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ScienceDirect Emerging themes in bacterial autophagy Matthew T Sorbara1 and Stephen E Girardin2 The role of autophagy in the control of intracellular bacterial pathogens, also known as xenophagy, is well documented. Here, we highlight recent advances in the field of xenophagy. We review the importance of bacterial targeting by ubiquitination, diacylglycerol (DAG) or proteins such as Nod1, Nod2, NDP52, p62, NBR1, optineurin, LRSAM1 and parkin in the process of xenophagy. The importance of metabolic sensors, such as mTOR and AMPK, in xenophagy induction is also discussed. We also review the in vitro and in vivo evidence that demonstrate a global role for xenophagy in the control of bacterial growth. Finally, the mechanisms evolved by bacteria to escape xenophagy are presented. Addresses 1 Department of Immunology, University of Toronto, Toronto M5S 1A8, Canada 2 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto M5S 1A8, Canada Corresponding author: Girardin, Stephen E ([email protected])
Current Opinion in Microbiology 2015, 23:163–170 This review comes from a themed issue on Host-microbe interactions: bacteria Edited by David Holden and Dana Philpott
http://dx.doi.org/10.1016/j.mib.2014.11.020 1369-5274/# 2014 Elsevier Ltd. All rights reserved.
the cytosolic side of late endosomes and lysosomes, known as the mTOR complex 1 (mTORC1) [2]. mTORC1 is assembled and functional only when cellular nutrients or cofactors (including amino acids, growth factors, oxygen and ATP) are not limiting, and active mTORC1 in turns phosphorylates several cellular proteins involved in cell proliferation, growth, ribosomal biogenesis and mRNA translation [2]; in addition, active mTORC1 potently inhibits autophagy by promoting the phosphorylation of the ULK1 complex, including ULK1 itself and ATG13 [3]. The kinase AMPK, which is another sensor of cellular energy (and in particular of the AMP/ATP ratio), also regulates autophagy through the phosphorylation of the ULK1 complex, although the global effect of AMPK on autophagy might depend on the trigger (glucose versus amino acid starvation) [3]. In addition to its role in cellular metabolism, numerous lines of evidence indicate that autophagy also plays a key role in innate immunity, by promoting the entrapment and lysosome-mediated degradation of bacterial pathogens, including bacteria that escape into the host cytosol (such as Shigella or Listeria) or those that reside in a modified intracellular vacuole (such as Salmonella and Mycobacterium). While several comprehensive reviews have detailed the role of autophagy in host defense also known as ‘xenophagy’ [4–6], we will highlight here the most recent advances in this rapidly expanding field of research, and present our views on the most important questions that remain to be solved in the coming years.
Bacterial targeting in xenophagy
Introduction Autophagy is an essential cellular process through which cellular materials, such as large macromolecular complexes or defective organelles, are progressively engulfed by a double membrane, or phagophore, resulting in the formation of an autophagosome [1]. After fusion with lysosomes, the constituents are degraded and metabolites recycled. While a constitutive level of autophagy is necessary for normal cellular housekeeping functions, autophagy can be strongly upregulated in conditions of metabolic starvation in order to provide a spare source of metabolites for a limited time before cell death ensues. Several signaling pathways contribute to the regulation of autophagy by metabolic checkpoints [1]. One of the most conserved of those is dependent on the kinase Tor (mTOR in mammals), which forms a large complex on www.sciencedirect.com
An active area of research in the field of xenophagy relates to the question of how infected cells can specifically direct the phagophore to bacteria that are either free in the cytosol or confined into a vacuole, while avoiding to target other cellular constituents, such as organelles. Four main molecular mechanisms are currently known to confer bacterial targeting during xenophagy, which involve: Nod proteins, ubiquitination, galectin-dependent pathway and diacylglycerol (DAG). Nod1 and Nod2: the Nod-like receptor (NLR) proteins Nod1 and Nod2 are cytosolic proteins that detect specific fragments derived from bacterial peptidoglycan and initiate pro-inflammatory signaling (NF-kB and MAP kinase pathways) following the recruitment of the adaptor protein Rip2 [7]. In the initial phase of bacterial invasion, Nod1/2 also directly interacts with the autophagy protein ATG16L1 in a Rip2-independent manner, thereby initiating the recruitment of the autophagic machinery at the site of bacterial entry [8]. However, it remains unclear if Current Opinion in Microbiology 2015, 23:163–170
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Nod1/2 can target bacteria (such as Shigella or Listeria) once they are free in the cytosol. Ubiquitination: it was observed that cytosolic and vacuoleconfined bacteria are rapidly decorated by ubiquitination [9], and different types of ubiquitin chains constitute the ubiquitin coat surrounding bacteria [10,11,12]. Several adaptor proteins, including NDP52, p62, NBR1 and optineurin, have been shown to serve as a bridge between ubiquitin groups and the autophagy protein LC3 [13]. At least some of these adaptor proteins likely serve nonredundant functions, as evidenced by the reported effect of targeting NDP52 [14], p62 [15] or optineurin [16] on bacterial restriction. The nature of the proteins targeted by ubiquitination, and even if those are from the host or the bacteria, remains unclear. However, recent progress was made with the identification of E3 ligases implicated in this ubiquitination process. LRSAM1 was identified as an important E3 ligase in the case of infection with Salmonella [17]. More recently, the ubiquitin ligase parkin was shown to be required for the formation of the ubiquitin coat surrounding Mycobacterium tuberculosis, and for the restriction of M. tuberculosis growth in macrophages [11]. Interestingly, the ubiquitin ligase activity of parkin has a well-established role in mitophagy, and mutations in the human gene encoding parkin lead to increased susceptibility to Parkinson’s disease, but also to infections by Mycobacterium Leprae and Salmonella enterica serovar Typhi, suggesting links between xenophagy and mitophagy, and between susceptibility to neurodegenerative disorders and to infectious diseases [13]. In addition, a role for the intracellular DNA sensor STING in the formation of the ubiquitin coat surrounding M. tuberculosis was identified [18], suggesting that STING and parkin might operate in the same pathway. Finally, S-guanylation of cysteine residues on the surface of intracellular Group A streptococcus by 8-nitro-cGMP was shown to drive polyubiquitination and autophagy [19]. Exposure of galectin proteins: the membrane remnants of vacuolar rupture in Shigella-infected cells were found to accumulate ubiquitinated proteins and autophagy markers [20], suggesting that the event of membrane damage could trigger xenophagy. In agreement, Thurston et al. demonstrated that galectin proteins, such as galectin-8/3/9, which are glycan-binding lectin proteins that can be found in the cytosol, accumulated at sites of membrane damage induced by either Salmonella or by glycyl-L-phenylalanine 2-naphthylamide (GPN), a molecule inducing sterile lysosomal damage [14]. Moreover, galectin-8 recruited NDP52 to the Salmonella-containing vacuole (SCV) through a mechanism distinct and more rapid than the ubiquitinmediated recruitment of NDP52 to the SCV [14]. Thus, galectin proteins might serve as important sensors of danger signals initiated at the damaged membrane by the exposure of glycans to the cytosol, while those glycans are normally only exposed to the luminal side of vesicles. Current Opinion in Microbiology 2015, 23:163–170
DAG: in Salmonella-infected cells, Shahnazari et al. observed early accumulation of DAG around the SCV, and inhibition of DAG formation using pharmacological or genetic approaches resulted in inhibition of bacterial autophagy [21]. While the authors proposed that DAGdependent and ubiquitin-dependent pathways of autophagy targeting to the SCV were independent, the role of DAG in Nod1/2-dependent or galectin/NDP52-dependent xenophagy remains to be addressed.
Mechanisms of xenophagy induction While much attention was given to the mechanisms responsible for bacterial targeting by autophagy (see above), little is known about how autophagy pathways are turned on during infection. Until recently, it was assumed that the constitutive levels of housekeeping autophagy were likely sufficient to drive xenophagy in infected cells. However, it seems counter-intuitive that a critical innate immune mechanism of bacterial restriction would be limited by nutrient sufficiency and the metabolic status of the infected cells; indeed, metabolic sensors, such as mTOR, play key roles in limiting autophagy. Studies in epithelial cell lines demonstrated that Shigella, Listeria and Salmonella all triggered a rapid inhibition of mTOR signaling, which is associated with a burst of autophagy in infected cells, thus showing that xenophagy induction can occur irrespective of the nutrient status of the infected cells [22–24]. Mechanistically, the inhibition of mTOR was driven by amino acid starvation (AAS) caused by membrane damage in the infected cells, resulting in mTOR dissociation from late endosome/lysosome membranes [23]. While Shigella caused a sustained inhibition of mTOR and induction of AAS pathways, infection with Salmonella or Listeria resulted in transient AAS stress and mTOR inhibition, a mechanism that likely accounts, at least in part, for the capacity of these bacteria to escape xenophagy-mediated bacterial restriction [23]. Although the mechanism by which bacteriainduced membrane damage causes AAS is unknown, it is interesting to note that bacterial pore-forming toxins, which induce severe membrane damage, have also been shown to inhibit mTOR and to turn on AAS pathways [25,26]. Moreover, recent evidence showed that the tyrosine kinase focal adhesion kinase (FAK) was recruited to the surface of the SCV in macrophages, resulting in increased activity of the mTORC1 through an unknown mechanism dependent on the Salmonella Pathogenicity Island 2 (SPI-2) [27]. This subversion mechanism thereby amplified autophagy inhibition through mTOR induction.mTOR is not the only critical regulator of nutrient status that influences the autophagy pathway. As mentioned above, AMPK also emerges as a potential key player in these pathways, but the role of this energy sensor in the context of host responses to bacterial pathogens remains poorly characterized. A recent study demonstrated that, in M. tuberculosis infected macrophages, www.sciencedirect.com
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AMPK induction by the drug AICAR was sufficient to promote antibacterial autophagy, in part through the transcriptional upregulation of autophagy-associated genes [28]. However, it remains unknown if bacterial pathogens can directly activate AMPK in infected cells, thereby affecting xenophagy.
Role of xenophagy in bacterial restriction Substantial work in cell lines and primary cell cultures has identified and supported a bactericidal role for autophagy (see Table 1). Interestingly, several groups have found that wild type strains of bacteria that have evolved to replicate in the cytoplasm, including S. flexneri, Francisella tularensis, and L. monocytogenes are not restricted by autophagy. Strikingly, only attenuated strains, lacking specific virulence factors, are efficiently targeted and degraded in autolysosomes. In contrast, bacteria species that reside and replicate within damaged-vacuoles, including M. tuberculosis or S. typhimurium, generally appear to be more efficiently restricted by autophagy (Table 1). In in vitro infection models where loss or stimulation of autophagy does impact on bacterial replication, the loss of autophagy generally increases bacterial replication by less than an order of magnitude with typical values of 2–5 the replication observed in autophagy competent cells. Furthermore, pathogens adapted to an intracellular lifestyle are still able to replicate in autophagy competent cells, and, in the case of bacteria such as Shigella and Listeria, spread from cell to cell. Therefore, a major question in the field has been whether the slight delay or decrease in intracellular replication within a cell translates to a substantial effect at the whole organism level. Several studies in non-mammalian model organisms have identified a critical role for autophagy in the control of bacterial infection. Seminal work from Yano et al. in Drosophila demonstrated that inhibition of autophagy through loss of either ATG5 or the peptidoglycan recognition protein, PGRL-LE, results in a dramatic increase in susceptibility to L. monocytogenes infection [29]. More recently, a critical role for the ubiquitin ligase parkin has been identified in clearance of S. typhimurium, M. marinum and L. monocytogenes in Drosophila [11]. Mostowy et al. recently proposed a zebrafish larvae model for studying the role of autophagy in Shigella infection, and found that loss of the autophagy adaptor p62 results in increased susceptibility to S. flexneri infection [30]. Finally, loss of core autophagy components in either Caenorhabditis elegans or the single cell Dictyostelium triggered increased susceptibility to S. typhimurium infection [31]. Therefore, in non-mammalian systems both the core components of the autophagy machinery (ATG proteins) and the components specific to xenophagy or subsets of autophagy (PGRP-LE, parkin, p62) have been implicated in host defense. However, it must be noted that these infection models in which xenophagy was shown to confer control of intracellular bacteria relied on the interaction between www.sciencedirect.com
bacterial pathogens and host cells that are not the natural targets of these bacteria, raising the possibility that xenophagy could mainly restrict pathogens that are not adapted to their host environment. Recent experiments using murine models of infection have supported a role for autophagy in bacterial pathogen clearance. Mice lacking expression of ATG5 or ATG16L1 in intestinal epithelial cells had increased Salmonella translocation to systemic sites and increased bacterial load [32,33]. Furthermore, mice lacking macrophage expression of ATG5 were more susceptible to M. tuberculosis or L. monocytogenes infection [18,34]. One caveat of these studies is that they do not exclude contributions from the autophagy machinery beyond direct bactericidal activity, such as maintenance of host cell fitness by controlling the turnover of cellular organelles and macromolecular complexes. Interestingly, during infection of mice with the extracellular pathogen Citrobacter rodentium, decreased expression of ATG16L1 triggered a protective hyper-inflammatory response, and a similar phenotype was observed following UPEC infection (see Table 1). In agreement, ATG16L1 was found to negatively regulate Nod1/2-dependent induction of proinflammatory pathways in an autophagy-independent manner [35]. Therefore, further work is needed to clarify the relative contributions of direct bactericidal activity and ancillary functions in the immune response.
Bacterial escape mechanisms from xenophagy One of the most compelling pieces of evidence that xenophagy is a critical innate immune mechanism of bacterial restriction comes from the identification of bacterial strategies that were evolved to escape host autophagy. Schematically, two main escape mechanisms seem to have been designed by bacterial pathogens: autophagy disarming and camouflage. Autophagy disarming: several bacteria have evolved means to inhibit autophagy to favor their intracellular replication. This is the case of M. tuberculosis, which is targeted by autophagy only after addition of rapamycin [36], suggesting that mTOR activity in infected cells is sufficient to turn down xenophagy. In addition, the ESX-1 secretion system was shown to specifically inhibit autophagosome-lysosome fusion, a key step in xenophagy [37]. Salmonella and Listeria also escape autophagy at least in part through the rapid reactivation of mTOR after an initial phase of inhibition caused by membrane damage (see above) [22,23]. A sophisticated example of autophagy disarming was provided in the case of Legionella pneumophila with the discovery that the effector RavZ could cleave the C-terminal end of lipid-conjugated LC3, thus irreversibly preventing the reconjugation of the protein to autophagosomes and thereby inhibiting autophagy [37]. Finally, Listeria phospholipases C were shown to stall Current Opinion in Microbiology 2015, 23:163–170
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Table 1 Autophagy-dependent restriction of bacterial replication. Evidence of a role for autophagy in restricting the proliferation of different model pathogens is indicated with tissue culture models of infection shaded in green, and in vivo infection models shaded in gray Species Group A Streptococcus (GAS)
Primary observation Restriction in xenophagy competent cells
Increased restriction through autophagy stimulation Increased mortality and bacterial load with increased autophagy Mycobacterium species
Restriction in xenophagy competent cells
Restriction with artificial autophagy induction Decreased survival, increased pathology and bacterial load Decreased survival Increased bacterial load, inflammation
Shigella flexneri
Restriction of DIcsB strain No restriction of wild type Shigella No restriction of wild type with autophagy induction Restriction of wild type Shigella, increased difference with DIcsB strain Increased mortality
Salmonella typhimurium
Restriction in xenophagy competent cells
Protection from GAS infection in RNF5 / [46]
mice
Knockdown in macrophage/monocyte lines: IRGM [51], Rubicon [52], parkin [11], NDP52 [18] Knockout cells: PARK2 / BMDMs [11], STING / , ATG5 / , TBK1 / BMDMs [18] RAW264.7 and primary macrophages [36], ATG5 fl/ fl and ATG5 fl/fl x Lyz-Cre BMDM [34] M. tuberculosis infection of ATG5 fl/fl LyzM-Cre mice [18] M. marinum infection of parkin-deficient Drosophila [11] M. marinum infection of p62, or DRAM1 deficient (morpholino treated) zebrafish embryos. Decreased bacterial load with DRAM1 overexpression [53] Wild type and ATG5 / MEFs [38], Tecpr1 siRNA in HeLa, Tecpr1 / MEFs [44] LC3C siRNA, NDP52 siRNA in HeLa [54] Rapamycin treatment HeLa [23] Control compared to ATG16L1 T300A MEFs, primary intestinal epithelial cells (24821797) p62 deficient (morpholino treated) zebrafish embryos [30]
Increased bacterial translocation, and intestinal inflammation Increased intestinal inflammation with ATG16L1 variant expression
MEFs: wild type compared to ATG5 / [55], PKCd / [21], ATG7 / [56], ATG16L1-deficient [57] HeLa knockdown studies: control compared to NDP52, Gal8 [14], WIPI2 [58], LC3C, NDP52 [54], LRSAM1, ATG16L1 [17], OPTN [16], NDP52, TBK1 [59], p62 [15], PKCd [21], FNBP1L [60] silenced cells U2OS knockdown studies: control compared to Barkor siRNA [56] Henle-407 knockdown studies: control compared to ATG12 or P22 knockdowns [61] LC3, p62, ATG5 siRNAs in HeLa [62] Rapamycin treatment and ATG16L1 shRNA in HeLa [23], MDP treatment and Nod2, Rip2, ATG16L1, or ULK1 knockdown in HCT116 [63,64] ATG7, BEC-1, or IGG-1 RNAi-fed C. elegans. Atg1 , Atg6 , or Atg7 Dictyostelium [31] Parkin-deficient Drosophila [11] Oral Salmonella infection of WT and FAK fl/fl LyzMCre mice [27] Oral Salmonella infection of ATG5fl/fl x villin Cre mice [32] Salmonella streptomycin-pretreatment infection model in ATG16L1fl/fl x villin Cre mice [33] Salmonella streptomycin-pretreatment infection model in ATG16L1 T300A mice [65]
Restriction of O-antigen mutants
ATG5 fl/fl Lyz-Cre macrophages [66]
Enhances replication Increased restriction through autophagy stimulation Decreased survival, increased bacterial load
Decreased bacterial load and pathology with increased autophagy Increased bacterial translocation
F. tularensis
Model system Knockout cells: wild type and ATG5 / MEFs [45]. Wild type and RNF5 / BMDMs [46] Knockdown in HeLa: Rab9a, Rab23 [47], Rab7 [48], ATG5, CD46 (cyt-1 isoform), GOPC [49], VAMP8 VTI1b [50] 8-Nitro-cGMP treatment in RAW264.7 cells [19]
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Table 1 (Continued ) Species L. monocytogenes
Primary observation
Model system
Restriction of wild type Listeria in xenophagy competent cells
PTPN2 knockdown in T84 and primary human colon lamina propria fibroblasts with PTPN2 variant [67], wild type and ATG5 / MEFs [68], Rubicon knockdown in THP-1, BMDM, and RAW264.7 [52], NOD2 / or TLR2 / BMDM [69] DPrfA infection of wild type and ATG5 / MEFs [70]. DActA infection of wild type and ATG5 / MEFs or p62 / and p62 / + p62 MEFs [39] Dependent on host MVP expression (siRNA in RAW264.7) [40] Wildtype and autophagy deficient ATG16L1 DCCD MEFs [22] Parkin-deficient [11], PGRP-LE deficient, or ATG5 siRNA [29] Drosophila Intraperitoneal challenge of ATG5 fl/fl and ATG5 fl/fl x Lyz-Cre mice [34]
No restriction of wild type Listeria but restriction of mutant Listeria in xenophagy competent cells. Decreased autophagy with InlK expression in DActA strain Wild type Listeria protects co-infecting DPlcA/B bacteria in-trans Increased bacterial load, decreased survival Increased bacterial load, decreased survival C. rodentium Yersinia enterocolitica
Decreased replication in autophagy deficient Restriction in xenophagy competent cells
ATG16L1 hypomorphic mice [71] ATG16L1 T300A human macrophages, or T316A murine macrophages [72]
E. coli
Restriction in xenophagy competent cells
AIEC infection in wild type and ATG5 / MEFs, ATG16L1 knockdown HeLa cells [73], ATG16L1 siRNA in THP-1 macrophages, wild type or NOD2 / murine primary macrophages [74]. miR-30c or miR-130a upregulation following AIEC infection in T84 [75] ATG16L1 hypomorphic mice infected with UPEC [76]
Increased replication with autophagy downregulation Decreased replication in autophagy deficient
early phagophore structures by inhibiting autophagic flux, resulting in bacterial escape from autophagy [22]. Camouflage: one of the first examples of xenophagy escape by camouflage came with the observation that Shigella protein IcsB efficiently prevented the entrapment of Shigella into autophagosomes [38]. The proposed mechanism suggested that IcsB interacted with VirG, another Shigella protein, thereby competitively inhibiting the interaction of the autophagy protein ATG5 to VirG [38]. In a similar fashion, Listeria protein ActA was shown to favor the recruitment of the Arp2/3 complex and VASP around bacteria, thereby masking bacterial surface and restricting access to autophagy adaptor proteins such as p62, resulting in decreased autophagy targeting [39]. The Listeria protein InlK also contributes to autophagy escape by recruiting the host protein Major Vault Protein at the bacterial surface [40]. Finally, the serotype M1T1 of Group A Streptococcus expresses a protease that cleaves p62, NDP52 and NBR1, thus limiting targeting by the autophagic machinery [41].
Conclusions and outstanding questions It is now clear that targeting of intracellular bacteria by the autophagic system, or xenophagy, plays an essential role in innate immunity. Despite considerable recent progress in the field of xenophagy, many unanswered questions remain: Why are there several bacterial targeting mechanisms for xenophagy (Nods/ubiquitin/galectins/DAG) and are they www.sciencedirect.com
inter-connected to increase sensitivity or specificity? Similarly, what are the relative contributions of the adaptor proteins that display domains of interaction with ubiquitinated proteins and LC3, such as p62, NDP52, NBR1 and optineurin? Finally, since ATG5-12-16L1 is required to target LC3 to the bacterial autophagosome, how critical is the capacity of p62, NDP52, NBR1 and optineurin to recruit LC3? Besides Nod1 and Nod2, are there any factors that are truly specific for xenophagy, as opposed to other processes of selective autophagy? Indeed, NBR1, p62 and ubiquitination are critical for the clearance of aggregated proteins [42,43], NDP52 and galectin-8 are recruited to damaged membranes even in the absence of infection [14], optineurin associates with LC3-positive vesicles upon nutrient deprivation [16], and Tecpr1 is involved in selective autophagy targeting pathogens, aggresomes and mitochondria [44]. Therefore, is the role of Nod proteins to direct towards bacteria an autophagic machinery that would be triggered by non-microbial signals, such as membrane damage and accumulation of ubiquitinated molecules? In any given cell, autophagy typically only targets a small fraction of the intracellular bacteria. If the same cause produces the same effect, why would not all the bacteria be targeted? Is there an unknown feature that would make a specific bacterium more prone to targeting by autophagy than others? Current Opinion in Microbiology 2015, 23:163–170
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How important xenophagy is for bacterial growth restriction as compared to other innate immune defense mechanisms, such as for instance phagocytosis? This is a difficult question since cellular models of defective xenophagy usually either target general or selective autophagy proteins, which both result in altered cellular fitness, potentially leading to indirect effects on the cell’s capacity to control bacterial pathogens. Answering this question will require the identification of factors involved in xenophagy, but not in general or other selective autophagy pathways.
Acknowledgements Research in the lab of SEG is supported by grants from Canadian Institutes of Health Research (CIHR), Natural Sciences and Engineering Research Council (NSERC), Canadian Cancer Society Research Institutes and Crohn’s and Colitis Canada. MTS. was supported by fellowships from the NSERC (CGS-M), the Ontario Graduate Scholarship (OGS), CIHR (CGSD) and by CIHR Strategic Training Fellowship STP-53877.
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ScienceDirect Emerging themes in bacterial autophagy Matthew T Sorbara1 and Stephen E Girardin2 The role of autophagy in the control of intracellular bacterial pathogens, also known as xenophagy, is well documented. Here, we highlight recent advances in the field of xenophagy. We review the importance of bacterial targeting by ubiquitination, diacylglycerol (DAG) or proteins such as Nod1, Nod2, NDP52, p62, NBR1, optineurin, LRSAM1 and parkin in the process of xenophagy. The importance of metabolic sensors, such as mTOR and AMPK, in xenophagy induction is also discussed. We also review the in vitro and in vivo evidence that demonstrate a global role for xenophagy in the control of bacterial growth. Finally, the mechanisms evolved by bacteria to escape xenophagy are presented. Addresses 1 Department of Immunology, University of Toronto, Toronto M5S 1A8, Canada 2 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto M5S 1A8, Canada Corresponding author: Girardin, Stephen E ([email protected])
Current Opinion in Microbiology 2015, 23:163–170 This review comes from a themed issue on Host-microbe interactions: bacteria Edited by David Holden and Dana Philpott
http://dx.doi.org/10.1016/j.mib.2014.11.020 1369-5274/# 2014 Elsevier Ltd. All rights reserved.
the cytosolic side of late endosomes and lysosomes, known as the mTOR complex 1 (mTORC1) [2]. mTORC1 is assembled and functional only when cellular nutrients or cofactors (including amino acids, growth factors, oxygen and ATP) are not limiting, and active mTORC1 in turns phosphorylates several cellular proteins involved in cell proliferation, growth, ribosomal biogenesis and mRNA translation [2]; in addition, active mTORC1 potently inhibits autophagy by promoting the phosphorylation of the ULK1 complex, including ULK1 itself and ATG13 [3]. The kinase AMPK, which is another sensor of cellular energy (and in particular of the AMP/ATP ratio), also regulates autophagy through the phosphorylation of the ULK1 complex, although the global effect of AMPK on autophagy might depend on the trigger (glucose versus amino acid starvation) [3]. In addition to its role in cellular metabolism, numerous lines of evidence indicate that autophagy also plays a key role in innate immunity, by promoting the entrapment and lysosome-mediated degradation of bacterial pathogens, including bacteria that escape into the host cytosol (such as Shigella or Listeria) or those that reside in a modified intracellular vacuole (such as Salmonella and Mycobacterium). While several comprehensive reviews have detailed the role of autophagy in host defense also known as ‘xenophagy’ [4–6], we will highlight here the most recent advances in this rapidly expanding field of research, and present our views on the most important questions that remain to be solved in the coming years.
Bacterial targeting in xenophagy
Introduction Autophagy is an essential cellular process through which cellular materials, such as large macromolecular complexes or defective organelles, are progressively engulfed by a double membrane, or phagophore, resulting in the formation of an autophagosome [1]. After fusion with lysosomes, the constituents are degraded and metabolites recycled. While a constitutive level of autophagy is necessary for normal cellular housekeeping functions, autophagy can be strongly upregulated in conditions of metabolic starvation in order to provide a spare source of metabolites for a limited time before cell death ensues. Several signaling pathways contribute to the regulation of autophagy by metabolic checkpoints [1]. One of the most conserved of those is dependent on the kinase Tor (mTOR in mammals), which forms a large complex on www.sciencedirect.com
An active area of research in the field of xenophagy relates to the question of how infected cells can specifically direct the phagophore to bacteria that are either free in the cytosol or confined into a vacuole, while avoiding to target other cellular constituents, such as organelles. Four main molecular mechanisms are currently known to confer bacterial targeting during xenophagy, which involve: Nod proteins, ubiquitination, galectin-dependent pathway and diacylglycerol (DAG). Nod1 and Nod2: the Nod-like receptor (NLR) proteins Nod1 and Nod2 are cytosolic proteins that detect specific fragments derived from bacterial peptidoglycan and initiate pro-inflammatory signaling (NF-kB and MAP kinase pathways) following the recruitment of the adaptor protein Rip2 [7]. In the initial phase of bacterial invasion, Nod1/2 also directly interacts with the autophagy protein ATG16L1 in a Rip2-independent manner, thereby initiating the recruitment of the autophagic machinery at the site of bacterial entry [8]. However, it remains unclear if Current Opinion in Microbiology 2015, 23:163–170
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Nod1/2 can target bacteria (such as Shigella or Listeria) once they are free in the cytosol. Ubiquitination: it was observed that cytosolic and vacuoleconfined bacteria are rapidly decorated by ubiquitination [9], and different types of ubiquitin chains constitute the ubiquitin coat surrounding bacteria [10,11,12]. Several adaptor proteins, including NDP52, p62, NBR1 and optineurin, have been shown to serve as a bridge between ubiquitin groups and the autophagy protein LC3 [13]. At least some of these adaptor proteins likely serve nonredundant functions, as evidenced by the reported effect of targeting NDP52 [14], p62 [15] or optineurin [16] on bacterial restriction. The nature of the proteins targeted by ubiquitination, and even if those are from the host or the bacteria, remains unclear. However, recent progress was made with the identification of E3 ligases implicated in this ubiquitination process. LRSAM1 was identified as an important E3 ligase in the case of infection with Salmonella [17]. More recently, the ubiquitin ligase parkin was shown to be required for the formation of the ubiquitin coat surrounding Mycobacterium tuberculosis, and for the restriction of M. tuberculosis growth in macrophages [11]. Interestingly, the ubiquitin ligase activity of parkin has a well-established role in mitophagy, and mutations in the human gene encoding parkin lead to increased susceptibility to Parkinson’s disease, but also to infections by Mycobacterium Leprae and Salmonella enterica serovar Typhi, suggesting links between xenophagy and mitophagy, and between susceptibility to neurodegenerative disorders and to infectious diseases [13]. In addition, a role for the intracellular DNA sensor STING in the formation of the ubiquitin coat surrounding M. tuberculosis was identified [18], suggesting that STING and parkin might operate in the same pathway. Finally, S-guanylation of cysteine residues on the surface of intracellular Group A streptococcus by 8-nitro-cGMP was shown to drive polyubiquitination and autophagy [19]. Exposure of galectin proteins: the membrane remnants of vacuolar rupture in Shigella-infected cells were found to accumulate ubiquitinated proteins and autophagy markers [20], suggesting that the event of membrane damage could trigger xenophagy. In agreement, Thurston et al. demonstrated that galectin proteins, such as galectin-8/3/9, which are glycan-binding lectin proteins that can be found in the cytosol, accumulated at sites of membrane damage induced by either Salmonella or by glycyl-L-phenylalanine 2-naphthylamide (GPN), a molecule inducing sterile lysosomal damage [14]. Moreover, galectin-8 recruited NDP52 to the Salmonella-containing vacuole (SCV) through a mechanism distinct and more rapid than the ubiquitinmediated recruitment of NDP52 to the SCV [14]. Thus, galectin proteins might serve as important sensors of danger signals initiated at the damaged membrane by the exposure of glycans to the cytosol, while those glycans are normally only exposed to the luminal side of vesicles. Current Opinion in Microbiology 2015, 23:163–170
DAG: in Salmonella-infected cells, Shahnazari et al. observed early accumulation of DAG around the SCV, and inhibition of DAG formation using pharmacological or genetic approaches resulted in inhibition of bacterial autophagy [21]. While the authors proposed that DAGdependent and ubiquitin-dependent pathways of autophagy targeting to the SCV were independent, the role of DAG in Nod1/2-dependent or galectin/NDP52-dependent xenophagy remains to be addressed.
Mechanisms of xenophagy induction While much attention was given to the mechanisms responsible for bacterial targeting by autophagy (see above), little is known about how autophagy pathways are turned on during infection. Until recently, it was assumed that the constitutive levels of housekeeping autophagy were likely sufficient to drive xenophagy in infected cells. However, it seems counter-intuitive that a critical innate immune mechanism of bacterial restriction would be limited by nutrient sufficiency and the metabolic status of the infected cells; indeed, metabolic sensors, such as mTOR, play key roles in limiting autophagy. Studies in epithelial cell lines demonstrated that Shigella, Listeria and Salmonella all triggered a rapid inhibition of mTOR signaling, which is associated with a burst of autophagy in infected cells, thus showing that xenophagy induction can occur irrespective of the nutrient status of the infected cells [22–24]. Mechanistically, the inhibition of mTOR was driven by amino acid starvation (AAS) caused by membrane damage in the infected cells, resulting in mTOR dissociation from late endosome/lysosome membranes [23]. While Shigella caused a sustained inhibition of mTOR and induction of AAS pathways, infection with Salmonella or Listeria resulted in transient AAS stress and mTOR inhibition, a mechanism that likely accounts, at least in part, for the capacity of these bacteria to escape xenophagy-mediated bacterial restriction [23]. Although the mechanism by which bacteriainduced membrane damage causes AAS is unknown, it is interesting to note that bacterial pore-forming toxins, which induce severe membrane damage, have also been shown to inhibit mTOR and to turn on AAS pathways [25,26]. Moreover, recent evidence showed that the tyrosine kinase focal adhesion kinase (FAK) was recruited to the surface of the SCV in macrophages, resulting in increased activity of the mTORC1 through an unknown mechanism dependent on the Salmonella Pathogenicity Island 2 (SPI-2) [27]. This subversion mechanism thereby amplified autophagy inhibition through mTOR induction.mTOR is not the only critical regulator of nutrient status that influences the autophagy pathway. As mentioned above, AMPK also emerges as a potential key player in these pathways, but the role of this energy sensor in the context of host responses to bacterial pathogens remains poorly characterized. A recent study demonstrated that, in M. tuberculosis infected macrophages, www.sciencedirect.com
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AMPK induction by the drug AICAR was sufficient to promote antibacterial autophagy, in part through the transcriptional upregulation of autophagy-associated genes [28]. However, it remains unknown if bacterial pathogens can directly activate AMPK in infected cells, thereby affecting xenophagy.
Role of xenophagy in bacterial restriction Substantial work in cell lines and primary cell cultures has identified and supported a bactericidal role for autophagy (see Table 1). Interestingly, several groups have found that wild type strains of bacteria that have evolved to replicate in the cytoplasm, including S. flexneri, Francisella tularensis, and L. monocytogenes are not restricted by autophagy. Strikingly, only attenuated strains, lacking specific virulence factors, are efficiently targeted and degraded in autolysosomes. In contrast, bacteria species that reside and replicate within damaged-vacuoles, including M. tuberculosis or S. typhimurium, generally appear to be more efficiently restricted by autophagy (Table 1). In in vitro infection models where loss or stimulation of autophagy does impact on bacterial replication, the loss of autophagy generally increases bacterial replication by less than an order of magnitude with typical values of 2–5 the replication observed in autophagy competent cells. Furthermore, pathogens adapted to an intracellular lifestyle are still able to replicate in autophagy competent cells, and, in the case of bacteria such as Shigella and Listeria, spread from cell to cell. Therefore, a major question in the field has been whether the slight delay or decrease in intracellular replication within a cell translates to a substantial effect at the whole organism level. Several studies in non-mammalian model organisms have identified a critical role for autophagy in the control of bacterial infection. Seminal work from Yano et al. in Drosophila demonstrated that inhibition of autophagy through loss of either ATG5 or the peptidoglycan recognition protein, PGRL-LE, results in a dramatic increase in susceptibility to L. monocytogenes infection [29]. More recently, a critical role for the ubiquitin ligase parkin has been identified in clearance of S. typhimurium, M. marinum and L. monocytogenes in Drosophila [11]. Mostowy et al. recently proposed a zebrafish larvae model for studying the role of autophagy in Shigella infection, and found that loss of the autophagy adaptor p62 results in increased susceptibility to S. flexneri infection [30]. Finally, loss of core autophagy components in either Caenorhabditis elegans or the single cell Dictyostelium triggered increased susceptibility to S. typhimurium infection [31]. Therefore, in non-mammalian systems both the core components of the autophagy machinery (ATG proteins) and the components specific to xenophagy or subsets of autophagy (PGRP-LE, parkin, p62) have been implicated in host defense. However, it must be noted that these infection models in which xenophagy was shown to confer control of intracellular bacteria relied on the interaction between www.sciencedirect.com
bacterial pathogens and host cells that are not the natural targets of these bacteria, raising the possibility that xenophagy could mainly restrict pathogens that are not adapted to their host environment. Recent experiments using murine models of infection have supported a role for autophagy in bacterial pathogen clearance. Mice lacking expression of ATG5 or ATG16L1 in intestinal epithelial cells had increased Salmonella translocation to systemic sites and increased bacterial load [32,33]. Furthermore, mice lacking macrophage expression of ATG5 were more susceptible to M. tuberculosis or L. monocytogenes infection [18,34]. One caveat of these studies is that they do not exclude contributions from the autophagy machinery beyond direct bactericidal activity, such as maintenance of host cell fitness by controlling the turnover of cellular organelles and macromolecular complexes. Interestingly, during infection of mice with the extracellular pathogen Citrobacter rodentium, decreased expression of ATG16L1 triggered a protective hyper-inflammatory response, and a similar phenotype was observed following UPEC infection (see Table 1). In agreement, ATG16L1 was found to negatively regulate Nod1/2-dependent induction of proinflammatory pathways in an autophagy-independent manner [35]. Therefore, further work is needed to clarify the relative contributions of direct bactericidal activity and ancillary functions in the immune response.
Bacterial escape mechanisms from xenophagy One of the most compelling pieces of evidence that xenophagy is a critical innate immune mechanism of bacterial restriction comes from the identification of bacterial strategies that were evolved to escape host autophagy. Schematically, two main escape mechanisms seem to have been designed by bacterial pathogens: autophagy disarming and camouflage. Autophagy disarming: several bacteria have evolved means to inhibit autophagy to favor their intracellular replication. This is the case of M. tuberculosis, which is targeted by autophagy only after addition of rapamycin [36], suggesting that mTOR activity in infected cells is sufficient to turn down xenophagy. In addition, the ESX-1 secretion system was shown to specifically inhibit autophagosome-lysosome fusion, a key step in xenophagy [37]. Salmonella and Listeria also escape autophagy at least in part through the rapid reactivation of mTOR after an initial phase of inhibition caused by membrane damage (see above) [22,23]. A sophisticated example of autophagy disarming was provided in the case of Legionella pneumophila with the discovery that the effector RavZ could cleave the C-terminal end of lipid-conjugated LC3, thus irreversibly preventing the reconjugation of the protein to autophagosomes and thereby inhibiting autophagy [37]. Finally, Listeria phospholipases C were shown to stall Current Opinion in Microbiology 2015, 23:163–170
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Table 1 Autophagy-dependent restriction of bacterial replication. Evidence of a role for autophagy in restricting the proliferation of different model pathogens is indicated with tissue culture models of infection shaded in green, and in vivo infection models shaded in gray Species Group A Streptococcus (GAS)
Primary observation Restriction in xenophagy competent cells
Increased restriction through autophagy stimulation Increased mortality and bacterial load with increased autophagy Mycobacterium species
Restriction in xenophagy competent cells
Restriction with artificial autophagy induction Decreased survival, increased pathology and bacterial load Decreased survival Increased bacterial load, inflammation
Shigella flexneri
Restriction of DIcsB strain No restriction of wild type Shigella No restriction of wild type with autophagy induction Restriction of wild type Shigella, increased difference with DIcsB strain Increased mortality
Salmonella typhimurium
Restriction in xenophagy competent cells
Protection from GAS infection in RNF5 / [46]
mice
Knockdown in macrophage/monocyte lines: IRGM [51], Rubicon [52], parkin [11], NDP52 [18] Knockout cells: PARK2 / BMDMs [11], STING / , ATG5 / , TBK1 / BMDMs [18] RAW264.7 and primary macrophages [36], ATG5 fl/ fl and ATG5 fl/fl x Lyz-Cre BMDM [34] M. tuberculosis infection of ATG5 fl/fl LyzM-Cre mice [18] M. marinum infection of parkin-deficient Drosophila [11] M. marinum infection of p62, or DRAM1 deficient (morpholino treated) zebrafish embryos. Decreased bacterial load with DRAM1 overexpression [53] Wild type and ATG5 / MEFs [38], Tecpr1 siRNA in HeLa, Tecpr1 / MEFs [44] LC3C siRNA, NDP52 siRNA in HeLa [54] Rapamycin treatment HeLa [23] Control compared to ATG16L1 T300A MEFs, primary intestinal epithelial cells (24821797) p62 deficient (morpholino treated) zebrafish embryos [30]
Increased bacterial translocation, and intestinal inflammation Increased intestinal inflammation with ATG16L1 variant expression
MEFs: wild type compared to ATG5 / [55], PKCd / [21], ATG7 / [56], ATG16L1-deficient [57] HeLa knockdown studies: control compared to NDP52, Gal8 [14], WIPI2 [58], LC3C, NDP52 [54], LRSAM1, ATG16L1 [17], OPTN [16], NDP52, TBK1 [59], p62 [15], PKCd [21], FNBP1L [60] silenced cells U2OS knockdown studies: control compared to Barkor siRNA [56] Henle-407 knockdown studies: control compared to ATG12 or P22 knockdowns [61] LC3, p62, ATG5 siRNAs in HeLa [62] Rapamycin treatment and ATG16L1 shRNA in HeLa [23], MDP treatment and Nod2, Rip2, ATG16L1, or ULK1 knockdown in HCT116 [63,64] ATG7, BEC-1, or IGG-1 RNAi-fed C. elegans. Atg1 , Atg6 , or Atg7 Dictyostelium [31] Parkin-deficient Drosophila [11] Oral Salmonella infection of WT and FAK fl/fl LyzMCre mice [27] Oral Salmonella infection of ATG5fl/fl x villin Cre mice [32] Salmonella streptomycin-pretreatment infection model in ATG16L1fl/fl x villin Cre mice [33] Salmonella streptomycin-pretreatment infection model in ATG16L1 T300A mice [65]
Restriction of O-antigen mutants
ATG5 fl/fl Lyz-Cre macrophages [66]
Enhances replication Increased restriction through autophagy stimulation Decreased survival, increased bacterial load
Decreased bacterial load and pathology with increased autophagy Increased bacterial translocation
F. tularensis
Model system Knockout cells: wild type and ATG5 / MEFs [45]. Wild type and RNF5 / BMDMs [46] Knockdown in HeLa: Rab9a, Rab23 [47], Rab7 [48], ATG5, CD46 (cyt-1 isoform), GOPC [49], VAMP8 VTI1b [50] 8-Nitro-cGMP treatment in RAW264.7 cells [19]
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Table 1 (Continued ) Species L. monocytogenes
Primary observation
Model system
Restriction of wild type Listeria in xenophagy competent cells
PTPN2 knockdown in T84 and primary human colon lamina propria fibroblasts with PTPN2 variant [67], wild type and ATG5 / MEFs [68], Rubicon knockdown in THP-1, BMDM, and RAW264.7 [52], NOD2 / or TLR2 / BMDM [69] DPrfA infection of wild type and ATG5 / MEFs [70]. DActA infection of wild type and ATG5 / MEFs or p62 / and p62 / + p62 MEFs [39] Dependent on host MVP expression (siRNA in RAW264.7) [40] Wildtype and autophagy deficient ATG16L1 DCCD MEFs [22] Parkin-deficient [11], PGRP-LE deficient, or ATG5 siRNA [29] Drosophila Intraperitoneal challenge of ATG5 fl/fl and ATG5 fl/fl x Lyz-Cre mice [34]
No restriction of wild type Listeria but restriction of mutant Listeria in xenophagy competent cells. Decreased autophagy with InlK expression in DActA strain Wild type Listeria protects co-infecting DPlcA/B bacteria in-trans Increased bacterial load, decreased survival Increased bacterial load, decreased survival C. rodentium Yersinia enterocolitica
Decreased replication in autophagy deficient Restriction in xenophagy competent cells
ATG16L1 hypomorphic mice [71] ATG16L1 T300A human macrophages, or T316A murine macrophages [72]
E. coli
Restriction in xenophagy competent cells
AIEC infection in wild type and ATG5 / MEFs, ATG16L1 knockdown HeLa cells [73], ATG16L1 siRNA in THP-1 macrophages, wild type or NOD2 / murine primary macrophages [74]. miR-30c or miR-130a upregulation following AIEC infection in T84 [75] ATG16L1 hypomorphic mice infected with UPEC [76]
Increased replication with autophagy downregulation Decreased replication in autophagy deficient
early phagophore structures by inhibiting autophagic flux, resulting in bacterial escape from autophagy [22]. Camouflage: one of the first examples of xenophagy escape by camouflage came with the observation that Shigella protein IcsB efficiently prevented the entrapment of Shigella into autophagosomes [38]. The proposed mechanism suggested that IcsB interacted with VirG, another Shigella protein, thereby competitively inhibiting the interaction of the autophagy protein ATG5 to VirG [38]. In a similar fashion, Listeria protein ActA was shown to favor the recruitment of the Arp2/3 complex and VASP around bacteria, thereby masking bacterial surface and restricting access to autophagy adaptor proteins such as p62, resulting in decreased autophagy targeting [39]. The Listeria protein InlK also contributes to autophagy escape by recruiting the host protein Major Vault Protein at the bacterial surface [40]. Finally, the serotype M1T1 of Group A Streptococcus expresses a protease that cleaves p62, NDP52 and NBR1, thus limiting targeting by the autophagic machinery [41].
Conclusions and outstanding questions It is now clear that targeting of intracellular bacteria by the autophagic system, or xenophagy, plays an essential role in innate immunity. Despite considerable recent progress in the field of xenophagy, many unanswered questions remain: Why are there several bacterial targeting mechanisms for xenophagy (Nods/ubiquitin/galectins/DAG) and are they www.sciencedirect.com
inter-connected to increase sensitivity or specificity? Similarly, what are the relative contributions of the adaptor proteins that display domains of interaction with ubiquitinated proteins and LC3, such as p62, NDP52, NBR1 and optineurin? Finally, since ATG5-12-16L1 is required to target LC3 to the bacterial autophagosome, how critical is the capacity of p62, NDP52, NBR1 and optineurin to recruit LC3? Besides Nod1 and Nod2, are there any factors that are truly specific for xenophagy, as opposed to other processes of selective autophagy? Indeed, NBR1, p62 and ubiquitination are critical for the clearance of aggregated proteins [42,43], NDP52 and galectin-8 are recruited to damaged membranes even in the absence of infection [14], optineurin associates with LC3-positive vesicles upon nutrient deprivation [16], and Tecpr1 is involved in selective autophagy targeting pathogens, aggresomes and mitochondria [44]. Therefore, is the role of Nod proteins to direct towards bacteria an autophagic machinery that would be triggered by non-microbial signals, such as membrane damage and accumulation of ubiquitinated molecules? In any given cell, autophagy typically only targets a small fraction of the intracellular bacteria. If the same cause produces the same effect, why would not all the bacteria be targeted? Is there an unknown feature that would make a specific bacterium more prone to targeting by autophagy than others? Current Opinion in Microbiology 2015, 23:163–170
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How important xenophagy is for bacterial growth restriction as compared to other innate immune defense mechanisms, such as for instance phagocytosis? This is a difficult question since cellular models of defective xenophagy usually either target general or selective autophagy proteins, which both result in altered cellular fitness, potentially leading to indirect effects on the cell’s capacity to control bacterial pathogens. Answering this question will require the identification of factors involved in xenophagy, but not in general or other selective autophagy pathways.
Acknowledgements Research in the lab of SEG is supported by grants from Canadian Institutes of Health Research (CIHR), Natural Sciences and Engineering Research Council (NSERC), Canadian Cancer Society Research Institutes and Crohn’s and Colitis Canada. MTS. was supported by fellowships from the NSERC (CGS-M), the Ontario Graduate Scholarship (OGS), CIHR (CGSD) and by CIHR Strategic Training Fellowship STP-53877.
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