Expanding the host cell ubiquitylation machinery targeting cytosolic Salmonella
2017; Springer Nature; Volume: 18; Issue: 9 Linguagem: Inglês
10.15252/embr.201643851
ISSN1469-3178
AutoresMira Polajnar, Marina S. Dietz, Mike Heilemann, Christian Behrends,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoArticle7 August 2017free access Transparent process Expanding the host cell ubiquitylation machinery targeting cytosolic Salmonella Mira Polajnar Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt am Main, Germany German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany Munich Cluster for Systems Neurology (SyNergy), Medical Faculty, Ludwig-Maximilians-University München, München, Germany Search for more papers by this author Marina S Dietz Institute of Physical and Theoretical Chemistry, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Mike Heilemann orcid.org/0000-0002-9821-3578 Institute of Physical and Theoretical Chemistry, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Christian Behrends Corresponding Author [email protected] orcid.org/0000-0002-9184-7607 Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt am Main, Germany Munich Cluster for Systems Neurology (SyNergy), Medical Faculty, Ludwig-Maximilians-University München, München, Germany Search for more papers by this author Mira Polajnar Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt am Main, Germany German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany Munich Cluster for Systems Neurology (SyNergy), Medical Faculty, Ludwig-Maximilians-University München, München, Germany Search for more papers by this author Marina S Dietz Institute of Physical and Theoretical Chemistry, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Mike Heilemann orcid.org/0000-0002-9821-3578 Institute of Physical and Theoretical Chemistry, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Christian Behrends Corresponding Author [email protected]i-muenchen.de orcid.org/0000-0002-9184-7607 Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt am Main, Germany Munich Cluster for Systems Neurology (SyNergy), Medical Faculty, Ludwig-Maximilians-University München, München, Germany Search for more papers by this author Author Information Mira Polajnar1,2,3, Marina S Dietz4, Mike Heilemann4 and Christian Behrends *,1,3 1Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt am Main, Germany 2German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany 3Munich Cluster for Systems Neurology (SyNergy), Medical Faculty, Ludwig-Maximilians-University München, München, Germany 4Institute of Physical and Theoretical Chemistry, Goethe University, Frankfurt am Main, Germany *Corresponding author. Tel: +49 89 440046509; E-mail: [email protected] EMBO Rep (2017)18:1572-1585https://doi.org/10.15252/embr.201643851 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 Ubiquitylation is one of the cardinal post-translational modifications in the cell, balancing several distinct biological processes and acting as a pathogen recognition receptor during bacterial pathogen invasion. A dense layer of polyubiquitin chains marks invading bacteria that gain access to the host cytosol for their selective clearance via xenophagy. However, the enzymes that mediate recognition of cytosolic bacteria and generate this ubiquitin (Ub) coat remain largely elusive. To address this, we employed an image-based RNAi screening approach to monitor the loss of Ub on Salmonella upon depletion of human Ub E3 ligases in cells. Using this approach, we identified ARIH1 as one of the ligases involved in the formation of Ub coat on cytosolic bacteria. In addition, we provide evidence that the RING-between-RING ligase ARIH1, together with LRSAM1 and HOIP, forms part of a network of ligases that orchestrates recognition of intracellular Salmonella and participates in the activation of the host cell immune response. Synopsis ARIH1 contributes to the formation of an ubiquitin coat on cytosolic S. Typhimurium. Together with LRSAM1 and HOIP, ARIH1 forms a network of E3 ligases that recognize cytosolic bacteria and mediate xenophagic degradation and host immune response. ARIH1 ubiquitylates cytosolic S. Typhimurium and contributes K48-linked polyubiquitin chains to the bacterial ubiquitin coat. ARIH1 is recruited to S. Typhimurium where it colocalizes with LRSAM1. ARIH1 and LRSAM1 have xenophagy-dependent and -independent functions. ARIH1 and LRSAM1 depletion is compensated by the recruitment of M1-linked polyubiquitin chains to cytosolic bacteria. ARIH1, LRSAM1 and HOIP constitute a regulatory anti-bacterial network. Introduction Salmonella enterica are gram-negative facultative anaerobic bacteria that can be divided into several subspecies and thousands of serovars based on the lipopolysaccharide and flagellar antigens. These serovars can be roughly categorized as typhoidal and non-typhoidal. Salmonella enterica ssp. enterica ser. Typhimurium (S. Typhimurium) belongs to the latter type that generally leads to food poisoning and gastrointestinal disease in a wide range of hosts. In mice, the bacteria are capable of breaching the epithelial barrier of the gastrointestinal tract by phagocytic engulfment or through endocytosis using M cells 1 and cross the gut vascular barrier to disseminate into the liver and the spleen 2. Invasion and intracellular proliferation are facilitated by various effector proteins delivered into host cells by two distinct type III secretion systems (T3SSs) encoded on the Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) 3. In general, the SPI-1 T3SS enables invasion and stimulates the initial inflammatory response while the SPI-2 T3SS contributes to the intracellular proliferation within the Salmonella-containing vacuole (SCV) 4. Macroautophagy (hereafter referred to as autophagy) is a fundamental, evolutionarily conserved, cellular process that enables cells to engulf and digest portions of their cytoplasm in a regulated manner 5. Anti-bacterial autophagy (xenophagy) serves as a cell-autonomous immune mechanism against invasive intracellular bacteria 6. This defensive response includes the formation of a dense coat of polyubiquitin chains that serves as pathogen recognition receptor and directs intracellular bacteria for autophagic degradation 78. Specifically, S. Typhimurium was shown to be targeted and subsequently degraded by the host's ubiquitin (Ub) and autophagy systems, respectively 9. Ubiquitylated bacteria are recruited to autophagosomes by concerted action of the mammalian Atg8 homologues of the LC3 subfamily, which are anchored in the membrane of the forming autophagosome, and several autophagy cargo receptors, namely NDP52, OPTN, and p62 that bind ubiquitylated proteins via their respective Ub binding domains and LC3 through their LC3-interacting regions 10. Ubiquitin is a small protein modifier that labels proteins in a highly specific manner. Conjugation of Ub to a targeted lysine of a protein is regulated by sequential activity of Ub activating (E1), conjugating (E2), and ligating (E3) enzymes. The presence of seven lysines in Ub allows formation of seven homotypic linkage types and multiple possible heterotypic chains 11. Moreover, Ub can form linear (M1) chains in which Ub moieties are connected in a head-to-tail orientation involving the N-terminal methionine 12. The proteins directly involved in detecting bacteria in the host cytosol and generating the ubiquitylation signal required for their subsequent autophagosomal degradation remain loosely defined. For example, RNF166 was recently shown to localize to S. Typhimurium and recruit autophagy receptors and LC3 13. However, ubiquitylation of S. Typhimurium by RNF166 was not tested in this study. To date, the E3 ligases LRSAM1 14 and HOIP 15, which is the catalytic subunit of the linear ubiquitin chain assembly complex (LUBAC), have been demonstrated to be involved in bacteria-associated ubiquitylation during infection with S. Typhimurium. Intriguingly, ubiquitylation of bacteria is only reduced but not abolished in LRSAM1-depleted or LRSAM1-deficient cells 14, and the recruitment of LUBAC requires a pre-existing ubiquitin signal 15. Moreover, in addition to the M1-linked polyUb layer that is regulated by the deubiquitinase OTULIN 16, lysine 63 (K63)-linked polyUb chains have also been detected in the Ub coat surrounding S. Typhimurium 17, while on the other hand LRSAM1 was shown to mediate K6- and K27-linked polyubiquitylation in vitro 14. Thus, it seems highly plausible that other E3 ligases than LRSAM1 and HOIP are involved in this process. The observation that Parkin mediates ubiquitylation of Mycobacterium tuberculosis 18 led us to speculate whether other members of this RING-between-RING (RBR) E3 ligase family besides HOIP play a role in antagonizing S. Typhimurium infections. Results ARIH1 is required to ubiquitylate cytosolic bacteria during S. Typhimurium infection To systematically identify host machineries involved in the ubiquitylation events directed to eliminate S. Typhimurium, we employed an image-based RNAi screening approach using siRNA pools individually targeting all 14 members of the human RBR E3 ligase family 19. Reverse transfection of HeLa cells with validated siRNAs (Fig EV1A) was followed by infection with a GFP-expressing wild-type S. Typhimurium or a strain lacking the bacterial effector SifA (ΔsifA). The latter bacteria undergo more frequent escape from SCVs 20, leading to increased numbers of ubiquitylated cytosolic S. Typhimurium. Importantly, in both strains the expression of GFP was under the control of the glucose 6-phosphate-responsive promoter 21 (hereafter referred to as cytoGFP), which allowed exclusive detection of S. Typhimurium that escaped their SCVs. Immunostaining of fixed cells with an Ub-specific antibody (FK2) was employed to monitor cytosolic bacteria for the loss of their Ub coat upon depletion of RBR family members 2 hours (h) post-infection (p.i.). High-content image acquisition with an automated high-throughput laser-spinning confocal microscope and an algorithm-based image analysis suite was employed to unbiasedly determine the number of GFP-positive S. Typhimurium in cells and the percentage of these bacteria to which Ub colocalized. Importantly, RNAi transfection per se did not affect bacterial invasion or SCV integrity, since transfection with a non-targeting control siRNA did not alter the number of cytosolic S. Typhimurium compared to mock transfection (Fig EV1E). Our screening effort revealed a substantial increase in the number of GFP-positive ΔsifA S. Typhimurium with a marked concurrent decrease in the Ub-positive fraction of these bacteria upon depletion of ARIH1 (also known as HHARI) (Fig 1A). When determining z-scores for the number of GFP-positive as well as Ub- and GFP-positive bacteria in cells depleted for RBR family members, only knockdown of ARIH1 resulted in values that passed the significance threshold of +2 and -2, respectively (Fig 1B and C). Notably, we were unable to detect significant changes upon depletion of any RBR ligase using wild-type S. Typhimurium (Fig EV1B–D). For deconvolution of the pooled ARIH1 siRNAs, we employed four individual siRNAs, which all efficiently depleted ARIH1 from cells (Fig EV1F). Upon infection with cytoGFP-expressing ∆sifA S. Typhimurium, three out of four siRNAs resulted in significantly increased levels of GFP-positive bacteria with a concomitant decrease in bacterial Ub colocalization. This phenotype was comparable to the effect of LRSAM1 depletion (Figs 1D–F and EV1G). Importantly, a similar accumulation of GFP-positive S. Typhimurium was also observed upon knockdown of the core autophagy genes ATG5 and ATG7 (Fig EV1H–J). Together, our findings indicate that ARIH1 is involved in ubiquitylating cytosolic S. Typhimurium and restricting the bacterial load in the cytosol. Click here to expand this figure. Figure EV1. Infection, knockdown, and wild-type S. Typhimurium controls (related to Fig 1) A. Lysates from HeLa cells transfected with indicated pooled siRNAs for 72 h were analyzed by SDS–PAGE and immunoblotting. Due to low specificity of commercially available antibodies, the efficiency of RNAi-mediated depletion of NKLAM and RNF144A could not be assessed by immunoblotting. Notably, Parkin is not expressed in HeLa cells. B. HeLa cells reversely transfected with sicontrol or pooled siRNAs targeting all 14 known RBR Ub E3 ligases for 72 h were infected with wild-type cytoGFP-expressing S. Typhimurium for 2 h prior to fixation and immunolabeling with anti-polyUb antibody (FK2). Number of GFP-positive (GFP+) and Ub-positive and GFP-positive (Ub+/GFP+) bacteria was determined using an automated quantification software and normalized to sicontrol counting on average 800 cells/sample (GFP-positive S. Typhimurium/cell = 3.90 ± 0.42, ubiquitylated S. Typhimurium [%] = 11.76 ± 2.10). Data represent mean ± SD. n = 2 biological replicates. C, D. z-scores of GFP+ (C) and Ub+/GFP+ (D) bacteria from (B). E. HeLa cells transfected with sicontrol or left untreated (mock) for 72 h were infected with ΔsifA cytoGFP-expressing S. Typhimurium for 2 h prior to fixation. Number of GFP+ bacteria in at least 250 cells/sample was determined using automated quantification. Data represent mean ± SD. Significance was determined using unpaired Student's t-test. ns = not significant. n = 3 biological replicates. F–I. Lysates from HeLa cells transfected with indicated single siRNAs for 72 h were analyzed by SDS–PAGE and immunoblotting. J. HeLa cells transfected with indicated single siRNAs for 72 h were infected as in (B) followed by fixation and confocal microscopy. Number of GFP+ bacteria was determined by automated quantification in 250 cells/sample on average. Data represent mean ± SD. Significance was determined using one-way ANOVA. *P < 0.05, **P < 0.01. n = 3 biological replicates. Download figure Download PowerPoint Figure 1. Image-based RNAi screening of RBR E3 ligases involved in bacterial Ub coat formation upon S. Typhimurium infection A. HeLa cells reversely transfected for 72 h with non-targeting control (sicontrol) or pooled siRNAs targeting all 14 known RBR Ub E3 ligases were infected with cytoGFP-expressing ΔsifA S. Typhimurium for 2 h prior to fixation and immunolabeling with anti-Ub antibody (FK2). Number of GFP-positive (GFP+) and Ub- and GFP-positive (Ub+/GFP+) bacteria was determined using automated quantification software and normalized to sicontrol counting on average 800 cells/sample (GFP-positive S. Typhimurium/cell = 3.50 ± 0.13, ubiquitylated S. Typhimurium [%] = 8.32 ± 1.10). Data represent mean ± SD. n = 2 biological replicates. B, C. z-scores of GFP+ (B) and Ub+/GFP+ (C) bacteria from (A). D. HeLa cells were transfected with indicated single siRNAs, infected, fixed, and immunolabeled as in (A). Scale bar: 10 μm. Arrowheads indicate colocalization events. E, F. Automated quantification of GFP+ (E) and Ub+/GFP+ (F) bacteria in on average 250 cells/sample. Data represent mean ± SD. Significance was determined using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant. n = 3 biological replicates. Data information: See also Fig EV1. Download figure Download PowerPoint ARIH1 precedes LRSAM1 in the recruitment to cytosolic S. Typhimurium where both ligases colocalize To examine whether ARIH1 is recruited to cytosolic bacteria during infection, we performed immunofluorescent labeling of cells infected with cytoGFP-expressing ∆sifA S. Typhimurium. Consistent with its ability to ubiquitylate bacteria, we indeed detected endogenous ARIH1 on GFP-positive S. Typhimurium that were also decorated with Ub (Fig 2A), while a reduced colocalization was observed upon ARIH1 knockdown (Fig EV2A). Importantly, a similar recruitment was also observed for the wild-type cytoGFP strain (Fig EV2B). Single-molecule localization microscopy revealed a nanoscale patchlike localization pattern of ARIH1 on these bacteria (Fig 2B). Intriguingly, LRSAM1 showed a similar clustered colocalization on the surface of cytosolic bacteria (Fig 2C). By examining the recruitment kinetics of both E3 ligases to cytosolic bacteria at endogenous levels, ARIH1 was found to localize to S. Typhimurium within the first 30 min p.i. while LRSAM1 was recruited later (2 h p.i.) and to a lesser extent than ARIH1 (Fig 2D and E). However, both ligases were found to colocalize on some of the SCV-escaped bacteria (Fig 2D). At 6 h p.i., bacterial colocalization of ARIH1 and LRSAM1 was heavily diminished, but some LRSAM1-positive S. Typhimurium could still be observed (Fig 2D and E). These results show that ARIH1 is recruited faster to cytosolic S. Typhimurium than LRSAM1 while LRSAM1 persists longer on these bacteria. Figure 2. ARIH1 is recruited to the cytosolic S. Typhimurium, localizes with LRSAM1, and contributes K48-linked polyUb chains to their Ub coat A. HeLa cells were infected with cytoGFP ΔsifA S. Typhimurium for 2 h followed by fixation, immunolabeling with anti-Ub (FK2) and anti-ARIH1 antibodies, and confocal microscopy. Scale bar: 5 μm. B, C. HeLa cells were infected and fixed as in (A) and immunolabeled with anti-ARIH1 (B) or anti-LRSAM1 (C) antibodies followed by additional fixation and super-resolution dSTORM imaging. Scale bar: 2 μm. Arrowheads indicate nanoscale protein patches. D. HeLa cells were infected for indicated p.i. time points and fixed as in (A) prior to immunolabeling with anti-ARIH1 and anti-LRSAM1 antibodies and confocal microscopy. Scale bar: 5 μm. Arrowheads indicate colocalization events. E. Automated quantification of LRSAM1-positive and ARIH1-positive S. Typhimurium from (D) in at least 100 cells/sample. Data represent mean ± SD. n = 2 biological replicates. F, G. HeLa cells transfected with indicated single siRNAs for 72 h and infected as in (A) prior to fixation and immunolabeling with anti-Ub (FK2) and anti-K48 (F) or -K63 polyUb (G) antibodies. Scale bar: 10 μm. Arrowheads indicate colocalization events. H, I. Automated quantification of K48+/GFP+ (F) or K63+/GFP+ (G) bacteria in at least 100 cells/sample from (F and G). Data represent mean ± SD. n = 2 biological replicates. Data information: See also Fig EV2. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Neddylated CRLs are not required for ubiquitylation of cytosolic S. Typhimurium (related to Figs 2, 3, 4) HeLa cells transfected with indicated single siRNAs for 72 h were infected with cytoGFP-expressing ΔsifA S. Typhimurium for 2 h followed by fixation and anti-ARIH1 immunolabeling. Number of ARIH1+/GFP+ bacteria in at least 250 cells/sample was determined using automated quantification. Data represent mean ± SD. n = 2 biological replicates. HeLa cells were infected with cytoGFP wild-type S. Typhimurium for 2 h followed by fixation, immunolabeling with anti-ARIH1 antibody, and confocal microscopy. Arrowheads indicate colocalization events. Scale bar: 5 μm. Integrity control of bacteria. While performing the in vitro ubiquitylation reaction, bacterial supernatants were sampled right before (s1) and immediately after (s2) the reaction. Intact bacteria were used as a positive control. HeLa cells infected as in (A) or left uninfected were treated with 2 μM MLN4924 or DMSO during the course of the infection prior to lysis. Lysates were analyzed by SDS–PAGE and immunoblotting. HeLa cells infected as in (A) were treated with MLN4924 or DMSO as in (D) prior to fixation and immunolabeling with anti-Ub antibody (FK2). Arrowheads indicate colocalization events. Scale bar: 5 μm. HeLa cells were transfected with indicated pooled siRNAs and infected as in (A) prior to fixation and anti-Ub (FK2) immunolabeling. Arrowheads indicate colocalization events. Scale bar: 5 μm. HeLa cells were reversely transfected with indicated pooled siRNAs for 72 h and lysed. Lysates were analyzed by SDS–PAGE and immunoblotting. Download figure Download PowerPoint ARIH1 contributes K48-linked chains to the Ub coat surrounding cytosolic S. Typhimurium M1- and K63-linked polyUb chains were reported to localize to S. Typhimurium 17. Since linear ubiquitylation is exclusively conferred by LUBAC 1215, we sought to address whether ARIH1 modifies cytosolic bacteria with K63-linked polyUb using a chain-specific antibody. As K48-linked polyUb was found to surround M. tuberculosis 18 and cytosolic Mycobacterium marinum in macrophages 22, we also included a K48-specific antibody in our immunofluorescence analysis. To our surprise, we observed that cytosolic S. Typhimurium were positive for both chain types (Fig 2F and G). Unexpectedly, only K48-linked Ub chains were substantially decreased on cytosolic bacteria when ARIH1 was depleted in cells (Fig 2H and I). Thus, these results indicate that the Ub coat surrounding S. Typhimurium in the cytosol is composed of at least three different Ub chain types of which ARIH1 regulates the levels of K48-linked Ub. ARIH1 ubiquitylates S. Typhimurium in vitro To examine whether ARIH1 alone is sufficient to ubiquitylate S. Typhimurium, we set up an in vitro ubiquitylation assay with purified components (Fig 3A). Consistent with previous reports 23 incubation of an ARIH1 variant lacking the autoinhibitory C-terminal Ariadne domain (∆Ariadne) with HA-tagged Ub, UBA1 as E1, and UBCH7 (alias UBE2L3) as E2 enzymes for 1 h at 37°C resulted in robust autoubiquitylation of ARIH1 in an ATP-dependent manner while full-length ARIH1 failed to ubiquitylate itself (Fig 3B). Next, we repeated the above reaction in the presence of S. Typhimurium (cytoGFP ΔsifA). Once ubiquitylation was stopped by EDTA, bacteria were separated from ARIH1 and other ubiquitylation reaction components by repeated centrifugation and washing. Subsequent immunoblot analysis revealed that these bacteria were modified by Ub in a manner dependent on active ARIH1 and ATP (Fig 3C). Notably, the buffer conditions of the in vitro ubiquitylation reaction did not affect the integrity of bacteria (Fig EV2C). To assess the specificity of this in vitro reaction, we examined whether ARIH1 also ubiquitylates purified mitochondria from the filamentous, ascomycete fungus Podospora anserina as an alternative substrate. Although ARIH1 was active in these reactions, we failed to detect any Ub signal on these mitochondria (Fig 3D). Since recent ubiquitinome profiling revealed that outer membrane proteins (OMPs) of S. Typhimurium become ubiquitylated upon their escape to the cytosol 24, we assessed whether ARIH1-mediated S. Typhimurium ubiquitylation requires the presence of OMPs. As expected, ARIH1 was unable to ubiquitylate S. Typhimurium that were stripped off their OMPs by proteinase K pretreatment (Fig 3E, two left lanes). As evident from ARIH1 autoubiquitylation, ubiquitylation reaction components were fully functional in these reactions due to the addition of a serine protease inhibitor (Fig 3E, two right lanes). Collectively, these results show that ARIH1 is sufficient to specifically ubiquitylate S. Typhimurium OMPs. Figure 3. In vitro ubiquitylation of S. Typhimurium by ARIH1 A. In vitro ubiquitylation reaction scheme. B. Purified inactive full-length or C-terminally truncated, active ARIH1 (Δariadne) were incubated with HA-Ub, UBA1, and UBCH7 in the absence or presence of ATP at 37°C for 1 h. Reactions were stopped by addition of EDTA and subjected to SDS–PAGE and immunoblot analysis. C–E. Reactions were carried out as in (B) in the presence of cytoGFP ΔsifA S. Typhimurium (C), purified mitochondria from P. anserina (D), or proteinase K-pretreated S. Typhimurium (E). Once the reactions were stopped, bacteria or mitochondria were repeatedly pelleted by centrifugation and washed followed by separation on SDS–PAGE and immunoblotting. Notably, PMSF was added to the ubiquitylation buffer in (E) to inhibit proteinase K activity during the ubiquitylation reaction. Data information: See also Fig EV2. Download figure Download PowerPoint Ubiquitylation of cytosolic S. Typhimurium does not require cullin-RING ligases Since ARIH1 was shown to require activation by neddylated members of the cullin-RING ligases (CRLs) family in vitro 25 and cooperatively binds the latter to monoubiquitylate CRL substrates 26, we assessed whether activated CRLs were involved in ubiquitylation of S. Typhimurium. To this end, we performed infection experiments in the absence and presence of the neddylation inhibitor MLN4924 27, which interferes with the NEDD8 conjugation cascade and leads to a rapid loss of cullin neddylation (Fig EV2D). Contrary to ARIH1 depletion, the number of Ub-positive cytosolic bacteria was unchanged upon MLN4924 treatment (Figs 4A and EV2E). Consistently, ubiquitylation of cytoplasmic S. Typhimurium was similarly unaffected upon depletion of CUL1 or CUL3 (Figs 4B and EV2F and G), which were both shown to interact and cooperate with ARIH1 in the ubiquitylation of CRL substrates 26. Thus, these results suggest that in response to S. Typhimurium infection ARIH1 is activated by a mechanism that is independent of neddylated CRLs. Figure 4. ARIH1 function during bacterial infection does not require activation by CRLs and encompasses both autophagy-dependent and autophagy-independent roles A. HeLa cells infected with cytoGFP-expressing ΔsifA S. Typhimurium for 2 h or left uninfected were treated with 2 μM MLN4924 or DMSO during the course of infection, fixed, and immunolabeled with anti-Ub antibody (FK2). Number of Ub+/GFP+ bacteria in at least 250 cells/sample was determined by using automated quantification. Data represent mean ± SD. Significance was determined using unpaired Student's t-test. ns = not significant. n = 3 biological replicates. B. HeLa cells were transfected with indicated pooled siRNAs for 72 h, infected, fixed, and immunolabeled as described in (A) prior to fixation. Number of Ub+/GFP+ bacteria was determined by automated quantification in at least 100 cells/sample. Data represent mean ± SD. n = 2 biological replicates. C–H. Wild-type or ATG7 CRISPR/Cas9 knockout HeLa cells transfected for 72 h with indicated siRNAs were infected during indicated p.i. time points as in (A) prior to fixation (C, D, and G) or lysis and serial dilution plating (E, F, and H). Number of GFP+ bacteria in at least 250 cells/sample was determined using automated quantification (C, D, and G). Results in (C–F) were normalized to the 30 min p.i. time point. Data represent mean ± SD. Significance was determined using two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant. n = 3 (wild-type HeLa) or n = 2 (ATG7 knockout HeLa) biological replicates. Data information: See also Fig EV2. Download figure Download PowerPoint ARIH1 and LRSAM1 have xenophagy-dependent and xenophagy-independent functions To examine whether ARIH1 exclusively functions in xenophagy during S. Typhimurium infection, we monitored the amount of intracellular bacteria in wild-type and ATG7 CRISPR/Cas9 knockout HeLa cells at different p.i. time points upon ARIH1 depletion by quantifying GFP-positive bacteria and colony formation units (CFUs). As a positive control, we also analyzed the effect of LRSAM1 knockdown in both conditions. The loss of ATG7 (Fig EV3A) increased the numbers of cytosolic S. Typhimurium at 2 and 6 h p.i. to a similar level than depletion of ARIH1 or LRSAM1 in wild-type cells (Fig 4C and D). Intriguingly, depletion of LRSAM1 or ARIH1 in cells lacking ATG7 led to substantially further increased numbers of GFP-positive bacteria at both p.i. time points (Fig 4C and D). Similar results were obtained for both ligases in wild-type and ATG7-deleted cells at 6 h p.i. by monitoring CFUs (Fig 4E and F). These additive effects of bacterial counts in ATG7 knockout cells upon depletion of either one of the ligases indicate that ARIH1 and LRSAM1 potentially protect the cell against pathogens by mechanisms both dependent and independent of autophagy. Click here to expand this figure. Figure EV3. Colocalization of xenophagy components to cytosolic S. Typhimurium upon ARIH1 depletion (related to Fig 4) A. Lysates from wild-type and ATG7 CRISPR/Cas9 knockout HeLa cells were analyzed by SDS–PAGE and immunoblotting. B–I. HeLa cells transfected with indicated siRNAs for 72 h were infected with ΔsifA cytoGFP-expressing S. Typhimurium for 2 h, fixed, and immunolabeled with anti-LC3B (B), anti-NDP52 (C), anti-p62 (D), or anti-OPTN (E) antibodies. Number of GFP+ bacteria that colocalized with LC3B (F), NDP52 (G), p62 (H), or OPTN (I) was determined by automated quantification in at least 100 cells/sample. Data represent mean ± SD. n = 2 biological replicates. Download figure Download PowerPoint ARIH1 and LRSAM1 play different roles but function in the same anti-bacterial pathway Given that ARIH1 and LRSAM1 both colocalize on bacteria, ubiquitylate cytosolic bacteria and limit the cytosolic pool of S. Typhimurium, we sought to address whether these two ligases share a genetic interaction. To this end, we compared the effect of single and double depletion of ARIH1 and LRSAM1 on the numbers of cytosolic bacteria and CFUs. Noteworthy, there were no significant changes observed in any of the experiments during the first 30 min p.i., indicating that neither single nor double knockdowns affect infection l
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