF-α mRNA expression in the HKO infected macrophage was compared with and infected cells to infection that the HKO infected macrophage cells a of TNF-α expression compared with and mRNA and protein of TNF-α expression in To these analysis was and and the we were to the data. these suggest that the LmGAPDH the host TNF-α expression the translation To the of the EVs in the host TNF-α expression, we the TNF-α expression in EVs or macrophages by A and and that the EVs from HKO macrophage in TNF-α expression compared with the EVs from or cells to incubation In of the EVs from or cells the TNF-α expression, the EVs from HKO cells to TNF-α expression To these analysis was and the were to the data. these suggest that the GAPDH in EVs TNF-α expression when with the host It is that EV has a of we LmGAPDH the host TNF-α To LmGAPDH within the the macrophages were with as and A and confirmed the of LmGAPDH in the analysis confirmed that cells lower of TNF-α expression in the of and compared with that LmGAPDH is the for the suppression of TNF-α we in vitro protein translation of TNF-α in the or of LmGAPDH and analysis that the protein in is indicating that in vitro protein translation in the or of with TNF-α the the TNF-α expression is to the LmGAPDH and In to protein in vitro TNF-α protein GAPDH also as a protein that with or 3′-UTR of mRNA expression or the posttranscriptional E. Glyceraldehyde-3-phosphate dehydrogenase selectively binds AU-rich RNA in the region Biol. 1995; PubMed Scopus Google Scholar). indicate that LmGAPDH is responsible for the in TNF-α protein To demonstrate that this in is to posttranscriptional we the binding of or 3′-UTR of TNF-α mRNA by LmGAPDH an RNA A to the 3′-UTR of TNF-α mRNA a with LmGAPDH that an of with a of the was to the the 3′-UTR of TNF-α the indicating that the was to In with 3′-UTR of TNF-α mRNA, the was in of of TNF-α mRNA that the binding of 3′-UTR of TNF-α mRNA with LmGAPDH is of TNF-α mRNA with LmGAPDH also findings suggest that LmGAPDH binds with the 3′-UTR of TNF-α mRNA, the from being and TNF-α production. To the interaction of LmGAPDH with 3′-UTR of TNF-α mRNA, we the with or in by RNA of and GAPDH binding indicate that of LmGAPDH is responsible for 3′-UTR studies that GAPDH is to different for nonglycolytic function M.A. glyceraldehyde-3-phosphate dehydrogenase: protein and nucleic in cells and in human Biochem. Biol. PubMed Scopus Google Scholar). a the of LmGAPDH in the role of this protein and to its functions with of other proteins that in a different cellular signaling pathway for parasite It is that the and the cytosolic GAPDH a role in glycolytic function in we have for the by various and activity of that the of LmGAPDH is in the as as in the EV to the protein is found M.A. I. M. McMaster R.W. M. Leishmania host signaling through protein PubMed Scopus Google Scholar). EV mediate LmGAPDH is to the host of an EV mediate LmGAPDH the this protein is to LmGAPDH an secretion and is thus to secreted via A of was found in on Leishmania, of the secreted proteins the B. 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Furthermore, on macrophages and also indicate that LmGAPDH is for the of parasites within macrophage and virulence in EVs from have to interact with host by fusion with the or through LmGAPDH in host cells on the of the host in the or within the host macrophage a lower of TNF-α expression in the of in compared with cells in LmGAPDH TNF-α expression in macrophages in suggest that LmGAPDH has to the host membrane for TNF-α is that the virulence secreted by of target the cytosolic for host cell function M. M. E. virulence and proteins to the host PubMed Scopus Google Scholar, S. M. S. A. M. of the with the of by Leishmania Microbiol. 1: PubMed Scopus Google Scholar). that is is involved in TNF-α GAPDH interact with a host transcription or to the region of TNF-α and as a host TNF-α the of is that the of TNF-α expression the posttranscriptional suggest that macrophage a of TNF-α mRNA compared with from the studies various of GAPDH Leishmania macrophages suggest that the transcription of TNF-α mRNA from host DNA on LmGAPDH we can the the other and immunoblot suggest that the protein expression of TNF-α in infected host cells is to the LmGAPDH no in TNF-α mRNA, infected host cells a significant in TNF-α protein the of posttranscriptional of TNF-α was that GAPDH with AU-rich of the region of mRNA that is responsible for posttranscriptional of B. D. J. J. control of cell function by PubMed Scopus Google Scholar) and TNF-α expression, translation and limiting and TNF-α D. B. K. R. S. A. Identification of a in the region of mRNA Natl. Acad. Sci. U. S. A. 1986; PubMed Scopus Google Scholar, AU-rich and in mRNA Biochem. Sci. 1995; PubMed Scopus Google Scholar, J. G. of mRNA in mammalian PubMed Scopus Google Scholar). mRNA of that LmGAPDH can with AU-rich of of the as to region of LmGAPDH is in binding to AU-rich of of It is that incubation of GAPDH with its activity E. Glyceraldehyde-3-phosphate dehydrogenase selectively binds AU-rich RNA in the region Biol. 1995; PubMed Scopus Google Scholar). studies suggest that and were to the binding of LmGAPDH indicating that the region in the of LmGAPDH as a that the of LmGAPDH regulated between its binding in and binding in within the glycolytic activity of LmGAPDH in the parasite the of is LmGAPDH host TNF-α mRNA during Leishmania infection to of human in phosphorylation to and to lower of J. A. A. of human macrophages Leishmania and PubMed Scopus Google Scholar). the mRNA translation of the and limiting TNF-α In vitro protein translation suggest that the protein expression of TNF-α with LmGAPDH the other to in vitro TNF-α protein that the mRNA translation of TNF-α In to the possible for the suppression of TNF-α in macrophage we a in on a virulence function of LmGAPDH contained in EV that is from parasite to host macrophage during infection. for the that the glycolytic enzyme LmGAPDH macrophage function by binding to the AU-rich region in the of TNF-α mRNA and protein translation and host immune LmGAPDH in EV a for in the in the was from the Leishmania of and were from or S. A. A. S. S. of Leishmania kinase by J. PubMed Scopus Google Scholar, A. S. A. S. S. kinase in Leishmania in and cell J. PubMed Scopus Google Scholar, A. A. S. S. S. of virulence in Leishmania PubMed Scopus Google Scholar). BALB/c mice and were from and in was by the and with the for the of and on on of BALB/c mice and were to parasites were in with and macrophage cell in in a was a cell and in a of when DNA was from by DNA was the and the entire of the GAPDH was to a that was and within the vector in DNA was to the were into and were in of was in of the an of to was and were for were by for and with of the LmGAPDH was by a protein of from the various are in a protein of other protein detected in the with the LmGAPDH were by in was by of LmGAPDH with were the and were and for analysis LmGAPDH activity was by was of and glyceraldehyde in a for to and the was with the of enzyme. from to was and were to the of GAPDH by was into the of was in by S. S. S. Leishmania cells Biol. 2008; PubMed Scopus Google Scholar). cells were vector was in by cells were and were to the HKO of LmGAPDH and were for and and were for of the and DNA were on and of the and of and were with and to of and and were into as S. S. S. from Leishmania the virulence of of by 2010; PubMed Scopus Google Scholar). was in the of and To LmGAPDH in the HKO parasites, LmGAPDH was an as and the and was the of and of the was into the HKO were with of and were in the of and For was was by of the for For EV cells were in from cell cells was and for for by for were was through a was on a and for the the was a of with EVs the EVs were with and for was as the was from C. F. C. and of from Leishmania and PubMed Google Scholar) with were by for and were in of the cell was in and was Cell was confirmed by cell was for and to cell and cell was for to was in and was on of a to on and for were from the of the were to the was in and to the of the was by the was to and cytosolic the EVs and were by the of was by analysis with were as GAPDH kinase and mice was a A of cells were in of with were a by cell in an for were in for cell and were to of the macrophage cell on in of a parasite to cell of the parasites were by with and cells were in A incubation was for the of parasite were to a for an infection of and were in and with were and BALB/c mice to were infected with in mice was by of in of mice and HKO parasites were determined by limiting with were in and of in with and was in the in a of parasites of was determined from the to to of incubation was to the of cytokines TNF-α cells were in for were with and with or different of Leishmania different RNA was from the cells to the was quantitative was on the Green expression of mRNA were control cells as and as the control a as by the and for TNF-α and and for were cell from the and macrophage was and to parasites and of TNF-α was TNF-α to the were infection for and in protein in the were a protein of proteins was to and proteins were to a membrane and for in to binding of was by in TNF-α were in by incubation with to with were by RNA was from the cells to the mRNA expression in cells was to in vitro translation by of of and of with or LmGAPDH in a for A was to with TNF-α and protein in a proteins were on To the translation a that was to was by were by and the on as cells were on in and LmGAPDH protein was to the protein in a was to the cells through cells on were in and the cells were by were with in with were and with for were with GAPDH the the were with and with a of for were on a with and were and and For we AU-rich RNA from TNF-α and an region from TNF-α and by by was with the RNA to the For RNA binding LmGAPDH protein and of in a of were for with in of binding For TNF-α RNA were to the of the is with LmGAPDH and LmGAPDH for the of and were to the to on LmGAPDH-mediated of TNF-α production. were on in that were for the was into in RNA on the membrane was by a and detected were by For EVs were with and a of the was on by incubation for was with of to and were in or to was a with a in with a of to were was into was to were with a between and a of were by was EVs were and into a and the was into the to and were the For by the was the with the to the of an of was for and an of was to the For were into a cell and from an of were from the a by the were as the from analysis for was by or analysis of A of was considered generated and during this are contained within the that have no of with the of this S. 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