Toxoplasma GRA 15 limits parasite growth in IFN γ‐activated fibroblasts through TRAF ubiquitin ligases
2020; Springer Nature; Volume: 39; Issue: 10 Linguagem: Inglês
10.15252/embj.2019103758
ISSN1460-2075
AutoresDebanjan Mukhopadhyay, Lamba Omar Sangaré, Laurence Braun, Mohamed‐Ali Hakimi, Jeroen P. J. Saeij,
Tópico(s)Herpesvirus Infections and Treatments
ResumoArticle15 April 2020free access Source DataTransparent process Toxoplasma GRA15 limits parasite growth in IFNγ-activated fibroblasts through TRAF ubiquitin ligases Debanjan Mukhopadhyay Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California Davis, Davis, CA, USA Search for more papers by this author Lamba Omar Sangaré Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California Davis, Davis, CA, USA Search for more papers by this author Laurence Braun Institute for Advanced Biosciences, Team Host-Pathogen Interactions and Immunity to Infection, INSERM U1209, CNRS, UMR5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Mohamed-Ali Hakimi orcid.org/0000-0002-2547-8233 Institute for Advanced Biosciences, Team Host-Pathogen Interactions and Immunity to Infection, INSERM U1209, CNRS, UMR5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Jeroen PJ Saeij Corresponding Author [email protected] orcid.org/0000-0003-0289-7109 Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California Davis, Davis, CA, USA Search for more papers by this author Debanjan Mukhopadhyay Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California Davis, Davis, CA, USA Search for more papers by this author Lamba Omar Sangaré Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California Davis, Davis, CA, USA Search for more papers by this author Laurence Braun Institute for Advanced Biosciences, Team Host-Pathogen Interactions and Immunity to Infection, INSERM U1209, CNRS, UMR5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Mohamed-Ali Hakimi orcid.org/0000-0002-2547-8233 Institute for Advanced Biosciences, Team Host-Pathogen Interactions and Immunity to Infection, INSERM U1209, CNRS, UMR5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Jeroen PJ Saeij Corresponding Author [email protected] orcid.org/0000-0003-0289-7109 Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California Davis, Davis, CA, USA Search for more papers by this author Author Information Debanjan Mukhopadhyay1, Lamba Omar Sangaré1, Laurence Braun2, Mohamed-Ali Hakimi2 and Jeroen PJ Saeij *,1 1Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California Davis, Davis, CA, USA 2Institute for Advanced Biosciences, Team Host-Pathogen Interactions and Immunity to Infection, INSERM U1209, CNRS, UMR5309, Université Grenoble Alpes, Grenoble, France *Corresponding author. Tel: +1 530 752 1401; E-mail: [email protected] EMBO J (2020)39:e103758https://doi.org/10.15252/embj.2019103758 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The protozoan parasite Toxoplasma gondii lives inside a vacuole in the host cytosol where it is protected from host cytoplasmic innate immune responses. However, IFNγ-dependent cell-autonomous immunity can destroy the vacuole and the parasite inside. Toxoplasma strain differences in susceptibility to human IFNγ exist, but the Toxoplasma effector(s) that determine these differences are unknown. We show that in human primary fibroblasts, the polymorphic Toxoplasma-secreted effector GRA15 mediates the recruitment of ubiquitin ligases, including TRAF2 and TRAF6, to the vacuole membrane, which enhances recruitment of ubiquitin receptors (p62/NDP52) and ubiquitin-like molecules (LC3B, GABARAP). This ultimately leads to lysosomal degradation of the vacuole. In murine fibroblasts, GRA15-mediated TRAF6 recruitment mediates the recruitment of immunity-related GTPases and destruction of the vacuole. Thus, we have identified how the Toxoplasma effector GRA15 affects cell-autonomous immunity in human and murine cells. Synopsis The polymorphic Toxoplasma effector GRA15 determines parasite strain differences in susceptibility to IFNγ by recruiting TRAF ubiquitin ligases that mediate the recruitment of p62, LC3B and LAMP1 to the parasitophorous vacuole in IFNγ-stimulated human fibroblasts and the recruitment of IRGs/GBPs in IFNγ-stimulated murine fibroblasts. The polymorphic effector GRA15 enhances Toxoplasma susceptibility to IFNγ-mediated growth inhibition in HFFs and MEFs and determines parasite strain differences in susceptibility to IFNγ in HFFs and MEFs. In IFNγ-stimulated HFFs parasites are eliminated through endolysosomal fusion with the parasitophorous vacuole, which is mediated by GRA15 recruitment of TRAF ubiquitin ligases and ubiquitin-like molecules and receptors to the parasitophorous vacuole. In IFNγ-stimulated MEFs parasites are eliminated through recruitment of IRGs/GBPs to parasitophorous vacuole, which is mediated by GRA15 recruitment of the TRAF6 ubiquitin ligase to the parasitophorous vacuole. Introduction Toxoplasma is a highly successful obligate intracellular parasite that can establish lifelong chronic infections in a wide range of warm-blooded animals. In humans, it causes opportunistic infections in immunosuppressed patients, congenital infections (Hill & Dubey, 2002), and blindness (Pleyer et al, 2014). Many different Toxoplasma strains exist, but in Europe and North America, human infections are dominated by the type I and type II clonal lineages (Howe & Sibley, 1995; Saeij et al, 2005). Upon host cell invasion, Toxoplasma wraps itself with the host cell plasma membrane, which becomes the nascent parasitophorous vacuole membrane. The vacuole membrane does not fuse with the endo-lysosome system, and without immune pressure, the vacuole does not acidify thus providing Toxoplasma with a niche for replication (Jones et al, 1972; Mordue & Sibley, 1997). Like Toxoplasma, many intracellular pathogens reside within a vacuole (pathogen-containing vacuole or PV) in the host cytoplasm (Liehl et al, 2015). The PV membrane (PVM) protects these pathogens from detection by host cytosolic pathogen recognition receptor (PRR). However, the host has developed mechanisms to destroy the PV thereby exposing the pathogen (Liehl et al, 2015; Saeij & Frickel, 2017). For example, in mice interferons upregulate the expression of two families of large dynamin-like GTPases: the immunity-related GTPases (IRGs) and the guanylate-binding proteins (GBPs) (Howard et al, 2011), which mediate the destruction of the PV of Toxoplasma and of many gram-negative bacteria (Liehl et al, 2015). Once the pathogen is in the cytoplasm, GBPs and IRGs can also mediate the vesiculation of the pathogen itself thereby exposing pathogen-associated molecular patterns (PAMPs) to cytosolic PRRs, which can lead to the activation of the inflammasome (Man et al, 2017) and the induction of a form of cell death called pyroptosis (Broz & Dixit, 2016). Because host cell death removes the replication niche of intracellular pathogens, this is an efficient way of inhibiting pathogen growth (Krishnamurthy et al, 2017). The mechanism of IRG and GBP recruitment to the Toxoplasma PVM and the identity of Toxoplasma effectors influencing IRG/GBP recruitment, or their activity, are intense areas of research. These GTPases are normally held inactive on host endomembranes by regulatory “GMS motif”-type IRGs (Haldar et al, 2013). Initially, “pioneer” effector “GKS” motif-type IRGs are recruited to the Toxoplasma PVM (Hunn et al, 2008), which is largely devoid of regulatory IRGs, where they oligomerize and become activated. What exact signal initiates the recruitment of these pioneer IRGs to the PVM is unclear. In murine cells, the initial conjugation of a ubiquitin-like protein (e.g., microtubule-associated protein light chain 3 [LC3] or γ-aminobutyric acid receptor-associated proteins [GABARAPs]) to the PVM was proposed to be the signal that initiates recruitment of “pioneer” IRGs (Choi et al, 2014; Sasai et al, 2017). PVM recruitment of pioneer IRGs somehow promotes the ubiquitination of the PVM which subsequently leads to the recruitment of the ubiquitin-binding protein p62 (also called sequestosome or SQSTM1) and E3 ubiquitin ligases (e.g., TNF receptor-associated factor 6 or TRAF6 and tripartite motif-containing 21 or TRIM21) in a co-dependent manner (Haldar et al, 2015; Lee et al, 2015; Foltz et al, 2017). PVM ubiquitination by these ubiquitin ligases can lead to further recruitment of p62 and TRAF6, thereby creating an amplification loop ensuring the full ubiquitination of the PV. GBPs also get recruited to the PVM and eventually vesiculation of the PVM by GBPs and IRGs exposes Toxoplasma, which can lead to its destruction by GBPs (Degrandi et al, 2013; Kravets et al, 2016) and pyronecrosis of host cells (Zhao et al, 2009). To counter the host defense mechanisms, Toxoplasma secretes ROP and GRA effector proteins into the host cell from organelles called rhoptries and dense granules, respectively. In both murine and human cells, type II strains are more susceptible to IFNγ-mediated growth inhibition than type I strains (Haldar et al, 2015; Selleck et al, 2015; Clough et al, 2016; Qin et al, 2017). Resistance of type I strains in murine cells is determined primarily by polymorphic ROP5 and ROP18, which, together with ROP17 and GRA7, cooperatively block IRG and GBP loading on the PVM and subsequent events (Khaminets et al, 2010; Steinfeldt et al, 2010; Niedelman et al, 2012; Etheridge et al, 2014; Behnke et al, 2015; Haldar et al, 2015). ROP16 and GRA15 also affect GBP loading on the PVM in murine cells through an unknown mechanism (Virreira Winter et al, 2011). IFNγ-stimulated human cells control Toxoplasma using diverse mechanisms dependent on the cell type (Krishnamurthy et al, 2017). For example, IFNγ-mediated induction of indoleamine 2,3 dioxygenase (IDO) causes breakdown of L-tryptophan, for which Toxoplasma is auxotrophic, which mediates inhibition of parasite growth in HeLa, HAP1, and fibroblast cells (Pfefferkorn, 1984; Pfefferkorn et al, 1986; Niedelman et al, 2013; Qin et al, 2017; Bando et al, 2018). In some human cell lines, another mechanism for parasite control is ubiquitination of the vacuole, which leads to lysosomal fusion in HUVEC cells, while in HeLa cells, an autophagic double membrane forms around the vacuole and parasite growth is stunted without lysosomal fusion (Selleck et al, 2015; Clough et al, 2016). In certain human cells, GBP1 also seems important for restriction of Toxoplasma growth but the mechanism of growth restriction is unclear. In a lung epithelial cell line (A549), GBP1 restricts parasite growth without its recruitment to the PVM (Johnston et al, 2016), while in human mesenchymal stem cells (MSCs), growth restriction was associated with GBP1 PVM recruitment (Qin et al, 2017). Much less is known about what initiates targeting of human immune effectors to the PVM and how this leads to parasite elimination. In contrast to mice, humans lack IFNγ-inducible IRGs, likely explaining why ROP5, ROP18, and ROP17 do not seem to play an important role in conferring protection against IFNγ-mediated growth inhibition in human cells (Niedelman et al, 2012; Selleck et al, 2015; Clough et al, 2016). Currently, no parasite proteins that determine strain differences in susceptibility to IFNγ-mediated cell-autonomous immunity in human cells have been identified. A secreted parasite effector, Toxoplasma inhibitor of STAT1-dependent transcription (TgIST), which blocks the STAT1 transcriptional response, was recently described, but this effector functions upstream of the upregulation of IFNγ-induced toxoplasmacidal mechanisms in both type I and type II strains (Gay et al, 2016; Olias et al, 2016). Herein, we report that the PVM-localized Toxoplasma GRA15 effector (Rosowski et al, 2011) enhances parasite susceptibility to IFNγ in primary human foreskin fibroblasts (HFFs) and murine embryonic fibroblasts (MEFs). GRA15 binds several ubiquitin ligases, including TRAF2 and TRAF6, which in HFFs is associated with enhanced recruitment of p62, LC3, and GABARAP to the PVM, enhanced endo-lysosomal fusion, and parasite destruction. In MEFs, GRA15 also interacts with TRAF2 and TRAF6, and TRAF6 recruitment leads to enhanced PVM loading with IRGs and GBPs and parasite destruction. Thus, we determined that the Toxoplasma effector GRA15 mediates strain differences in susceptibility to cell-autonomous immunity in human cells and determined the mechanism by which GRA15 enhances parasite susceptibility to IFNγ in both human and murine fibroblasts. Results The polymorphic effector GRA15 enhances Toxoplasma susceptibility to IFNγ-mediated growth inhibition in HFFs To determine whether the type I RH and the type II Pru strain differ in susceptibility to IFNγ-mediated growth inhibition in HFFs, we measured the IFNγ-mediated reduction in plaque numbers and plaque area. The relative reduction in the number of plaques formed in IFNγ-stimulated vs. unstimulated cells reflects killing of Toxoplasma, while the relative reduction in the area of the plaques is a sensitive assay that reflects overall inhibition of parasite growth over multiple lysis cycles (Niedelman et al, 2012, 2013). Compared to RH, Pru had a larger IFNγ-induced loss in plaque numbers and plaque area (Figs 1A and EV1A). We used parasites expressing luciferase to determine IFNγ-mediated growth inhibition 24 h post-infection (p.i.) and observed that Pru growth was more inhibited by IFNγ compared to RH growth (Fig 1B). IFNγ stimulation resulted in a decrease in the relative number of vacuoles (Fig 1C) as well as a decrease in the number of parasites per vacuole (Fig 1D), both of which were more pronounced for the Pru strain. Figure 1. The type II (Pru) Toxoplasma strain is more susceptible to IFNγ-mediated growth inhibition in primary human foreskin fibroblasts (HFFs) than the type I RH strain due to the presence of the polymorphic effector GRA15 A. HFFs were pre-stimulated with IFNγ (10 U/ml) for 24 h. Plaque assays were performed for each strain and each condition. Plaque number and area loss were calculated 4 days p.i. for RH and 6 days p.i. for Pru. Assays were performed with RH (n = 4) and Pru (n = 4). B. Relative parasite growth was measured 24 h p.i. in IFNγ-stimulated and unstimulated HFFs by luciferase assay. Growth of each strain in IFNγ-stimulated HFFs is expressed relative to growth in unstimulated HFFs. Experiments were performed with RH (n = 4) and Pru (n = 4). C. Number of PVs was calculated using HFFs grown on coverslips and stimulated with 10 U/ml IFNγ for 24 h. Following stimulation, HFFs were infected with either RH (MOI = 1) or Pru (MOI = 3) for another 24 h. Coverslips were fixed and stained with GRA7 for parasite PVM and Hoechst 33258 for nuclei. The number of PVs in 5–6 fields of the coverslips was counted for each condition and normalized with the number of host cells in each field. Experiments were performed with RH (n = 3) and Pru (n = 3). Representative images of percentage infected cells for each strain and condition are provided. Scale bar is 100 μm. D. Parasites per vacuole were determined 24 h p.i. with similar conditions and staining as described in (C) except MOIs used were 0.5 and 1 for RH (n = 3) and Pru (n = 3), respectively. E. Plaque assays were performed similarly as described for (a) with Pru WT (n = 20 for plaque number and n = 7 for plaque area), PruΔgra15 (n = 20 for plaque number and n = 7 for plaque area), and PruΔgra15 + GRA15-complemented (n = 12 for plaque number and n = 7 for plaque area). F. Plaque assays were performed similarly as described for (A) with RH (n = 5 for plaque number and n = 3 for plaque area) and RH + GRA15II (n = 5 for plaque number and n = 3 for plaque area). G. Relative parasite growth was measured as described in (B) with Pru WT (n = 12), PruΔgra15 (n = 12), and PruΔgra15 + GRA15-complemented (n = 8). H. Number of PVs was calculated as described in (C) with Pru WT (n = 3), PruΔgra15 (n = 3), RH (n = 3), and RH + GRA15II (n = 3) and normalized with the number of host cells in each field. Data information: Statistical analysis was done by two-way ANOVA followed by Tukey's multiple comparison test. Data are represented as mean ± standard error of mean (SEM). Source data are available online for this figure. Source Data for Figure 1 [embj2019103758-sup-0004-SDataFig1.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. IFNγ-mediated susceptibility of the type II Pru strain is neither dependent on MYR1-dependent parasite secreted factors nor on L-Trp breakdown by primary human fibroblastsHFFs were pre-stimulated for 24 h with 10 U/ml IFNγ followed by infection with the indicated strains. A. Representative images of the plaque assay described for Fig 1A. Left panel is for unstimulated HFFs infected with either RH (top) or Pru (bottom), and right panel is for IFNγ-stimulated HFFs infected with RH (top) and Pru (bottom). Scale bar is 100 μm. B. Plaque assays were performed with PruΔku80Δhpt (Pru) and PruΔku80ΔhptΔmyr1 (PruΔmyr1) strains, and plaque numbers were counted and plaque areas were measured after 6 days. Plaque numbers and plaque areas are averages from eight and six biological replicates, respectively. C. Representative images of the plaque assay described for Fig 1E. Left panel is for unstimulated HFFs infected with Pru WT (top), Pru Δgra15 (middle), or Pru Δgra15 + GRA15 (bottom) and right panel is for IFNγ-stimulated HFFs infected with Pru WT (top), Pru Δgra15 (middle), or Pru Δgra15 + GRA15 (bottom). Scale bar is 100 μm. D. Representative images of the plaque assay described for Fig 1F. Left panel is for unstimulated HFFs infected with RH (top) or RH + GRA15II (bottom), and right panel is for IFNγ-stimulated HFFs infected with RH (top) and RH + GRA15II (bottom). Scale bar is 100 μm. E. Cells were supplemented with media containing 0.6 mM L-Trp, stimulated with IFNγ for 24 h, and infected with RH or Pru parasites. Plaque number was measured 4 (RH) or 6 days (Pru) p.i. Experiments were performed two times. F. L-Kynurenine was measured from HFF culture supernatant as a marker of IDO activity. Experiment was done two times. G. Number of total host nuclei counted per experiment for Fig 1C and H for each indicated strains and conditions. Experiments were performed six times with RH and Pru WT and three times with RH + GRA15II and Pru Δgra15 H. Measurement of infectivity of Pru WT and Pru Δgra15 by plaque assay in unstimulated HFFs using equal number of parasites (250). Data information: Data are represented as mean ± SEM. Source data are available online for this figure. Download figure Download PowerPoint After invasion, Toxoplasma resides within the host cytosol in a PV and starts secreting GRAs into the PV lumen where they stay or get transported to the PVM or beyond the PVM into the host cell (Hakimi et al, 2017). The transport of GRAs beyond the PVM, but not onto the PVM, is mediated by a putative translocon containing the proteins MYR1/2/3 (Franco et al, 2016; Naor et al, 2018). We wanted to determine whether GRAs secreted beyond PVM affect susceptibility of Pru parasites to IFNγ. However, Pru and PruΔmyr1 parasites showed similar IFNγ-mediated reductions in plaque number and area (Fig EV1B), indicating that these phenotypes are not influenced by GRAs secreted beyond the PVM. We therefore hypothesized that maybe a GRA in the PVM facing the host cytosol might be involved as these are not affected by MYR1. GRA15 is a polymorphic Toxoplasma effector protein present in the PVM that activates the NF-κB transcription factor (Rosowski et al, 2011), a master regulator of cell signaling and cell death (Dutta et al, 2006), independent of MYR1 (Franco et al, 2016). The type I RH strain has an early stop codon in GRA15 leading to a nonfunctional GRA15 (Rosowski et al, 2011). To determine whether GRA15 plays a role in the susceptibility of Pru to IFNγ-mediated growth inhibition, we infected IFNγ-stimulated or naïve HFFs with Pru, PruΔgra15, and the PruΔgra15 strain complemented with an HA-tagged copy of GRA15 (PruΔgra15 + GRA15) and measured plaque number and area after 6 days. PruΔgra15 parasites showed significantly less plaque number and area loss compared to wild-type parasites in IFNγ-stimulated HFFs (Figs 1E and EV1C). Complementation of Δgra15 parasites with GRA15 restored growth inhibition to wild-type levels (Figs 1E and EV1C). RH parasites expressing type II GRA15 (RH + GRA15II) (Rosowski et al, 2011) showed significantly more plaque loss compared to the wild-type RH strain (Figs 1F and EV1D). Furthermore, we observed that the enhanced GRA15-mediated susceptibility of Pru to IFNγ was already apparent 24 h p.i. (Fig 1G). GRA15 also enhanced the IFNγ-mediated elimination of vacuoles (Fig 1H and EV1E) but did not affect parasite infection of host cells (Fig EV1F). It was recently shown that in immortalized HFFs, IFNγ-induced IDO1 expression determines the IFNγ-mediated growth inhibition of Toxoplasma. The MYR1-dependent secreted Toxoplasma effector TgIST was shown to protect Toxoplasma from IDO1-mediated growth inhibition in cells stimulated after infection by inhibiting STAT1-mediated IDO1 expression but not in cells pre-stimulated with IFNγ (Bando et al, 2018). In contrast, we previously showed that IDO-mediated L-Trp degradation only plays a minor role in inhibition of parasite growth in IFNγ-stimulated primary HFFs (Niedelman et al, 2013). To rule out the role of IDO in the increased susceptibility of Pru parasites to IFNγ, we show that L-Trp supplementation did not restore the reduction of plaque loss in RH and Pru strains (Fig EV1G). Furthermore, there is no difference in IDO activity in RH- vs. Pru-infected IFNγ-stimulated HFFs (Fig EV1H). Thus, in IFNγ-stimulated HFFs parasite expression of GRA15 leads to reduced parasite growth and enhanced disappearance of vacuoles. GRA15 enhances IFNγ-induced endo-lysosomal fusion with the vacuole In HUVEC cells, ubiquitin, ubiquitin-like proteins (LC3/GABARAP), and ubiquitin receptors (p62/NDP52) are recruited to the Toxoplasma PVM, eventually leading to its destruction by fusion with endo-lysosomes (Clough et al, 2016). To determine whether this occurs in HFFs and the potential role of GRA15 in this process, we pre-stimulated HFFs with IFNγ, infected cells with RH, Pru, or PruΔgra15 parasites, and measured accumulation of ubiquitin, p62, NDP52, LC3B, GABARAP, and LAMP1 around the PVM 3 h p.i. Surprisingly, and unlike what has been observed in HeLa, HUVEC, and murine cells (Haldar et al, 2015; Lee et al, 2015; Selleck et al, 2015; Clough et al, 2016), we observed that although PVMs of both RH and Pru strains were coated with ubiquitin in IFNγ-stimulated HFFs, a larger fraction of RH vacuoles was coated (Fig 2A). We did not observe any difference in the ubiquitin coating intensity among the different parasite strains (Fig EV2A). Deletion of GRA15 had no effect on ubiquitination of Pru vacuoles (Fig 2A). The type of ubiquitin linkage recruited to the PVM can influence the subsequent outcome (Swatek & Komander, 2016). We observed K63-linked, and no K48-linked (Fig EV2B), ubiquitin localized to the PVM (Fig 2B). We observed a significantly larger fraction of Pru PVMs coated with p62 compared to RH (Fig 2C). Deletion of GRA15 from Pru resulted in significantly fewer PVMs coated with p62, while a similar fraction of PVMs of the GRA15-complemented strains as wild-type Pru were coated with p62 (Fig 2C). Unlike the type I RH strain, the type I GT1 strain contains a functional GRA15, which we previously showed determines RH vs. GT1 differences in activation of NF-κB (Yang et al, 2013). Consistent with a role for GRA15 in mediating p62 PVM recruitment, a significantly larger fraction of the vacuoles from the RH + GRA15II and the GT1 strain stained positive for p62 compared to RH vacuoles (Fig EV2C). Additionally, we observed that GT1 was significantly more susceptible to IFNγ-mediated parasite elimination compared to RH (Fig EV2D). A similar fraction of PVMs of RH and Pru vacuoles contained NDP52 in IFNγ-stimulated HFFs (Fig 2D). However, deletion of GRA15 in Pru resulted in reduction of NDP52 recruitment to the PVM (Fig 2D). Like p62, both LC3B and GABARAP were recruited to a larger fraction of the PVs of Pru compared to RH (Fig 2E and F) in IFNγ-stimulated HFFs. The PruΔgra15 strain had ~2-fold less vacuoles that were coated with LC3B and GABARAP compared to wild-type Pru (Fig 2E and F). The GRA15-complemented strain had a larger fraction of PVs coated with LC3B compared to the GRA15-deleted strain (Fig 2E). To determine whether the recruitment of LC3B, GABARAP, and p62 is associated with lysosomal destruction of the vacuole, we infected IFNγ-stimulated HFFs and counted LAMP1-positive vacuoles 3 h p.i. IFNγ enhanced the recruitment of LAMP1 to vacuoles of all strains, but significantly more LAMP1-positive vacuoles were seen in Pru-infected, compared to RH-infected, cells. Deletion of GRA15 significantly reduced the number of LAMP1-positive vacuoles (Fig 2G). In many LAMP1-positive vacuoles, the parasites were distorted, and they often did not stain positive for GRA7, used as parasite PV marker. However, by using the DNA-binding dye Hoechst, these vacuoles still clearly contained parasite DNA but were in advanced stages of parasite degradation (Fig EV3). The lysosomal inhibitor BafA1 significantly inhibited the disappearance of vacuoles in IFNγ-stimulated cells (Fig 2H). Figure 2. GRA15 enhances IFNγ-induced PVM decoration with autophagy-related proteins and endo-lysosomal-mediated vacuole destruction in HFFs A–G. HFFs were stimulated with for 24 h with 10 U/ml IFNγ or left unstimulated and subsequently infected with RH, Pru, or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (A) total ubiquitin (n = 3 for RH, n = 5 for Pru, and n = 3 for PruΔgra15), (B) K63-linked ubiquitin (n = 3 for RH, n = 3 for Pru, and n = 3 for PruΔgra15), (C) p62 (n = 3 for RH, n = 10 for Pru, n = 7 for PruΔgra15, and n = 4 for Pru Δgra15 + GRA15), (D) NDP52 (n = 3 for RH, n = 3 for Pru, and n = 3 for PruΔgra15), (E) LC3B (n = 3 for RH, n = 3 for Pru, n = 3 for PruΔgra15, and n = 3 for Pru Δgra15 + GRA15), (F) GABARAP (n = 3 for RH, n = 3 for Pru, and n = 3 for PruΔgra15), and (G) LAMP1 (n = 3 for RH, n = 3 for Pru, and n = 3 for PruΔgra15) is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 μm. The yellow box inside each representative image is shown as inset pictures with magnification. H. The number of PVs per 20× objective field was counted and compared between IFNγ-stimulated and IFNγ + bafilomycin A1 (100 nM)-treated HFFs 24 h p.i. with Pru strain. Images from at least six fields were taken for each condition (n = 3). Data information: Each dot represents one experiment. Each time, at least 100 different vacuoles were scored and analyzed. Statistical analysis was done by two-way ANOVA followed with Tukey's multiple comparison test (A–G) and one-way ANOVA for (H). Data are represented as mean ± SEM. Source data are available online for this figure. Source Data for Figure 2 [embj2019103758-sup-0005-SDataFig2.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. PVM ubiquitination is K48-linked ubiquitination-independent, and the type I GT1 strain, which endogenously expresses GRA15, is more susceptible to IFNγ-mediated parasite killing A. PVM ubiquitin coating intensity is similar between RH, Pru, and PruΔgra15. The measurement of fluorescence intensity of ubiquitin staining on the PVM was performed using NIS-Elements software version 4 (Nikon) from the experiments described in Fig 2. For each strain type, intensities of at least 50 vacuoles were measured. B. HFFs were stimulated with for 24 h with 10 U/ml IFNγ or left unstimulated and subsequently infected with RH, Pru, or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for K63-linked ubiquitin (n = 3 for RH, n = 3 for Pru, and n = 3 for PruΔgra15) is shown in the right bar diagram. On the left-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 μm. C. p62 coating was performed as described in Fig 2, except here it is performed with RH, GT-1, and RH + GRA15II. Experiment was performed three times with RH and RH + GRA15II and two times with GT-1. D. Plaque assay was performed as described in Fig 1A. Plaques were counted 4 and 6 days p.i. for RH and GT-1, respectively. Experiment was performed three times. Data information: Statistical analysis was done by one-way ANOVA followed with Tukey's multiple comparison test for (A) and two-sample Student's t-test for (C, D). Data are represented as mean ± SEM. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. IFNγ mediates parasite degradation through endo-lysosomal fusionPanel of Pru-infected HFFs
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