The ubiquitin ligation machinery in the defense against bacterial pathogens

Review13 September 2021Open Access The ubiquitin ligation machinery in the defense against bacterial pathogens Ishita Tripathi-Giesgen Ishita Tripathi-Giesgen Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Christian Behrends Corresponding Author Christian Behrends [email protected] orcid.org/0000-0002-9184-7607 Munich Cluster for Systems Neurology (SyNergy), Medical Faculty, Ludwig-Maximilians-University München, München, Germany Search for more papers by this author Arno F Alpi Corresponding Author Arno F Alpi [email protected] orcid.org/0000-0002-9572-7266 Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Ishita Tripathi-Giesgen Ishita Tripathi-Giesgen Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Christian Behrends Corresponding Author Christian Behrends [email protected] orcid.org/0000-0002-9184-7607 Munich Cluster for Systems Neurology (SyNergy), Medical Faculty, Ludwig-Maximilians-University München, München, Germany Search for more papers by this author Arno F Alpi Corresponding Author Arno F Alpi [email protected] orcid.org/0000-0002-9572-7266 Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Author Information Ishita Tripathi-Giesgen1, Christian Behrends *,2 and Arno F Alpi *,1 1Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany 2Munich Cluster for Systems Neurology (SyNergy), Medical Faculty, Ludwig-Maximilians-University München, München, Germany *Corresponding author. Tel: +49(0)89440046509; E-mail: [email protected] *Corresponding author. Tel: +49(0)8985782480; E-mail: [email protected] EMBO Reports (2021)22:e52864https://doi.org/10.15252/embr.202152864 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The ubiquitin system is an important part of the host cellular defense program during bacterial infection. This is in particular evident for a number of bacteria including Salmonella Typhimurium and Mycobacterium tuberculosis which—inventively as part of their invasion strategy or accidentally upon rupture of seized host endomembranes—become exposed to the host cytosol. Ubiquitylation is involved in the detection and clearance of these bacteria as well as in the activation of innate immune and inflammatory signaling. Remarkably, all these defense responses seem to emanate from a dense layer of ubiquitin which coats the invading pathogens. In this review, we focus on the diverse group of host cell E3 ubiquitin ligases that help to tailor this ubiquitin coat. In particular, we address how the divergent ubiquitin conjugation mechanisms of these ligases contribute to the complexity of the anti-bacterial coating and the recruitment of different ubiquitin-binding effectors. We also discuss the activation and coordination of the different E3 ligases and which strategies bacteria evolved to evade the activities of the host ubiquitin system. Glossary ARIH1 ARIadne 1 homolog ATG Autophagy-related BMDM Bone marrow-derived macrophages BMP Bone morphogenic protein CALCOCO Calcium-binding and coiled-coil domain-containing protein CC1 Coiled-coil 1 CRL Cullin RING ligase DUB Deubiquitinating enzyme GAS Group A streptococcus GIR Galectin interacting region GWAS Genomewide association study HECT Homologous to E6AP carboxyl terminus HHAR1 Homologues to human ARIadne 1 HOIL-IL Heme-oxidized IRP2 Ub ligase 1L HOIP HOIL-1-interacting protein IBR In between RING IKK IκB kinase LAP LC3-associated phagocytosis LDD Linear Ub-determining domain LIR LC3-interacting region LPS Lipopolysaccharide LRR leucine-rich repeat LRSAM1 Leucine-rich repeat and sterile alpha motif-containing protein 1 LUBAC Linear Ub assembly complex MAP1LC3B Microtubule-associated proteins 1A/1B light-chain 3B NBR1 Neighbor of BRCA1 NDP52 Nuclear dot protein 52 NEL Novel E3 ligase NEMO NF-kB essential modifier NF-kB Nuclear factor kappa-B NOD1 Nucleotide-binding oligomerization domain-containing protein 1 OMP Outer membrane protein OPTN Optineurin OTULIN OTU domain DUB with LINear linkage specificity PAMP Pathogen-associated molecular pattern PE Phosphatidylethanolamine PINK1 PTEN-induced kinase 1 RBR RING-between-RING RING Really interesting new gene RNF213 Ring finger protein 213 SAM Sterile alpha motif-containing domain SCV Salmonella-containing vacuole SHARPIN Shank-associated RH domain interacting protein SIM Structured illumination microscopy SLO Streptolysin O Smurf1 SMAD Ub regulator factor 1 SQSTM1 Sequestosome-1 TAK1 Transforming growth factor-β-activated kinase 1 TBK1 TANK-binding kinase 1 TGF-β transforming growth factor beta TLR Toll-like receptor TRAF2 TNF receptor-associated factor 2 TRIM21 Tripartite motif-containing protein 21 TXN Thioredoxin Ub Ubiquitin UBA Ubiquitin-associated UBL Ubiquitin-like domain UPD Unique Parkin domain UVRAG UV radiation resistance associated ZF Zinc finger Introduction Most mammalian cell types have the capacity to defend and protect themselves against pathogens, thereby contributing to cell-autonomous innate immunity. Over the last two decades, macroautophagy has emerged as a critical innate immunity pathway that detects a variety of cytosolic pathogens and initiates their disposal. Originally, macroautophagy has been described as a catabolic process that sequesters unwanted intracellular components, including misfolded proteins and dysfunctional organelles, and eliminates them through the lysosomal degradation machinery (Fig. 1A). A hallmark of macroautophagy is the de novo formation of a double-membrane vesicle called autophagosome (Nakatogawa, 2020). Autophagosome biogenesis proceeds from an isolated crescent-shaped membrane structure (called phagophore or isolation membrane) that is derived from preexisting endomembranes including the ER and that grows around, and ultimately encloses cytosolic components. To complete macroautophagy, autophagosome fuses with the lysosome, forming an autolysosome to allow disintegration of the inner membrane and the degradation of the sequestered material by diverse lysosomal hydrolases, such as lipases, proteases, glycosylases, and nucleases (Glick et al, 2010; Rubinsztein et al, 2012). To date, an increasing number of autophagy-related (ATG) proteins have been identified that are essential for the formation of the autophagosome and its fusion with the lysosome. Their molecular details and mechanistic interactions have been worked out over the last decades, their description, however, would exceed the scope of this review and the interested reader is referred to some excellent recent reviews (Noda & Inagaki, 2015; Nakatogawa, 2020). Figure 1. Selective macroautophagy (A) Key steps in selective macroautophagy. (1) The process of macroautophagy is initiated by isolation membranes and vesicles that gradually expand and mature to phagophores decorated with membrane-anchored LC3 and GABARAPs (orange hexagons). (2) These act as a binding site for autophagy cargo receptors (brown) allowing direct delivery and accumulation of cellular cargo. Finally, the phagophore closes forming a double-membraned vesicle called autophagosome (3), which subsequently fuses with the lysosome forming an autolysosome (4), where the content is degraded by lysosomal enzymes. (B) Xenophagy, selective macroautophagy of Salmonella. (1) Salmonella enters host cells by forming a Salmonella-containing vacuole (SCV) that protects it from the host surveillance system and serves as a replicative niche. (2) SCVs can rupture allowing access of host cytosolic proteins. (3) The exposed glycans, that are normally present on the outer side of the cell membrane, serve as a danger signals that are recognized by galectins (purple triangles). Besides, Salmonella is detected by E3 ligases that generate a dense Ub coat, consistent of different linkage-type Ub chains (red and orange circles), around Salmonella. (4) Both, galectins and Ub recruit a variety of autophagy receptors (brown) that mediate Salmonella capture by LC3-conjugated phagophores and Salmonella-containing autophagosomes (5) are targeted for lysosomal degradation (6). Download figure Download PowerPoint Macroautophagy comes in two flavors, depending on how degradation targets are sequestered. During bulk macroautophagy, the forming autophagosome engulfs randomly cytoplasmic components that are close by. This form of macroautophagy is active at a basal level in nutrient-rich conditions, but can be upregulated in response to several stress conditions, such as starvation, thereby playing an integral part in recycling nutrients to maintaining energy homeostasis. By contrast, during selective macroautophagy, certain molecules, structures, and organelles are recognized via eat-me signals by target-specific proteins named autophagy receptors, which in turn mediate a stepwise recruitment of different macroautophagy machinery components to initiate and scaffold autophagosome formation exclusively around a distinct cargo (Reggiori et al, 2012; Stolz et al, 2014; Zaffagnini & Martens, 2016). Selective macroautophagy can be classified according to its cargo, including mitophagy (mitochondria), ribophagy (ribosomes), ER-phagy (ER), lipophagy (lipid droplets), lysophagy (lysosomes), and pexophagy (peroxisomes) (Gatica et al, 2018). However, cells utilize selective macroautophagy not only to clear and recycle their own cellular material, but can also direct the macroautophagy machinery to eliminate invaded cellular pathogens, a process commonly known as antimicrobial macroautophagy or xenophagy—from Greek for 'foreign/strange' and 'eating' (Hu et al, 2020). Non-canonical forms of autophagy that do not involve the formation of double-membrane autophagosomes such as LC3-associated phagocytosis (LAP) also contribute to eliminate pathogens (Sanjuan et al, 2007; Mehta et al, 2014; Heckmann et al, 2017; Martinez, 2018). However, in this review, we will focus on our current mechanistic understanding of xenophagy and discuss recent advances on the role of the ubiquitin (Ub) machinery in targeting pathogenic bacteria for their clearance and how bacteria developed ways to fend off this process for survival or to even hijack it for their own benefit. Mechanism of ubiquitin-mediated xenophagy Intracellular pathogens, such as the bacteria Shigella flexneri (S. flexneri), Mycobacterium tuberculosis (M. tuberculosis), Salmonella enterica serovar Typhimurium (S. Typhimurium), and Listeria monocytogenes (L. monocytogenes), are able to reside and reproduce inside the host cell and are responsible for many severe human diseases. Besides passive engulfment by macrophages and neutrophils via phagocytosis, bacteria evolved different strategies to actively invade endothelial and epithelial host cells (Cossart & Sansonetti, 2004). Following their entry, pathogens can either reside inside membranous compartments (vacuoles) or escape to the cytosol. Nevertheless, xenophagy can target these bacteria to restrict their growth and replication. Since S. Typhimurium is one of the best-studied examples of bacteria that are targeted and eliminated by xenophagy (Herhaus & Dikic, 2018), we will use this model substrate to describe the different steps and key features of antimicrobial macroautophagy (Fig. 1B). After internalization, S. Typhimurium resides in an acidic compartment called the Salmonella-containing vacuole (SCV). SCVs are used by S. Typhimurium as a permissive niche for survival and growth (LaRock et al, 2015). Numerous bacterial genes, including the effector SifA, are required for the extension of the membranous surface of the SCV to accommodate the replicating bacteria. However, in the early phase of infection (usually within the first hour), a subset of SCV membranes is damaged and specific signature molecules become exposed on the bacterial cell wall as well as on the luminal surface of the SCV. Specifically, β-galactosides—which normally modify the extracellular side of the plasma membrane and the luminal surface of endosomes—are recognized on the damaged SCV membranes by the fast recruitment of the β-galactoside-binding lectin, galectin-8 which acts as an essential damage and/or pattern-recognition receptor and directly associates with the autophagy receptor NDP52 (also known as CALCOCO2) via its the galectin-interacting region (GIR) motif (Thurston et al, 2012). Besides β-galactosides/galectin-8-initiated autophagy, damaged SCVs and exposed Salmonella activate the cellular Ub machinery and trigger the formation of a dense Ub coat dressing the whole Salmonella (Perrin et al, 2004). Ubiquitylation, the covalent attachment of Ub to substrate proteins, requires the orchestrated action of at least three enzymes working in a relay, an Ub-activating enzyme (E1), an Ub-conjugating enzyme (E2), and an Ub ligase (E3) (Hershko et al, 2000; Buetow & Huang, 2016). The E3s play a central part in substrate specificity and can be classified into three main families depending on the presence of characteristic domains and on the mechanism of Ub transfer to usually lysine residues of substrate proteins: really interesting new gene (RING) E3 ligases catalyze via allosteric activation of the E2˜Ub conjugate (˜ indicating a thioester bond) the direct Ub transfer from the E2 to the substrate, whereas homologous to the E6AP carboxyl terminus (HECT) E3 ligases and Ring-Between-Ring (RBR) E3 ligase utilize a catalytic cysteine forming a transient E3˜Ub thioester before mediating Ub transfer onto substrates (Zheng & Shabek, 2017). Substrates can be modified by single Ub molecules on one or multiple lysins (mono- or multi-monoubiquitylation) and/or polyubiquitin chains (polyubiquitylation) can be formed by linking Ub molecules via one of their seven lysine residues (K6, K11, K27, K29, K33, K48, K63) or N-terminal methionine (M1). These Ub chains are structurally distinct and can come in different variations, homotypic (single linkage type) or heterotypic (multiple, mixed linkage types) including branched polyubiquitin chains, resulting in a complex Ub code (Komander & Rape, 2012). Identity and origin of ubiquitylation targets forming the Ub coat were assessed by quantitative proteomics of S. Typhimurium-infected epithelial cells and revealed ubiquitylation sites on several outer membrane-associated and integral outer membrane proteins of Salmonella (Fiskin et al, 2016). Moreover, several chain types are associated with the Ub coat of S. Typhimurium including M1- (also known as linear Ub chains), K48-, and K63-polyubiquitin chain linkages, suggesting a complex structure and signaling platform of the Ub coat (van Wijk et al, 2012; Manzanillo et al, 2013). Over the last two decades, several E3 ligases like Smurf1, Parkin, ARIH1, LRSAM1, RNF213, and LUBAC, were identified to ubiquitylate more than one type of bacteria, and in some cases, the bacteria are targeted simultaneously by multiple E3 ligases. For instance, ARIH1, LRSAM1, RNF213, and LUBAC are known to colocalize with damaged SCVs and promote cytosolic S. Typhimurium Ub coat formation. There is also a group of E3 ligases like RNF166 (Heath et al, 2016), MARCH8 (Jin et al, 2017), TRIM21 (Hos et al, 2020), TRIM22 (Lou et al, 2018), and TRIM16 (Chauhan et al, 2016) that do not recognize the pathogen itself but recognize other aspects of infection like exposed glycans (Chauhan et al, 2016). Similar to Galectin-8, the bacterial Ub coat functions as a xenophagic eat-me signal (Birmingham et al, 2006) and triggers the binding of autophagy receptors such as p62 (alias SQSTM1), NDP52 (alias CALCOCO2), and optineurin (OPTN) which are essential for the engulfment of bacteria by autophagosomes (Zheng et al, 2009; Wild et al, 2011; Thurston et al, 2012). These receptors are multifunctional proteins harboring distinct ubiquitin-binding domains (UBD) with inherent specificities toward different ubiquitin chain types, as well as protein–protein interaction motifs that mediate the recruitment of autophagy machinery components to induce the formation and elongation of phagophore membranes proximal to bacteria (Kirkin & Rogov, 2019). The modular nature of these receptors is best understood in the case of NDP52 which employs a GIR and an Ub-binding zinc finger (ZF) to associate with bacteria, a SKICH domain to recruit the autophagy promoting ULK1 and TBK1 kinases via their respective adaptors FIP200 and SINBAD/NAP1 as well as an LC3-interacting region (LIR) to guide the engulfment of bacteria by the nascent autophagosome via LC3C (von Muhlinen et al, 2012; Thurston et al, 2012; Ravenhill et al, 2019). While NDP52's ZF seems to promiscuously bind mono- and polyubiquitin (Xie et al, 2015), the UBAN domain of OPTN specifically recognizes M1- and K63-linked ubiquitin chains of the Salmonella Ub coat (Wild et al, 2011). TBK1-mediated phosphorylation of OPTN fosters its interaction with LC3, thereby supporting phagosome/autophagosome formation and restriction of Salmonella growth (Wild et al, 2011). Notably, the actions of multifunctional Ub-binding autophagy receptors are not limited to xenophagy, but shared among other selective autophagy processes including aggrephagy and mitophagy (Turco et al, 2019; Vargas et al, 2019; Shi et al, 2020; Yamano et al, 2020). There are also some special cases, like xenophagy of Mycobacterium tuberculosis, where cytosolic Ub is directly binding to the UBA domain of the M. tuberculosis surface protein Rv1468c leading to the recruitment of p62 and LC3-mediated delivery of the bacteria to lysosomes (Chai et al, 2019). Hence, Ub plays a central role as danger signal and sensor of cytosolic bacteria, highlighting the importance of the Ub machinery in xenophagy. E3 ubiquitin ligases associated with xenophagy The Ub coat on the surface of cytosolic bacteria including S. Typhimurium and the nonmotile ΔactA mutant of L. monocytogenes was first described by Perrin and colleagues in 2004 (Perrin et al, 2004). This seminal discovery initiated extensive research efforts in the past two decades to identify the Ub-targeting machinery that recognizes and targets cytosol bacteria for Ub-mediated xenophagy and innate immune responses. In this chapter, we will describe in a historic order the identification of xenophagy-associated E3 ligases. We will discuss our current knowledge of their E3-typical features and mechanisms in context of (i) sensing/recognition of cytosolic bacteria (ii) E3 ligase activation and (iii) type of Ub substrate modification. LRSAM1 In 2011, the first E3 ligase, leucine-rich repeat and sterile alpha motif-containing protein 1 (LRSAM1), was functionally linked to Ub coat synthesis and xenophagy (Ng et al, 2011). LRSAM1, first shown to play a role in the endosomal sorting machinery (Amit et al, 2004; McDonald & Martin-Serrano, 2008), belongs to the RING-type E3 ligases that share the presence of a RING domain (Fig. 2). Canonical RING domains comprise conserved cysteines and histidines that allow coordinate binding of two zinc ions thereby stabilizing an overall globular three-dimensional domain structure (Deshaies & Joazeiro, 2009). RING domains themselves lack Ub transfer activity, but accommodate the charged E2˜Ub in a distinct architecture known as the closed conformation that allosterically activates E2˜Ub for the nucleophilic attack by a suitable placed substrate lysine to form an isopeptide bond (Dou et al, 2012, 2013; Plechanovova et al, 2012; Pruneda et al, 2012; Branigan et al, 2020). Apart from the C-terminal RING domain, LRSAM1 contains a leucine-rich repeat (LRR) domain at the N terminus, a sterile alpha motif-containing domain (SAM domain), and two coiled-coil domains (CC1 and CC2), which are known to regulate the activity of E3 ligases by forming self-associated oligomers (Bian et al, 2017). Figure 2. The RING-type E3 ligase LRSAM1 in Ub coat formation of cytosolic Salmonella The domain architecture of LRSAM1 showing the leucine-rich repeats (LRR) domains (red dotted box) that detects PAMPs of Salmonella and mediates bacterial binding, coiled-coil (CC) domains, a sterile alpha motif (SAM) containing domain, and the C terminus RING domain with E3 ligase activity. LRSAM1-mediated ubiquitylation of cytosolic Salmonella, preferentially K6- and K27-linked chains, leads to the accumulation of autophagy cargo receptor NDP52 mediating the recruitment of the autophagy machinery. Download figure Download PowerPoint LRRs are typically involved in the detection of pathogen-associated molecular patterns (PAMPs) making LRSAM1 a suitably designed E3 for recognizing and ubiquitylating cytosolic bacteria. Notably, an LRR-mediated recognition mechanism by E3 ligases was already proposed at the time when the Ub coat was first observed (Perrin et al, 2004). LRSAM1 colocalizes with xenophagy-susceptible pathogens including S. Typhimurium, L. monocytogenes EGD-e, S. flexneri, and invasive Escherichia coli, and colocalization is mediated by the LRR domain (Huett et al, 2012). The nature of PAMPs that are recognized by LRR is not characterized yet (Fig. 2). The RING domain of LRSAM1 is dispensable for bacterial association; however, it is essential to promote bacteria-associated ubiquitylation with preference for K6- and K27-linked chain formation in vitro. Whether the Ub coat is formed by bacteria-bound autoubiquitylated LRSAM1 and/or by LRSAM1-mediated ubiquitylation of bacterial surface proteins is not entirely clear. LRSAM1 accumulation with cytosolic Salmonella is peaking 40 min post-infection coinciding with transient galectin-8/NDP52 colocalization. However, LRSAM1 and NDP52 mark spatially different subdomains around Salmonella and are recruited independently (Huett et al, 2012; Thurston et al, 2012). Ub detection follows LRSAM1 occurring an hour post-infection and is thought to foster NDP52 accumulation necessary for the recruitment of the autophagy machinery (Thurston et al, 2012). The functional association of LRSAM1 with xenophagy is essential to control bacterial infection. Depletion of LRSAM1 in epithelial cells promotes cytoplasmic growth of S. Typhimurium. Lymphoblasts derived from Charcot–Marie–Tooth disease patients, which lack expression of LRSAM1 due to a frameshift that truncates the protein including the entire RING domain, are less efficient in controlling bacterial replication of the less virulent S. Typhimurium strain (i.e. NTCC12023; (Huett et al, 2012)). Conversely, the bactericide Biochanin A, a plant isoflavone derivate, was shown to enhance killing of Salmonella in infected HeLa cells and macrophages by reinforced LRSAM1/NDP52 accumulation and autophagosome formation (Zhao et al, 2018). The underlying mechanism of Biochanin A's bactericidal activity is not known, though. LRSAM1, as a pattern-recognition E3, may play a central role in the early step of anti-bacterial defense by the establishment of the initial Ub coat on S. Typhimurium; however, it became clear that the Ub coat is further modified and re-modeled by other xenophagy-associated E3s to create an even more complex signaling platform on cytosolic bacteria. PARKIN Genomewide association studies (GWAS) identified some genetic polymorphism in the parkin gene (PRKN) that are also associated with increased susceptibility to intracellular pathogens, such as S. Typhimurium and Mycobacterium leprae, suggesting a potential connection between mitochondrial homeostasis and xenophagy via Parkin (Mira et al, 2004; Ali et al, 2006). However, Parkin is primarily known for its well-established role in mitophagy and got much attention as it is frequently mutated in autosomal recessive juvenile Parkinsonism, a neurological disorder that leads to the progressive loss of dopaminergic neurons. Parkin belongs to the RBR family of E3 ligases that are defined by common domain organization and catalytic mechanism (reviewed in (Cotton & Lechtenberg, 2020)). The catalytic RBR module consists of three zinc-binding motifs: a RING1 domain with a canonical RING fold mediating E2 binding, followed by an In-Between-RING (IBR) domain, and finally a Rcat (also known as RING2) domain containing the catalytic cysteine (Fig. 3A). Specific for Parkin is the presence of an N-terminal Ub-like (UBL) domain that shares a structural fold similar to Ub and an additional zinc-coordinating unique parkin domain (UPD) (also known as RING0) (Trempe et al, 2013). Both, UBL and UPD play a central role in regulating Parkin's E3 ligase activity. The apo-form of Parkin adopts a multilayered autoinhibited conformation, whereby the UBL and a repressor element (REP) occlude the E2 binding site in RING1, and the UPD domain masks the catalytic cysteine 431 (C431) in Rcat preventing the formation of the E3˜Ub thioester intermediate (Chaugule et al, 2011; Trempe et al, 2013; Wauer & Komander, 2013). Several elegant studies revealed a striking activation mechanism of Parkin in mitophagy. Parkin activation depends on PINK1 kinase, which itself accumulates and is activated on the outer membrane of damaged mitochondria (Matsuda et al, 2010; Narendra et al, 2010; Vives-Bauza et al, 2010; Kondapalli et al, 2012). PINK1 phosphorylates Ub on serine 65 (pS65-Ub) which increases Ub's affinity for an allosteric site—a pS65-Ub-binding pocket—in Parkin. Once bound, pS65-Ub induces a conformational change in the IBR domain resulting in the release of the inhibitory UBL from the RBR module, overall destabilizing the autoinhibitory conformation and making the Rcat accessible for transthiolation. Subsequently, freed UBL becomes a substrate of PINK1 and pS65-UBL further fosters an open, active conformation (Ordureau et al, 2014; Kazlauskaite et al, 2015; Kumar et al, 2015; Sauve et al, 2015; Wauer et al, 2015; Yamano et al, 2015; Aguirre et al, 2017; Gladkova et al, 2018). Figure 3. RBR-type E3 ligases involved in coating cytosolic bacteria with ubiquitin (A) Diagram of Parkin showing the Ub-like (UBL) domain, the unique parkin domain (UPD), and the repressor element (REP), which form the regulatory part of Parkin, and the catalytic part comprising the RBR module at the C terminus. The mechanism for activation of Parkin in response to bacterial infection remains to be addressed but upon Mycobacterium infection, activated Parkin decorates the cytosolic bacteria with an Ub coat (predominantly K63 chains) that recruits p62 and NDP52 autophagy adaptors triggering xenophagy. (B) Domain organization of the three subunits of LUBAC: HOIP, HOIL-IL, and SHARPIN. HOIL-IL and SHARPIN interact via their respective UBL domain with the UBA domain of HOIP (solid lines), with potential contribution of the double NZF (dNZF) domain (dotted line). The dNZF and RBR domains of HOIP (red dotted boxes) are required for binding Salmonella in a two-phase mechanism: in the first phase, LUBAC binds to preexisting Ub chains on Salmonella, in the second phase, enhanced HOIP activity mediates M1- and mixed M1-linked Ub chains synthesis on these preexisting chains and triggers further LUBAC binding. The M1-Ub chains recruit adapter proteins OPTN and NEMO which initiate xenophagy and pro-inflammatory response, respectively. DUB activity of OTULIN and CYLD may counterbalance LUBAC activity. (C) Schematic view of ARIH1 domains showing the acidic/glycine region, UBA-like domain (UBAL), the RBR module, and the regulatory Ariadne domain at the C terminus. ARIH1 decorates the Salmonella with K48-Ub chains. The mechanism of its activation, binding to the cytosolic Salmonella, and the downstream action of ARIH1-mediated K48-Ub chains are not understood. Download figure Download PowerPoint The PINK1/Parkin pathway is tightly linked with damaged mitochondria, however, and rather surprising, it was shown that Parkin is required for M. tuberculosis xenophagy in vitro. M. tuberculosis is a vacuolar pathogen that resides inside a phagosomal compartment after entering the cell. Due to the activity of mycobacterial ESX-1 secretion system, the phagosomal membrane becomes permeable allowing components of the Ub-xenophagy pathway to access M. tuberculosis (Watson et al, 2012). In infected murine bone marrow-derived macrophages (BMDMs), Parkin colocalizes with M. tuberculosis and mediates a predominantly K63-linked Ub coat surrounding M. tuberculosis phagosomes. This contrasts findings by Ordureau et al (2014) indicating that Parkin does not exhibit specificity for single chain types (Manzanillo et al, 2013; Ordureau et al, 2014). Parkin-mediated ubiquitylation subsequently leads to the recruitment of autophagy receptors p62 and NDP52 via UBA and UBZ Ub-binding domains, respectively, that facilitate the delivery of mycobacteria to autophagosomes (Ichimura et al, 2008; Watson et al, 2012; Manzanillo et al, 2013). In vivo data further support an important role of Parkin against a range of intracellular bacterial infections. Park2−/− mice have an extreme susceptibility to M. tuberculosis and L. monocytogenes infections. Moreover, D. melanogaster and C. elegans strains deficient for Parc2 homologues are highly susceptible to infection by pathogens including L. monocytogenes, S. Typhimurium, and Mycobacterium marinum, suggesting a conserv