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• W4381276339 abstract "Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Cells are perpetually challenged by pathogens, protein aggregates or chemicals, that induce plasma membrane or endolysosomal compartments damage. This severe stress is recognised and controlled by the endosomal sorting complex required for transport (ESCRT) and the autophagy machineries, which are recruited to damaged membranes to either repair or to remove membrane remnants. Yet, insight is limited about how damage is sensed and which effectors lead to extensive tagging of the damaged organelles with signals, such as K63-polyubiquitin, required for the recruitment of membrane repair or removal machineries. To explore the key factors responsible for detection and marking of damaged compartments, we use the professional phagocyte Dictyostelium discoideum. We found an evolutionary conserved E3-ligase, TrafE, that is robustly recruited to intracellular compartments disrupted after infection with Mycobacterium marinum or after sterile damage caused by chemical compounds. TrafE acts at the intersection of ESCRT and autophagy pathways and plays a key role in functional recruitment of the ESCRT subunits ALIX, Vps32 and Vps4 to damage sites. Importantly, we show that the absence of TrafE severely compromises the xenophagy restriction of mycobacteria as well as ESCRT-mediated and autophagy-mediated endolysosomal membrane damage repair, resulting in early cell death. Editor's evaluation This study presents important findings on the mechanism as to how Mycobacterium-containing vacuoles are recognized by host cell factors and subjected to membrane repairment or autophagic degradation using Dictyostelium discoideum as a useful model. The evidence for the role of TrafE in damaged membrane repair and xenophagy induction is convincing. This work will be of interest to cell biologists and microbiologists. https://doi.org/10.7554/eLife.85727.sa0 Decision letter Reviews on Sciety eLife's review process Introduction After its internalisation by professional phagocytes, the first strategy of Mycobacterium tuberculosis and its close relative Mycobacterium marinum to escape the bactericidal phagosome is to impede its maturation and tailor the Mycobacterium-Containing Vacuole (MCV) as a permissive niche allowing the proliferation of the pathogen (Soldati and Neyrolles, 2012). From the very beginning, bacteria progressively gain access to the cytosol through the action of the ESX-1 secretion system, via the secretion of the membranolytic effector EsxA and the mycobacterial branched apolar lipids phthiocerol dimycocerosates (PDIMs)( Pym et al., 2002; Lerner et al., 2018; Quigley et al., 2017; Augenstreich et al., 2019; Lienard et al., 2020; Osman et al., 2020). The ESX-1 secretion system plays a crucial role for M. marinum virulence and intracellular growth, and is primarily encoded by the genes within the ‘region of difference 1’ (RD1) locus. M. marinum mutants with non-functional ESX-1 inflict significantly less MCV membrane damage and fail to become exposed to or escape to the cytosol, hampering their detection by the host cell-autonomous defence machineries (Cardenal-Muñoz et al., 2017; Gao et al., 2004). Once in contact with the cytosol, the wild-type (WT) bacteria within damaged MCVs are detected and tagged with signals that promote MCV damage repair as well as selective anti-bacterial autophagy, termed xenophagy (López-Jiménez et al., 2018). To complete the infection cycle, the pathogen must reach the cytosol and be released by host-cell lysis, exocytosis or ejection in order to disseminate to adjacent bystander cells (Hagedorn et al., 2009; Queval et al., 2017; Cardenal-Muñoz et al., 2017; López-Jiménez et al., 2018). Xenophagy is fundamental for eukaryotic cell-autonomous immunity (Sharma et al., 2018; Deretic et al., 2013). It requires the coordinated action of about 15 autophagy-related genes (ATG) and is initiated by the recruitment of the core-autophagy factors Atg1 (ULK1 in mammals) kinase complex and Atg9 (ATG9 in mammals) to the nascent phagophore – a double membrane crescent-like structure. Its closure around the cargo depends on the Atg16-Atg12-Atg5 E3-like complex, and the lipidation of Atg8 (LC3 in mammals), which marks the phagophore (Mesquita et al., 2017). The resulting autophagosome fuses with lysosomes allowing the degradation of its content. Xenophagy requires detection and selective recruitment of the pathogen to the phagophore by means of autophagy cargo receptors such as p62 (SQSTM1 in mammals) that link ubiquitin-tagged bacteria and host components with Atg8 family proteins on the phagophore membranes, thereby enforcing proximity between the pathogen and the autophagosome (Boyle and Randow, 2013). Autophagy is involved in the removal of extensively damaged compartments by a specific macroautophagy termed lysophagy. Autophagy also cooperates with ESCRT to repair sterile- or pathogen-induced membrane damage (Skowyra et al., 2018; López-Jiménez et al., 2018; Papadopoulos et al., 2020). The ESCRT-III complex functions in membrane deformation away from the cytosol and plays a critical role in various membrane remodelling processes, including phagophore closure, autophagosome-lysosome fusion, plasma membrane, and lysosome membrane repair (Vietri et al., 2020; Schuck, 2020). One thoroughly studied ESCRT function is the coupling of cargo sorting and intralumenal vesicles formation required for degradation of receptors. The endolysosomal and MCV membrane ruptures generate morphological and topological landscape for the detection machineries which deposit ‘repair-me’ and ‘eat-me’ signals (Boyle and Randow, 2013; Koerver et al., 2019; Papadopoulos et al., 2020). Ubiquitination is a well-studied, highly conserved and versatile post-translational modification used as a signalling platform in diverse catabolic processes and playing a role during infection with various intracellular bacteria. In mammalian cells, the RING domain E3 ligases LRSAM1 and TRAF6 have been shown to promote ubiquitination of vacuoles containing Salmonella Typhimurium and Chlamydia trachomatis bacteria, and the protozoan parasite Toxoplasma gondii (Huett et al., 2012; Haldar et al., 2015), and NEDD4 (Pei et al., 2017), Parkin (Manzanillo et al., 2013), Smurf1 (Franco et al., 2017) to mediate ubiquitination of mycobacteria and host proteins during infection. In addition, the mammalian cytosolic lectins, galectins, recognize damage by binding exposed glycans on the lumenal membrane leaflets and components of bacteria cell wall, thereby serving as ‘eat-me’ tags for autophagy. Moreover, complexes such as TRIM16-Galectin3 also mediate autophagy removal of damaged phagosomes and lysosomes during pathogen invasion (Chauhan et al., 2016; Randow and Youle, 2014). The human tumour necrosis factor receptor-associated factors (TRAF1-7) function in a number of biological processes such as innate and adaptive immunity and cell death as E3 ubiquitin ligases and scaffold proteins (Xie, 2013). TRAFs are composed of several domains: the N-terminal domain containing a RING motif (except TRAF1), a series of Zn-finger motifs that connect the N- and C-terminal regions and the C-terminal TRAF domain (except for TRAF7) subdivided in TRAF-N coiled-coil domain and TRAF-C domain comprised of seven to eight antiparallel β-strands (Bradley and Pober, 2001; Park, 2018; Joazeiro and Weissman, 2000). The RING domain and the Zn-finger motifs of TRAF6 are required for the E3 ligase activity and together with the dimeric E2-conjugating enzyme complex Ubc13-UeV1A, TRAF6 catalyses the generation of poly-Ub chains linked via the lysine K63 of Ub, in a homo- or hetero-oligomerisation-dependent manner with other TRAF members (Middleton et al., 2017). The RING domain is also important for dimerization of trimeric TRAF proteins allowing the formation of signalling networks (Das et al., 2022). The TRAF-N domain enables TRAF proteins homo- and hetero-oligomerisation that is also crucial for their E3 ligase activity. The TRAF-C domain allows interaction with receptor and adaptor proteins (Yin et al., 2010). Endomembrane ruptures on one hand permit the pathogen to gain access to the cytosol, but on the other hand to be detected. Then, together with the vacuole membrane remnants, bacteria are targeted to the autophagy machinery. Defects in endomembrane repair and lysophagy correlate with weakened cellular defences against pathogens, ageing, cancer and neurodegeneration (Papadopoulos and Meyer, 2017). Even though the role of ‘eat-me’ signals in autophagy is well understood, little is known about their implication in detection and repair of pathogen-induced or sterile membrane damage. Furthermore, how pathogen and membrane damages are detected, leading to the deposition of such ‘eat-me’ signals, remains unknown. Using the social amoeba Dictyostelium discoideum – M. marinum model system for host-pathogen interactions, we have shown previously, that upon infection, M. marinum does colocalize with (Ub) and the autophagy markers Atg8 and p62, and that autophagy deficiency results in drastic increase in bacteria proliferation, and that failure to repair or recycle damaged MCVs results in increased cell death (Cardenal-Muñoz et al., 2017; Wang et al., 2018; Eriksson et al., 2020). Here, we have unravelled a novel function of a TRAF-like family protein RING domain-containing E3 ligase. Among half a dozen candidates explored, we found that the evolutionary conserved E3-ligase TrafE is recruited to MCVs or endolysosomes in a membrane damage- or tension-dependent manner. Importantly, we provide evidence that in the absence of TrafE, the autophagy restriction of M. marinum as well as the autophagy-mediated and ESCRT-mediated endolysosomal membrane damage repair are severely compromised, leading to precocious cell death. Results The E3-ligase TrafE is upregulated early-on after infection and is recruited to MCVs Using the sequence of the human TRAF6 as a reference we searched and identified bioinformatically (Figure 1—figure supplement 1A–C) a number of D. discoideum TRAF-like proteins, five of which, namely TrafA, TrafB, TrafC, TrafD, and TrafE harbour all the stereotypical TRAF protein family domains, share remarkable structural similarity with human TRAF6 (Figure 1—figure supplement 1D, E) and are expressed during the vegetative stage of the life cycle (Dunn et al., 2018; Stajdohar et al., 2017). In parallel, RNA-sequencing (RNA-seq) transcriptomic comparison between control and M. marinum-infected D. discoideum cells showed that out of the aforementioned candidates only trafE is upregulated early after infection (Figure 1—figure supplement 2; Hanna et al., 2019). TrafE is also enriched at the early MCV, as revealed by organelle proteomics (Guého et al., 2019). To validate a possible role in infection of the aforementioned TRAF-like proteins, we first sought to identify candidates that display remarkable relocalization during infection. To do so, we generated stably transformed cell lines ectopically expressing TrafA, TrafB, TrafC, TrafD and TrafE candidate proteins, N- or C-terminally fused to GFP, under a constitutive promoter. We monitored the subcellular localization of these GFP fusions by live confocal microscopy over a period of 24 hr in the context of an infection with M. marinum, compared to a mock-infected control. As early as 1 hour post infection (hpi), GFP-TrafE showed robust recruitment to MCVs, sustained over the infection time course, predominantly in the area of M. marinum bacteria poles (Figure 1A, B and C) consistent with previously demonstrated localization of ubiquitin and the autophagy-related Atg8 (López-Jiménez et al., 2018). GFP-TrafA and GFP-TrafB remained solely cytosolic or nuclear, respectively, in control and infection conditions at all time points (Figure 1—figure supplement 3A, B). We confirmed in silico that TrafB harbours a nuclear localization signal (NLS) similarly to human TRAF4, as predicted by both NLS Mapper and NLStradamus (Nguyen Ba et al., 2009; Kosugi et al., 2009). Interestingly, despite TrafC and TrafD being 95.56% similar in terms of amino acid sequence, they displayed different behaviour upon infection. GFP-TrafC like GFP-TrafE was recruited to MCV, which was not the case for GFP-TrafD (Figure 1—figure supplement 3C, D). Figure 1 with 4 supplements see all Download asset Open asset Upon infection TrafE is upregulated and recruited to MCVs. (A) GFP-TrafE-expressing D. discoideum cells (green) were mock-infected or infected and then assessed at 1.5, 3, 6, or 24 hr with mCherry-expressing M. marinum (red). Representative maximum projections of live time-lapse spinning disk confocal images with arrowheads pointing at GFP-TrafE recruitment to MCVs/bacteria. Scale bars correspond to 10 µm. Images are representative of at least three independent experiments. (B) Quantification of the percentage of intracellular MCVs/bacteria positive for GFP-TrafE during the infection time-course. Each point is representative of a 6 random multi-position field with n=2 from N=3 independent experiments. Bars represent SEM. (C) Quantification of GFP-TrafE in the vicinity of M. marinum poles from random images in N>3 independent experiments. (D) Normalized mRNA levels of trafE in mock-infected or M. marinum-infected D. discoideum cells at 1.5, 6, or 24 hr. Shown are mean and SEM of the fold change (FC) representing three independent experiments. Statistical differences were calculated with an unpaired t test (n.s.: non-significant, *: p-value ≤0.05). (E) The intracellular growth of M. marinum-lux was monitored every 1 hr inside WT and TrafE overexpressing cells, for 72 hr. Shown are mean and SEM of the fold change (FC) from three independent experiments. Statistical differences were calculated using Bonferroni multiple comparison test after ANOVA (***: p-value ≤0.001). (F) Recruitment of the endogenous TrafE-GFP (green) to MCV/bacteria (red) after 1.5 hpi. Scale bars correspond to 10 µm. Images are representative of at least three independent experiments. Figure 1—source data 1 Quantification data for Figure 1B. https://cdn.elifesciences.org/articles/85727/elife-85727-fig1-data1-v2.xlsx Download elife-85727-fig1-data1-v2.xlsx Figure 1—source data 2 Quantification data for Figure 1C. https://cdn.elifesciences.org/articles/85727/elife-85727-fig1-data2-v2.xlsx Download elife-85727-fig1-data2-v2.xlsx Figure 1—source data 3 Raw data for RT-qPCR in Figure 1D. https://cdn.elifesciences.org/articles/85727/elife-85727-fig1-data3-v2.xlsx Download elife-85727-fig1-data3-v2.xlsx Figure 1—source data 4 Raw data for intracellular growth assay in Figure 1E. https://cdn.elifesciences.org/articles/85727/elife-85727-fig1-data4-v2.xlsx Download elife-85727-fig1-data4-v2.xlsx Real-Time quantitative-PCR (RT q-PCR) carried out to monitor the transcriptional profile in control and infected cells confirmed up to fourfold upregulation of trafE already at 1 hpi, consistent with the RNA-seq data (Figure 1D and Figure 1—figure supplement 2; Hanna et al., 2019). We also evaluated a possible overexpression phenotype of TrafC, TrafD, and TrafE during infection with bioluminescent M. marinum (M. marinum-lux)(Sattler et al., 2007). Whilst the intracellular growth of M. marinum WT remained unaffected in cells overexpressing TrafC or TrafD, this growth was significantly hampered when TrafE was overexpressed (Figure 1—figure supplement 4A, Figure 1E). These results persuaded us to explore further the role of TrafE in infection as potentially the major E3-ligase candidate. In order to assess potential artefacts of overexpression, a TrafE-GFP chromosomal knock-in (TrafE-GFP KI) was generated and tested functionally by monitoring intracellular growth of M. marinum-lux. Importantly, bacteria growth in the TrafE-GFP KI strain was comparable to that in WT cells (Figure 1—figure supplement 4B), indicating that C-terminal GFP fusion of TrafE does not interfere with its function. In addition, the endogenous TrafE-GFP colocalized with MCVs already 1.5 hr after infection (Figure 1F) like the ectopically expressed GFP-TrafE (Figure 1A). Thus, our data points to TrafE involvement in the early MCV membrane damage detection and the MCV or M. marinum ubiquitination, necessary for repair or degradation. We conclude that TrafE is a conserved E3-ligase upregulated and recruited to the M. marinum compartment upon infection and likely involved in D. discoideum cell-autonomous defence. Loss of TrafE promotes M. marinum early release and replication, and is highly toxic for host cells To explore further whether TrafE is either involved in the host defence response or is exploited by bacteria to favour their proliferation, we generated cell lines in which the trafE coding sequence (CDS) was ablated (trafE-KO), and strains overexpressing TrafE. We observed that the intracellular load of M. marinum-lux was increased in trafE-KO cells in a way comparable to the growth of the pathogen in the autophagy-impaired atg1-KO mutant cells (Figure 2A). This result, together with the decreased M. marinum growth in WT cells overexpressing TrafE (Figure 1E), indicate that the endogenous amount and activity of TrafE is limiting for bacterial restriction. Figure 2 with 1 supplement see all Download asset Open asset Loss of TrafE is detrimental for D. discoideum cells after infection with M. marinum. (A) The intracellular growth of M. marinum-lux was monitored every 1 hr inside WT, atg1-KO, trafE-KO or GFP-TrafE complemented trafE-KO cells, for 72 hr. Shown are mean and SEM of the fold change (FC) representing three independent experiments. Statistical differences were calculated using Bonferroni multiple comparison test after ANOVA ***: p-value ≤0.001. (B) WT, atg1-KO or trafE-KO cells expressing mCherry-Plin were infected with GFP-expressing M. marinum. The percentage of cells with intracellular bacteria colocalizing with Plin was assessed manually at 6 and 24 hpi. Error bars indicate the SEM from 3≤n ≤ 4 relicates from N=3 independent experiments. Statistical differences are indicated with an asterisk and were calculated with an unpaired t test (n.s.: non-significant, *: p-value ≤0.05, **: p-value ≤0.01, ***: p-value ≤0.001). (C–D) WT or trafE-KO cells infected with DsRed-expressing M. marinum imaged by high-content microscopy every 1 hr. Number of infected cells (C) or number of extracellular bacteria (D) were monitored and the average of three replicates n≥200 cells per time point was plotted. Error bars represent SEM. Cell strain-time dependent statistical differences were calculated using Bonferroni multiple comparison test after two-way ANOVA (**: p-value ≤0.01). (E) WT, atg1-KO, trafE-KO or GFP-TrafE-complemented trafE-KO cells infected with mCherry-expressing M. marinum in microfluidic chip single-cell experiment were monitored every 1 hr. The number of live cells was counted at each time point for 20 hr and the counts from N=3 independent experiments were plotted as Kaplan-Meier probability of survival curves. Statistical differences were calculated using Bonferroni multiple comparison correction after two-way ANOVA (n.s.: non-significant, ***: p-value ≤0.001). Figure 2—source data 1 Raw data for intracellular growth assay in Figure 2A. https://cdn.elifesciences.org/articles/85727/elife-85727-fig2-data1-v2.xlsx Download elife-85727-fig2-data1-v2.xlsx Figure 2—source data 2 Quantification data for Figure 2B. https://cdn.elifesciences.org/articles/85727/elife-85727-fig2-data2-v2.xlsx Download elife-85727-fig2-data2-v2.xlsx Figure 2—source data 3 Raw data for high-content segmentation in Figure 2C. https://cdn.elifesciences.org/articles/85727/elife-85727-fig2-data3-v2.xlsx Download elife-85727-fig2-data3-v2.xlsx Figure 2—source data 4 Raw data for high-content segmentation in Figure 2D. https://cdn.elifesciences.org/articles/85727/elife-85727-fig2-data4-v2.xlsx Download elife-85727-fig2-data4-v2.xlsx Figure 2—source data 5 Quantification data for single-cell experiment in Figure 2E. https://cdn.elifesciences.org/articles/85727/elife-85727-fig2-data5-v2.xlsx Download elife-85727-fig2-data5-v2.xlsx It has been demonstrated that atg1-KO cells suffer from a complete loss of autophagy resulting in the absence of xenophagy and thus were strongly impaired in the restriction of cytosolic bacteria (Cardenal-Muñoz et al., 2017; López-Jiménez et al., 2018). To examine whether absence of TrafE also results in a complete autophagy block or in a more specific and limited phenotype, we monitored the progression of the two mutant strains through the developmental cycle (Otto et al., 2004). Upon starvation, atg1-KO cells displayed severe developmental phenotypes and did not proceed beyond aggregation. In contrast, trafE-KO cells successfully completed their developmental cycle and formed viable spores (Figure 2—figure supplement 1A), suggesting that TrafE may not function as a general autophagy regulator but may be more specifically linked to infection- or membrane damage-related activities. Earlier studies also indicated that in the absence of autophagy (e.g. in atg1-KO cells) or ESCRT-III-mediated membrane damage repair (e.g. in tsg101-KO cells), bacteria escape the MCV precociously to the cytosol (Cardenal-Muñoz et al., 2017). As a reporter for M. marinum escape to the cytosol, we monitored Plin, the D. discoideum homolog of the mammalian perilipins known to bind the hydrophobic cell wall of mycobacteria exposed to the cytosol (Barisch et al., 2015; López-Jiménez et al., 2018). The proportion of mCherry-Plin-positive bacteria indicated that, in trafE-KO host cells, M. marinum was exposed to the cytosol significantly earlier compared to WT cells, but similarly to atg1-KO cells (Figure 2B, Figure 2—figure supplement 1B). Importantly, in atg1-KO and tsg101-KO cells, M. marinum escapes precociously to the cytosol but has distinct and even opposite fates. In atg1-KO cells, cytosolic bacteria accumulate Ub but proliferate significantly due to a lack of functional xenophagy. In tsg101-KO cells M. marinum is ubiquitinated and delivered to the autophagy machinery, leading to higher restriction of bacterial growth than in WT cells (López-Jiménez et al., 2018). Interestingly, similarly to autophagy deficient atg1-KO cells, in trafE-KO host cells M. marinum escapes earlier and displays high proliferation, suggesting that both MCV damage repair and xenophagy restriction of bacteria are impaired. To monitor this, we infected WT and trafE-KO cells with mCherry-expressing bacteria and monitored the infection progression by high-content (HC) confocal microscopy. Surprisingly, after 30 hpi we observed a significant decrease in the number of infected trafE-KO cells compared to infected WT cells (Figure 2C). This was paralleled by a high number of extracellular M. marinum that had likely been released by lysis from host trafE mutant cells near 30 hpi (Figure 2D). In conclusion, we hypothesized that M. marinum escapes precociously to the cytosol of trafE-KO cells, proliferate unrestrictedly and, as a likely consequence, kills its host faster. To validate this hypothesis, we employed a microfluidic device, the InfectChip (Delincé et al., 2016), in which single infected cells are trapped to allow long-term recording of host and pathogen fate (Mottet et al., 2021). We confirmed that M. marinum is highly toxic for trafE-KO cells, a phenotype almost fully complemented by GFP-TrafE overexpression (Figure 2E). Surprisingly, the survival probability of infected autophagy-deficient atg1-KO host cells was not significantly affected under these conditions and time-frame (Figure 2E), even though M. marinum intracellular growth is also drastically increased in these mutants compared to WT (Cardenal-Muñoz et al., 2017). Increased cell death likely occurs later in atg1-KO (López-Jiménez et al., 2019) compared to trafE-KO cells. Altogether, these results reveal that as previously observed in atg1-KO cells, absence of TrafE leads to M. marinum early escape from the MCV to the cytosol, and strongly affects its targeting by the xenophagy machinery. Importantly, the acute toxicity of M. marinum in trafE-KO cells indicates that, in contrast to Atg1, TrafE is maybe more broadly involved in cell survival mechanisms. TrafE action is triggered by M. marinum-induced MCV membrane damage Previously, we have shown that mycobacteria escaping to the cytosol are captured and restricted by xenophagy (López-Jiménez et al., 2018). Taking into account that MCV membrane disruption prompts the recruitment of both the ESCRT and the autophagy machineries, we hypothesized that TrafE colocalization with M. marinum depends on membrane damage. Both, M. tuberculosis and M. marinum share the genomic locus Region of Difference 1 (RD1) encoding the ESX-I (type VII) secretion system required for the secretion of the membranolytic virulence factor EsxA and its chaperone EsxB (Hagedorn et al., 2009). As previously shown (Cardenal-Muñoz et al., 2017), in D. discoideum cells infected with M. marinum ∆RD1, which induces less MCV damages, ubiquitination of bacteria and their compartment is significantly decreased. In addition, nearly 80% of GFP-TrafE recruitment to MCVs was observed in the vicinity of bacteria poles (Figure 1C), where ESX-1 is localized and active in both M. tuberculosis and M. marinum (Carlsson et al., 2009). Interestingly, upon infection with M. marinum ∆RD1, TrafE-GFP recruitment to MCV was severely reduced (Figure 3A and B). Previously, we showed that both intracellular growth of WT M. marinum as well as autophagosome formation depend on a functional ESX-1 system. Consequently, the bacteria load of M. marinum ∆RD1 was significantly and similarly reduced in both WT and atg1-KO cells (Cardenal-Muñoz et al., 2017). Therefore, we measured the intracellular growth of WT and M. marinum ∆RD1 bacteria. Lack of the ESX-1 system drastically reduced bacterial proliferation in both WT and trafE-KO cells (Figure 3C), indicating that the growth advantage of WT M. marinum in trafE-KO cells is strictly dependent on MCV damage and escape to the cytosol. In addition, the same microfluidic device for long-term recording of single infected cells (Figure 2E) also revealed that the survival probability of both trafE-KO and atg1-KO host cells was not affected during infection with M. marinum ∆RD1 (Figure 3D). In conclusion, we propose that membrane damage is required for TrafE recruitment to MCVs and subsequent restriction of bacteria proliferation by xenophagy. Figure 3 Download asset Open asset TrafE recruitment to MCVs/bacteria is membrane damage-dependent. (A) D. discoideum cells expressing endogenous TrafE-GFP (green) were infected and then assessed at 1.5, 3, or 24 hr with M. marinum WT (red) or M. marinum ∆RD1 (red). Representative maximum projections of live images with arrowheads pointing at TrafE-GFP recruitment to MCVs/bacteria. Scale bars correspond to 10 µm. Images are representative of three independent experiments. (B) Quantification of the percentage of intracellular MCV/bacteria positive for TrafE-GFP during the infection time-course. SEM from two to four independent experiments. Statistical differences were calculated with an unpaired t test (****: p-value ≤0.0001). (C) The intracellular growth of M. marinum-lux WT or M. marinum ∆RD1 was monitored every hour inside WT or trafE-KO cells, for 72 hr. Shown are mean and SEM of the fold change (FC) representing three independent experiments. Statistical differences were calculated using Bonferroni multiple comparison test after one-way ANOVA (***: p-value ≤0.001). (D) WT, atg1-KO, trafE-KO, or GFP-TrafE-complemented trafE-KO cells infected with M. marinum-lux ∆RD1 in microfluidic chip single-cell experiment were monitored every 1 hr. The number of live cells was counted at each time point for 20 hr and the counts from three independent experiments were plotted as Kaplan-Meier survival curves. Statistical difference were calculated using Bonferroni multiple comparison correction after ANOVA (n.s.: non-significant). Figure 3—source data 1 Quantification data for Figure 3B. https://cdn.elifesciences.org/articles/85727/elife-85727-fig3-data1-v2.xlsx Download elife-85727-fig3-data1-v2.xlsx Figure 3—source data 2 Raw data for intracellular growth assay in Figure 3C. https://cdn.elifesciences.org/articles/85727/elife-85727-fig3-data2-v2.xlsx Download elife-85727-fig3-data2-v2.xlsx Figure 3—source data 3 Quantification data for single-cell experiment in Figure 3D. https://cdn.elifesciences.org/articles/85727/elife-85727-fig3-data3-v2.xlsx Download elife-85727-fig3-data3-v2.xlsx K63-ubiquitination levels are decreased in the absence of TrafE A hallmark of damaged MCVs is ubiquitination known to play an essential role in pathways such as cell survival (e.g. autophagy), intracellular pathogen clearance (e.g. xenophagy) and cell death (e.g. apoptosis, necrosis)(Cardenal-Muñoz et al., 2017; Papadopoulos and Meyer, 2017; Yoshida et al., 2017; López-Jiménez et al., 2018; Gómez-Díaz and Ikeda, 2019; Papadopoulos et al., 2020). We carried out immunofluorescence assays using antibodies specific to K63-linkage polyubiquitin and determined that GFP-TrafE colocalized with K63-Ub on MCVs (Figure 4A). Next, we compared the total levels of polyubiquitin-positive MCVs or K63-linked polyubiquitinylated MCVs in M. marinum-infected WT or trafE-KO cells, using a global anti-polyubiquitin antibody or the specific K63-linkage po" @default.
• W4381276339 created "2023-06-20" @default.
• W4381276339 creator A5038112377 @default.
• W4381276339 date "2023-02-16" @default.
• W4381276339 modified "2023-10-03" @default.
• W4381276339 title "Decision letter: A TRAF-like E3 ubiquitin ligase TrafE coordinates ESCRT and autophagy in endolysosomal damage response and cell-autonomous immunity to Mycobacterium marinum" @default.
• W4381276339 doi "https://doi.org/10.7554/elife.85727.sa1" @default.
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