Autophagic targeting and avoidance in intracellular bacterial infections

Abstract

Eukaryotic cells use autophagy to break down and recycle components such as aggregated proteins and damaged organelles. Research in the past decade, particularly using Salmonella enterica serovar Typhimurium as a model pathogen, has revealed that autophagy can also target invading intracellular bacterial pathogens for degradation. However, many bacterial pathogens have evolved mechanisms that allow for evasion of the autophagic pathway, such as motility or direct and irreversible cleavage of proteins that comprise the autophagic machinery. As a complete and detailed understanding of the autophagic pathway and its derivatives continues to develop, it is likely that other mechanisms of inhibition by bacterial pathogens will be discovered.

Introduction

Cells use a specialized form of autophagy, known as xenophagy, to direct intracellular pathogens for degradation in the lysosome. Studies of intracellular bacterial pathogens have described the degradation of some species by the autophagic pathway, and the mechanisms by which bacteria may escape, avoid, or inhibit autophagic targeting. Eukaryotic host cells appear to have evolved specific mechanisms to target bacterial pathogens for xenophagy, however, host pathways that recognize bacterial pathogens for autophagic targeting are not completely defined. Here, we review mechanisms of autophagic targeting and avoidance and discuss remaining questions surrounding how the cell senses and targets bacterial invaders to the autophagic pathway.

Autophagic targeting of bacterial pathogens

Eukaryotic cells recycle unneeded components, such as damaged organelles or protein aggregates, with autophagy. While various forms of autophagy exist, here we focus on macroautophagy, which is henceforth referred to as autophagy for simplicity. Multiple signals may induce autophagy, such as starvation or inactivation of mTORC1, via signaling events upstream of autophagy initiation [1,2]. When autophagy is induced, an isolation membrane envelops the cargo, resulting in formation of a double-membrane bound organelle called an autophagosome. The membrane is derived primarily from the endoplasmic reticulum (ER) [3,4], but may also originate from ER-mitochondria contact sites and/or ER-Golgi contact sites [5,6]. In canonical models of autophagy, cargo are ubiquitinated, and then adapter proteins bind these ubiquitin residues and also Atg8 proteins on the isolation membrane [7]. The most common Atg8 protein that is monitored to assess autophagy in mammalian cell lines is called microtubule-associated protein 1A/1B-light chain 3 (LC3) [8,9]. The autophagosome then fuses with a lysosome where the cargo is degraded.

Intracellular bacterial pathogens are one type of substrate that the autophagy machinery targets for degradation (Figure 1). When autophagy specifically targets an invading pathogen, the process is called xenophagy. The most studied model is infection with Salmonella enterica serovar Typhimurium. Proteins involved in autophagy have been shown to influence S. typhimurium replication and interact with the Salmonella-containing vacuole (SCV). LC3, ubiquitin, and a number of adapter proteins, such as NDP52, p62/SQSTM1, TAX1BP1, and optineurin, all localize to the SCV. Silencing or deletion of genes required for autophagy results in increased bacterial replication during infection in mice, Caenorhabditis elegans, Drosophila melanogaster, and tissue culture cell lines [10–15].

Figure 1.

Interactions of different bacterial species with autophagy. Autophagosomes form around S. typhimurium and M. tb containing phagosomes in response to either membrane damage or DNA sensing in each infection, respectively. L. monocytogenes and S. flexneri avoid xenophagy with actin tail motility. Nonmotile mutants or pclA mutants of L. monocytogenes that cannot break out of the LCV are targeted for xenophagy. Nonmotile mutants of S. flexneri are targeted for xenophagy through a mechanism involving septin caging. L. pneumophila avoids xenophagy with the T4SS effector protein RavZ. RavZ is a protease that irreversibly cleaves all Atg8 proteins. Nonmotile mutants of B. pseudomallei are targeted for LAP. Motile Burkholderia species escape the phagosome with actin tail motility.

Although the association of the autophagic machinery and involvement of it in reducing bacterial replication is well established, signals involved in sensing intracellular bacteria and targeting them for envelopment by autophagosomes are not fully understood. The E3 ubiquitin ligase, LRSAM1, is required for the ubiquitination involved in xenophagy of S. typhimurium [16]. How LRSAM1 may link to upstream cellular signaling pathways is not clear in all cases. There are likely other unidentified E3 ligases that participate in ubiquitination of S. typhimurium. Moreover, the exact site of ubiquitination, being either on the bacteria itself or the vacuole membrane, remains controversial. Importantly, ubiquitin-independent mechanisms for autophagic targeting also exist. In S. typhimurium infected cells, a subset of bacteria ruptures the vacuoles in which they reside. The host protein, Galectin-8, detects damaged membranes by binding to host glycans that are exposed upon vacuole rupture. The autophagy adapter protein NDP52 then binds Galectin-8 and Atg8 proteins on an isolation membrane that forms an autophagosome around the vacuole [17•]. Ubiquitin-mediated processes can then amplify detection that occurs through the Galectin-8 pathway. Additionally, sensing of diacylglycerol (DAG) to target S. typhimurium for autophagy has been proposed as an ubiquitin-independent sensing mechanism [18]. It remains unclear if other sensing mechanisms may exist.

Like S. typhimurium, there are other examples of pathogenic bacteria that can be targeted by autophagy during infection. Induction of autophagy with rapamycin, an inhibitor of mTOR, promotes restriction of Mycobacterium tuberculosis during infection in macrophages [19]. Mice containing myeloid cells deficient in Atg5 are more susceptible to M. tb infection and display dampened levels of inflammation [20]. LC3, adapter proteins, and ubiquitin all localize to the M. tb containing vacuole [21]. Furthermore, Ubiquilin 1 (UBQN1) and the E3 ubiquitin ligase Parkin independently direct the autophagic machinery to M. tb [22,23]. Similarly, Group A Streptococcus pyogenes (GAS) is enveloped by double-membrane bound autophagosomes after escape from the pathogen-containing vacuole resulting in bacterial degradation. Infection of Atg5−/− cells with GAS results in increased bacterial numbers [24••]. As during infection with S. typhimurium, other upstream signals that may be involved in autophagic targeting are undefined for M. tb and S. pyogenes.

In addition to selective autophagy, cargo can also be subject to degradation after targeting by a specialized form of autophagy known as LC3-associated phagocytosis (LAP). During LAP cargo is contained within a single membrane bound compartment after phagocytosis by the host cell. LC3 is attached to the membrane of the phagosome immediately after phagocytosis. The LC3-positive compartment then fuses with the lysosome [25••,26]. Importantly, the protein Rubicon is required for LAP but not autophagic-mediated degradation of the fungus, Aspergillus fumigatus [26]. However, the only known example of a bacterial pathogen being targeted by LAP is that of non-motile mutants of Burkholderia pseudomallei [27]. LAP can be triggered when cargo interacts with Toll-Like Receptors (TLR) prior to phagocytosis, suggesting a link between the innate immune system and this specialized form of autophagy [25••].

Thus, future investigations will be required to determine the extent to which LAP targets intracellular bacterial pathogens, the requirements for such targeting, and the extent to which this pathway may influence the overall host response to a given pathogen.

Mechanisms of autophagy avoidance and inhibition

One mechanism associated with the avoidance of autophagic targeting of bacterial pathogens is actin tail motility. ActA mutants of Listeria monocytogenes are unable to form actin tails and cannot spread cell-to-cell. ActA and PlcA mutants are targeted by machinery of the autophagic pathway and are eventually degraded, resulting in a decrease in bacterial replication [28,29]. It is assumed that the loss of motility causes these mutants to be more efficiently targeted by the autophagic machinery, and it is clear that this process is dependent on Atg5, and involves ubiquitin and the autophagy adapter proteins NDP52 and p62, suggesting a mechanism that is dependent on selective autophagy [30].

Similarly, bipA and bopA mutants of B. pseudomallei are targeted for degradation by autophagy. BipA is a translo-cator protein of the T3SS, and BopA is a T3SS effector protein. Both BipA and BopA are required for actin tail motility. Furthermore, induction of autophagy by rapamycin treatment increases autophagic degradation of B. pseudomallei [31]. Unlike L. monocytogenes, which appears to be targeted primarily by selective autophagy, nonmotile B. pseudomallei mutants are targeted by another autophagy pathway called LC3-associated phagocytosis (LAP). LAP involves the enclosure of cargo into a single-membrane bound compartment that is conjugated by LC3 almost immediately after phagocytosis. LC3 labeled B. pseudomallei containing-compartments are bound by a single membrane, suggesting that the autophagic mechanism targeting nonmotile mutants for degradation may be LAP-mediated [27].

In contrast to the autophagic targeting of L. monocytogenes and B. pseudomallei, in which loss of motility increases susceptibility to autophagic targeting, actin tail motility of Shigella flexneri increases targeting by autophagy. Septins are G-proteins that form cage-like structures around S. flexneri that have begun to form actin tails and are in the cytoplasm. Septins are associated with targeting of these bacteria by the autophagic machinery through a mechanism that is dependent on mitochondria [32,33••,34]. So far, targeting by septin caging appears to be specific to S. flexneri and is not necessarily a broad targeting mechanism for motile pathogens, as recruitment of autophagic markers NDP52 and p62 to L. monocytogenes is not dependent on septins or actin [30].

Another mechanism to avoid targeting by autophagic machinery is the direct inhibition of autophagy. To date, the Legionella pneumophila T4SS effector RavZ is the only identified bacterial factor that has a biochemical activity that directly inhibits components of the autophagy machinery. RavZ is a cysteine protease that localizes to autophagosomes and irreversibly cleaves Atg8 family proteins including the LC3 isoforms and GABARAP isoforms [35]. RavZ contains a novel PI3P binding domain, a protease domain, and an amphipathic loop that anchors the protein into the autophagosome membrane. RavZ targets autophagosomes through the coordination of these three features, which effectively allow the protein to sense the specific membrane curvature of autophagosomes [36].

Other possible mechanisms of autophagy avoidance or inhibition

Although it is clear that autophagy is capable of targeting bacterial pathogens for degradation, there are likely other yet undiscovered mechanisms that bacterial pathogens use to avoid or inhibit autophagy. One possibility is that host autophagy adaptor proteins may target specific bacterial pathogens differently. Currently, adaptor proteins are believed to serve as bridges that link ubiquitinated cargo to LC3, and thus an isolation membrane as it envelops this cargo, creating an autophagosome around the bacterium. However, the specificity of these adaptor proteins, as well as the different Atg8 orthologues is only beginning to be discovered. For example, NDP52 specifically binds to the Atg8 orthologue LC3C [12], OPTN has been linked to mitophagy [37,38•], and p62/SQSTM1, NBR1, and ALFY are linked to aggrephagy (autophagy of aggregated proteins) [39–41]. Little is known of the differences between the Atg8 orthologues or the requirement for one in place of another. However, the GABARAPs appear to be involved in later events of autophagosome formation, while the LC3s are involved earlier [42]. For bacterial infections, processes invoked by particular pathogens may be targeted differently depending on the specificity of the Atg8 and adapter proteins. Indeed of the autophagy adapters that have so far been discovered, not all of them have been tested or implicated in xenophagy and few have been tested outside of S. typhimurium infections (Table 1). Further cell biological studies on the functions of these proteins during different aspects of autophagy may inform understanding of how bacterial pathogens are targeted by autophagy during intracellular infections.

Table 1.

Autophagy adapter proteins. List of known autophagy adapters, which type(s) of autophagy they have been implicated in, and in which intracellular bacterial infections they have been studied. NBR1, TOLLIP, and ALFY have been suggested as autophagy adapters but have not been implicated in autophagy of any bacterial pathogens. Note that some of these proteins may have other cellular functions outside of autophagy

Name	Type of autophagy	Bacterial infections studied
NDP52	Xenophagy, Mitophagy	S. typhimurium, M. tuberculosis, L. monocytogenes, S. flexneri
OPTN	Xenophagy, Mitophagy	S. typhimurium
p62/SQSTM1	Xenophagy, Aggrephagy, Pexophagy	S. typhimurium, M. tuberculosis, L. monocytogenes, S. flexneri
TAX1BP1	Xenophagy	S. typhimurium
NBR1	Aggrephagy
TOLLIP	Aggrephagy
ALFY	Aggrephagy

It is possible that other mechanisms of autophagy avoidance rely on disrupting or targeting as of yet unknown mechanisms involved in autophagic targeting of bacterial pathogens. The identification of such mechanisms in the host will allow for genetic screening of other bacterial factors that may be involved in autophagy avoidance or disruption. It is also possible that autophagic machinery may not target certain types of bacterial pathogens. Hypothetically, such a pathogen would not initiate upstream signaling events, not due to any particular avoidance mechanism, but rather by not being sensed by the host cell to initiate any autophagic mechanism.

Complications of autophagy in bacterial infection scenarios

More recently, it has come to light that ATG genes, originally identified due to their association with autophagy, may function in other cellular pathways. Restriction of M. tb in the mouse model appears to be specifically associated with the deletion of Atg5 via a mechanism involving polymorphonuclear leukocytes, but not with the deletion of other Atgs that are required for a functional autophagy system. Future studies will be required to validate this phenomenon and determine if it is relevant in humans [43•]. Nevertheless, this finding poses a warning, and suggests that further controls may be necessary in order to establish firm connections with the autophagic pathway. ATG5 has been linked to a number of other functions in the cell, such as apoptosis and cellular immunity [44–46]. Atg8 orthologues are also involved in cytoskeletal functions and bind to many other proteins including GTPase GEFs and GAPs [46]. Furthermore, many chemical inhibitors that are widely used in the autophagy field influence other pathways, and thus may produce off target effects in some experimental models. To improve experiments in which the role of autophagy is tested, multiple controls in which autophagy is disrupted may include the introduction or expression of the protein RavZ, and/or multiple mutants of ATG genes.

Conclusion

It is clear that the autophagic pathway restricts growth and proliferation of certain bacterial pathogens. However, different bacterial pathogens may be targeted differently depending on their lifestyles and host pathways with which they interact. A more detailed understanding of the machinery involved in the initiation of autophagic targeting of bacterial pathogens will be required. Future cell biological studies will also be required to investigate the specificity of autophagy adaptor proteins and Atg8 orthologues. Together, these data would allow for further identification of mechanisms that bacterial pathogens use to avoid autophagy, and to what extent autophagic targeting counters bacterial replication during human infections.