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LC3-associated phagocytosis - The highway to hell for phagocytosed microbes
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LC3-associated phagocytosis - The highway to hell for phagocytosed microbes Marc Herb, Alexander Gluschko, Michael Schramm
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Institute for Medical Microbiology, Immunology and Hygiene, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: LC3-associated phagocytosis Non-canonical autophagy Immunity Macrophages
Phagocytes ingest, kill and degrade invading microbes in a process called phagocytosis. LC3-associated phagocytosis (LAP) combines the molecular machinery of phagocytosis with that of autophagy, the cellular pathway for ingestion of cytoplasmic components, resulting in the eponymous association of ‘microtubule-associated proteins 1 A/1B light chain 3’ (LC3) with the phagosomal membrane. The LC3-decorated phagosomes, or LAPosomes, show enhanced fusion with lysosomes resulting in enhanced killing and degradation of contained pathogens. Thus, LAP is a particularly microbicidal pathway. In this review, we discuss the molecular mechanisms involved in induction and execution of LAP and its crucial role in antimicrobial immunity against bacteria, fungi and parasites. As LAP has only recently been defined, we also point out the key open questions that remain to be answered.
1. Introduction Phagocytosis, the engulfment of extracellular particles into vesicles called phagosomes (Fig. 1), is one of the key components of cellular antimicrobial immunity [1]. Specialized immune cells, phagocytes, such as granulocytes, dendritic cells, monocytes and macrophages use phagocytosis to ingest, kill and degrade invading microbes. Of paramount importance for killing and subsequent degradation of phagocytosed microbes is maturation of the phagosome into a phagolysosome by fusion with lysosomes as this results in transfer of microbicidal acid hydrolases from lysosomes into the phagosome. Remarkably, a number of microbes have evolved strategies to avoid being killed in phagolysosomes [1]. Some pathogens such as Staphylococcus aureus or Yersinia spp. avoid being phagocytosed at all. Protozoans such as Leishmania and bacteria such as Mycobacteria, Legionella or Salmonella remodel phagosomes in their favor, usually by halting phagosome maturation into phagolysosomes. Other bacterial pathogens such as Listeria monocytogenes, Shigella flexneri or Burkholderia pseudomallei, destroy the phagosome before it fuses with lysosomes and escape into the cytosol. Analogous to the ingestion of extracellular particles by phagocytosis, cells have evolved a pathway for ingestion of intracellular structures: macroautophagy (although macroautophagy is only one of three subtypes of autophagy, the term ‘autophagy’ usually is used synonymously for macroautophagy; we follow this convention throughout this
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manuscript) [2]. Like phagocytosis, autophagy results in sequestration of the target structure in a vesicle, the autophagosome (Fig. 1). Autophagosomes, however, are surrounded not by a single membrane but by two (or multiple) membranes. This characteristic double-membrane is a consequence of the process leading to autophagosome formation: a cup-shaped structure called phagophore becomes elongated until it completely surrounds the target. Like phagosomes, autophagosomes mature and finally fuse with lysosomes into autolysosomes, resulting in degradation of their cargo. Basically, any component of the cytoplasm can be targeted by autophagy. In the context of microbial infection, cytosolic microbes as well as altered or damaged pathogen-containing vacuoles can be targeted by autophagy. In this case, i.e. when autophagy targets structures of non-self origin, it is also referred to as xenophagy (nonetheless, for the sake of simplicity, the term ‘autophagy’ is used instead of ‘xenophagy’ throughout this manuscript). As being targeted by autophagy usually means being killed and degraded, a number of pathogens with an intracellular lifestyle have evolved strategies to avoid being sequestered in autophagosomes [3]. For example, Listeria monocytogenes prevent being recognized as targets for autophagy, Salmonella block autophagy induction, Legionella proteolytically inactivate components of the autophagic machinery and Mycobacteria prevent autophagosome-lysosome fusion. In recent years, a third pathway for ingestion of particulate structures has been discovered: LC3-associated phagocytosis (LAP). During LAP, components of the autophagic machinery are recruited to
Corresponding author. E-mail address: [email protected] (M. Schramm).
https://doi.org/10.1016/j.semcdb.2019.04.016 Received 17 January 2019; Received in revised form 18 April 2019; Accepted 23 April 2019 1084-9521/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Marc Herb, Alexander Gluschko and Michael Schramm, Seminars in Cell and Developmental Biology, https://doi.org/10.1016/j.semcdb.2019.04.016
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Fig. 1. Graphical overview of phagocytosis, canonical autophagy and LC3-associated phagocytosis. During phagocytosis, extracellular particles such as microbes are recognized by specific surface receptors and ingested into vesicles called phagosomes. During canonical autophagy, a membrane structure called phagophore surrounds cytosolic components such as altered or damaged pathogen-containing vacuoles or microbes, giving rise to a double-membrane vesicle called autophagosome. Originally, the protein LC3 had been considered to be specifically recruited to autophagosomal membranes. However, by a non-canonical autophagy pathway called LC3-associated phagocytosis, LC3 can also be recruited to phagosomes that then are referred to as LAPosomes. Phagocytosis, canonical autophagy and LAP are initiated via distinct pathways and phagosomes, autophagosomes and LAPosomes form and mature by different mechanisms. The terminal step for all of them, however, is the fusion with lysosomes by which they acquire the lysosomal acid hydrolases that are essential for killing and degradation of phagocytosed microbes.
phagosomes leading to the eponymous association of ‘microtubule-associated proteins 1 A/1B light chain 3’ (LC3) with the phagosomal membrane (Fig. 1). Of note, ‘LC3’ is a collective term for the mammalian orthologs of yeast Atg8, i.e. LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2/GATE-16. To discriminate such LC3-decorated phagosomes from conventional phagosomes, they have been named LAPosomes. As LAPosome formation requires many, yet not all (!), components of the molecular machinery required for generation of canonical, double-membrane autophagosomes but uses them for conjugation of LC3 to single-membrane phagosomes, LAP is considered a non-canonical form of autophagy. Nonetheless, LAP also shares many features of conventional phagocytosis such as the initiation by surface receptors. In this review, we will delineate the molecular mechanisms of LAP and discuss its distinct function in antimicrobial immunity as well as how some pathogens manage to evade LAP. Moreover, we also point out the key open questions that remain to be answered about this relatively recently identified process.
membrane via one of three class III PI(3)K complexes (PI3KC3) [15]. All three PI3KC3 contain the core components VPS34, VPS15 and Beclin-1 but contain different Beclin-1-binding proteins that define specificity and function. The two different PI3KC3s that are involved in canonical autophagy contain either ATG14 L (for PI3KC3-C1) or UVRAG (for PI3KC3-C2). The PI3KC3 formed during LAP contains not only UVRAG but, importantly, also Rubicon [5]. While this Rubicon-containing PI3KC3 is essential for PI(3)P formation during LAP [5], Rubicon negatively regulates canonical autophagy at multiple steps [16,17]. How this differential effect is achieved is incompletely understood [18]. In the case of autophagy, a complex consisting of WIPI proteins and ATG2 binds to the PI(3)P generated in the target membrane. By binding to both PI(3)P and ATG16L1, the WIPI-ATG2 complex recruits the LC3 conjugation complex consisting of the ATG12 and the LC3 conjugation systems, resulting in attachment of LC3 to the target membrane. Also for LAP, ATG16L1 recruitment to the PI(3)P-containing target membrane is essential. However, the factor directly binding to both PI(3)P and ATG16L1 during LAP is still unknown. WIPI2, at least, is not involved [5]. Interestingly, a different domain of ATG16L1 seems to be responsible for its recruitment to PI(3)P-containing membranes during LAP than during autophagy [19,20] suggesting that a completely different factor than WIPI proteins may recruit ATG16L1 to the PI(3)Pcontaining membrane during LAP. The second fundamental difference in induction of LAP and autophagy is that generation of reactive oxygen species (ROS) by the phagocyte oxidase Nox2 is essential for LAP but dispensable for autophagy (redox regulation of canonical autophagy rather occurs via mitochondria-derived ROS [21]) [5,13]. For activation of ROS production by Nox2, the cytosolic subunits p67phox, p47phox, p40phox and Rac1/2 have to be recruited to gp91phox and p22phox, the integral membrane subunits that form the catalytic center. Rubicon regulates Nox2 activity in two ways: i) because Rubicon stabilizes p22phox (and hence also gp91phox) via direct binding [22] and ii) because p40phox binding to PI(3)P generated by the Rubicon-containing PI3PKC3 is required for full Nox2 activation [5]. Notably, why Nox2-derived ROS are so crucial for LAP is not understood at all. As mentioned above, the LC3 conjugation complex consisting of the ATG12 and the LC3 conjugation systems are the same for autophagy and LAP. Hence, only the mechanisms directing the LC3 conjugation complex to the target membrane differ between autophagy and LAP. This target membrane is the phagophore or the omegasome in the case of autophagy [2] and the phagosome in the case of LAP [4]. In summary, the defining characteristics of LAP are i) induction by surface receptors, ii) independence of ULK complex components, iii) involvement of the Rubicon-containing PI3PKC3 instead of PI3PKC3-C1 or -C2, iv) strict dependence on Nox2-derived ROS and v) the
2. Mechanisms of LAP induction The molecular mechanisms of LAPosome formation are by far not completely understood. What is known so far is that LAP shares many components of the molecular machinery responsible for LC3 conjugation to the target membrane with canonical autophagy (Fig. 2). For example, the two ubiquitin-like conjugation systems responsible for the conjugation of LC3 to the membrane lipid phosphatidylethanolamine, i.e. for the attachment of LC3 to the target membrane, are the same for LAP and canonical autophagy [4,5]. In both cases, the ATG12 conjugation system consists of ATG7 (E1-like) and ATG10 (E2-like), the LC3 conjugation system of ATG7 (E1-like), ATG3 (E2-like) and a complex of ATG16L1, ATG5 and ATG12 (E3-like as a complex). Yet, there also are some clear differences in the molecular machineries of LAP and canonical autophagy (Fig. 2). The most fundamental difference is the activation mechanism. Canonical autophagy is induced by the kinases mTORC1 and/or AMPK via a pre-initiation complex composed of ULK1 and/or ULK2, FIP200, ATG13 and ATG101 [6]. By contrast, induction of LAP is completely independent of this pre-initiation complex [5] and, instead, is triggered by surface receptors. Among those are pattern recognition receptors such as TLRs [4,7], Dectin-1 [8–10], Dectin-2 [11] and Mac-1/CR3/ integrin αmβ2 [12], IgG receptors such as FcγR [13] and receptors recognizing dead cells such as Tim4 [14]. Pre-initiation complex activation (during autophagy) or surface receptor activation (during LAP) then induces generation of the membrane lipid phosphatidylinositol-3-phosphate (PI(3)P) in the target 2
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Fig. 2. Molecular machineries leading to induction of LAP and canonical autophagy. The molecular machineries of LAP and canonical autophagy differ in some important aspects. While canonical autophagy is induced via a pre-initiation complex that is also referred to as ULK complex, LAP does not require any ULK complex components but is induced by specific surface receptors. Ulk complex or surface receptor activation then lead to formation of PI(3)P in the target membrane by class III PI(3)K complexes that are specific for either canonical autophagy or LAP. The two canonical autophagy-specific class III PI(3)K complexes contain either ATG14 L or UVRAG whereas the LAP-specific class III PI(3)K complex contains UVRAG and, importantly, Rubicon. During canonical autophagy, a complex of WIPI proteins (mainly WIPI2) and ATG2 binds to PI(3)P in the target membrane and. This complex recruits the LC3 conjugation system that mediates the conjugation of LC3 to the target membrane. LC3 conjugation to the target membrane during LAP is mediated by the same LC3 conjugation system. However, LC3 conjugation system recruitment to the target membrane during LAP does not require WIPI2. Instead, ROS generated by the Nox2 complex are absolutely crucial.
of factors such as membrane lipid composition, recruitment of a molecular machinery including Rab7, RILP, PLEKHM1 and the HOPS complex and SNARE complex formation [29]. To date, no data exist on specific membrane lipid composition of LAPosomes. Interestingly, LC3 proteins, and in particular the LC3 proteins of the GABARAP family [30], have been shown to directly regulate fusion with lysosomes by binding and recruiting PLEKHM1 [31], an adapter protein required for Rab7 and HOPS complex recruitment, to autophagosomes. It will be interesting to see if promotion of phagolysosomal fusion by LAP also is due to LC3-mediated recruitment of PLEKHM1. Another possibility is that LAP promotes phagolysosomal fusion via formation of a specific SNARE complex. The SNARE complex mediating fusion of autophagosomes with lysosomes consists of STX17 and SNAP29 or VTI1b on autophagosomal side and VAMP8 on lysosomal side [32,33]. The SNAREs mediating fusion of conventional phagosomes with lysosomes are not well defined, but include STX7, STX8, VTI1b, SNAP23 and VAMP7 [34]. On which SNAREs (autophagosomal, phagosomal or completely distinct) fusion of LAPosomes with lysosomes relies remains elusive, though. In summary, how LAP enhances phagolysosomal fusion on the molecular level remains one of the most interesting open questions.
recruitment of LC3 to single-membrane phagosomes. 3. Functions of LAP With some notable exceptions, the main function of LAP is to promote fusion of phagosomes with lysosomes resulting in enhanced degradation of the cargo. For example, fusion of phagosomes containing microbe-like particles such as TLR ligand-coated latex beads and zymosan [4] or live microbes such as Aspergillus fumigatus [5], Legionella dumoffii [23] and Listeria monocytogenes [12] with lysosomes is substantially enhanced by LAP. Furthermore, fusion with lysosomes of phagosomes containing dead cells is promoted by LAP [14]. Some stimuli such as Fcγ receptor-engaging particles may be an exception, as LAP did not further enhance lysosomal fusion of phagosomes containing IgG-opsonized zymosan or sheep red blood cells [24]. Moreover, LAP can even delay phagosome maturation resulting in prolonged antigen presentation by MHCII [25]. Indeed, LAP-deficient dendritic cells show impaired antigen processing [8,19,25]. in vivo, LAP does not seem to be required for tissue homeostasis as LAP-deficient mice do not show any respective defects [20]. With age, however, defective clearance of dead cells in LAP-deficient mice results in increased serum levels of pro-inflammatory cytokines, development of autoantibodies and of a systemic lupus erythematosus-like syndrome [26]. Moreover, LAP actually is detrimental for tumor control as it enhances immune tolerance [27]. For anti-microbial immunity, however, LAP is crucial [5,12,28]. The main reason why LAP is crucial for anti-microbial immunity is the promotion of phago-lysosomal fusion [12,28]. The molecular mechanisms how LAP enhances fusion with lysosomes are not understood at all, though. Fusion of vesicles with lysosomes depends on a number
4. LAP in anti-microbial immunity As elaborated above, engagement of LAP usually promotes phagolysosomal fusion resulting in enhanced microbial killing. However, a number of pathogens have evolved strategies to avoid being targeted and killed by LAP. Among these pathogens are bacteria, fungi and parasites. 3
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containing phagosomes promotes fusion with lysosomes and subsequent killing of L. monocytogenes [12]. Thus, sufficient production of ROS by Nox2 seems to be crucial to fully retrieve the anti-listerial potential of LAP. Importantly, Nox2-derived ROS do not seem to be listericidal by themselves but enhance anti-listerial activity of macrophages by inducing LAP [12]. As Nox2-derived ROS are not only crucial for LAP but also can have direct antimicrobial and signaling functions [40] it often is difficult to discriminate between these alternatives. Yet, inhibition of lysosomal acid hydrolase activity under conditions that preserve Nox2 activity as well as LC3 recruitment to L. monocytogenescontaining phagosomes strongly impairs anti-listerial activity of macrophages [12]. Therefore, it indeed is enhanced fusion of LAPosomes with lysosomes that underlies enhancement of bacterial killing by LAP. Like L. monocytogenes, Mycobacterium tuberculosis specialize in using macrophages as a proliferative niche. To this end, M. tuberculosis prevent fusion of phagosomes with lysosomes and targeting by canonical autophagy [41]. M. tuberculosis also possess a virulence factor, CpsA, that prevents targeting and killing by LAP [28] (Fig. 3). In contrast to phagosomes containing wildtype M. tuberculosis, phagosomes containing CpsA-deficient M. tuberculosis become positive for Nox2 and decorated with LC3 and fuse with lysosomes [28]. Both, deficiency for Nox2 or that for components of the autophagic machinery involved in LAP, strongly impairs killing of CpsA-deficient M. tuberculosis by macrophages in vitro and in vivo. By contrast, deficiency for components of the autophagic machinery specific for canonical autophagy does not markedly impair killing of wildtype or CpsA-deficient M. tuberculosis. Thus, M. tuberculosis clearly prevent being killed by LAP via CpsA. Mechanistically, CpsA prevents recruitment of Nox2 to M. tuberculosiscontaining phagosomes resulting in impaired ROS production and, thereby, induction of LAP. In the case of M. tuberculosis, direct evidence that Nox2 contributes to antibacterial activity of macrophages predominantly by inducing LAP is missing but deficiency for Nox2 resulted in a similar degree of impairment as deficiency for components of the autophagic machinery involved in LAP [28]. Burkholderia pseudomallei also has evolved a strategy to avoid targeting by LAP. Mutations affecting the type III secretion system (T3SS) or the effector protein BopA result in substantially increased localization of B. pseudomallei in LAPsomes [42]. Much more B. pseudomalleicontaining LAPosomes acquire the late phagosomal/lysosomal marker protein LAMP1 than conventional B. pseudomallei-containing phagosomes indicating that, also in this case, LAP promotes fusion with lysosomes. However, if targeting of B. pseudomallei by LAP results in enhanced killing was not investigated. Moreover, there is at least some contribution also of canonical autophagy to killing of B. pseudomallei as autophagy induction further enhances bacterial killing [43]. Hence, direct evidence for an involvement of LAP in immunity against B. pseudomallei remains to be presented. In contrast to the more commonly known Legionella pneumophila, Legionella dumoffii are targeted and killed by LAP [23]. The majority of L. dumoffii survive in macrophages by using their type IV secretion system (T4SS) to remodel the phagosome into an endoplasmatic reticulum-like vacuole that does not fuse with lysosomes. However, a subpopulation of L. dumoffii is targeted by LAP, which results in phagosome maturation and killing of the pathogen. Of note, while targeting of L. dumoffii by LAP is induced via TLR2 activation and ROS production, it also strictly requires expression of the T4SS. T4SS mutant L. dumoffii are not targeted by LAP at all. How macrophages use TLR2 to discriminate between T4SS-expressing and T4SS-deficient L. dumoffii remains elusive, though. Furthermore, if LAP also plays a role in immunity to L. dumoffii in vivo remains to be shown. Other bacteria such as Yersinia pseudotuberculosis [44] or Shigella flexneri [45] reside in LC3-decorated single-membrane vacuoles after infection of non-myeloid cells. However, if these vacuoles are decorated with LC3 via bona fide LAP or rather via a different form of non-canonical autophagy remains to be investigated.
Fig. 3. Targeting by LAP enhances killing of ingested bacteria. L. monocytogenes specialize in escaping from conventional phagosomes. In LAPosomes, however, L. monocytogenes are killed and degraded. LAP of L. monocytogenes is initiated by the surface receptor Mac-1, which induces ROS production by Nox2 and subsequent recruitment of LC3. L. monocytogenescontaining LAPosomes show enhanced fusion with lysosomes and, thus, acquire more bactericidal acid hydrolases. In consequence, L. monocytogenes are efficiently eliminated by LAP. M. tuberculosis uses the virulence factor CpsA to prevent Nox2 recruitment to the phagosome and, thus, induction of LAP. Phagosomes containing wildtype M. tuberculosis do not mature into degradative compartments. By contrast, phagosomes containing CpsA-deficient M. tuberculosis acquire Nox2, associate with LC3 and fuse with lysosomes resulting in elimination of the pathogen by LAP.
4.1. LAP vs. bacteria An excellent, and to date also the only, example of a pathogenic bacterium that is readily targeted and killed by LAP in vivo, is L. monocytogenes [12] (Fig. 3). Initially, L. monocytogenes had been suggested to exploit LAP to establish an intracellular proliferative niche [35]. However, the exact opposite is the case as tissue macrophages rely on promotion of fusion of L. monocytogenes -containing phagosomes with lysosomes by LAP for killing of L. monocytogenes [12]. While L. monocytogenes possess virulence factors to escape from phagosomes and to avoid targeting by canonical autophagy [12,36–39], they do not seem to have virulence factors to avoid LAP as heat-killed L. monocytogenes and L. monocytogenes deficient for the transcriptional regulator of most listerial virulence factors, prfA, are targeted by LAP to the same extent as wildtype L. monocytogenes [12]. In consequence, LAP, but not canonical autophagy, promotes killing of L. monocytogenes by macrophages and enhances immunity to L. monocytogenes infection in vivo [12]. Interestingly, L. monocytogenes are properly targeted and killed by LAP only in tissue macrophages that produce sufficient amounts of ROS by Nox2. In Nox2-deficient tissue macrophages and in in vitro-differentiated macrophages, which produce significantly less ROS by Nox2 than tissue macrophages [12], LC3 recruitment to L. monocytogenescontaining phagosomes is induced via an alternative pathway by the pore-forming toxin of L. monocytogenes, Listeriolysin O (LLO), [12,35]. However, this ROS-independent LC3 recruitment to L. monocytogenescontaining phagosomes does not confer anti-listerial properties as L. monocytogenes escape from these LC3-decorated phagosomes [37]. By contrast, ROS-dependent LC3 recruitment to L. monocytogenes4
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Fig. 4. Targeting by LAP enhances killing of ingested fungi. Recognition of ingested S. cerevisiae mainly by Dectin-1 and -2 induces ROS production by Nox2 and subsequent recruitment of LC3 to S. cerevisiae-containing phagosomes. These LAPosomes show enhanced fusion with lysosomes resulting in increased exposure of S. cerevisiae to lysosomal acid hydrolases and, thus, enhanced killing and degradation of S. cerevisiae. A. fumigatus are also targeted and killed by LAP. However, their conidia limit targeting by LAP via their cell wall component melanin. Melanin sequesters Ca2+ in the phagosome, which prevents activation of a Ca2+/calmodulin signaling pathway that leads to recruitment of Rubicon and Nox2 to the phagosome and, thereby, LAP. Accordingly, A. fumigatus deficient for melanin, for example because of germination, are targeted and degraded more efficiently by LAP.
receptors for induction of LAP here are Dectin-1 and -2 [8–11], with some contribution also of TLRs [4]. That deficiency for LAP, but not that for canonical autophagy, impairs immunity to the filamentous fungi Aspergillus fumigatus [5] demonstrates that LAP contributes to anti-fungal immunity also in vivo. However, A. fumigatus manage to limit the contribution of LAP via their cell wall component melanin [47] (Fig. 4). Conidia (the spores of fungi) of A. fumigatus are efficiently targeted by LAP only when devoid of melanin, either caused by genetic deficiency, chemical removal or swelling during germination. Consequently, melanin-deficient A. fumigatus are killed in vivo much more efficiently and in a LAP-dependent manner. Mechanistically, melanin sequesters Ca2+ in the phagosome [48]. This prevents activation of a Ca2+/calmodulin signaling pathway
Similarly, LC3 recruitment to Salmonella typhimurium infecting epithelial cells [13] or to adherent and invasive Escherichia coli and Staphylococcus aureus in macrophages [46] have been shown to require Nox2-derived ROS. However, other defining characteristics of LAP have not been investigated in these studies. Thus, whether these processes represent bona fide LAP remains to be demonstrated. 4.2. LAP vs. fungi LAP obviously has a fundamental role in immunity to fungal infections. Already in their landmark paper introducing LAP, Sanjuan et al. identified LAP as a mechanism that promotes killing of ingested Saccharomyces cerevisiae in vitro [4] (Fig. 4). The most important 5
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Fig. 5. Targeting by LAP enhances killing of L. major. L. major limit targeting by LAP by using their surface metalloprotease gp63 to cleave the SNARE protein VAMP8 that is necessary for recruitment of Nox2 and, thus, induction of LAP. Moreover, a subpopulation mimicking apoptotic host cells subverts LAP to dampen the host response. A non-canonical autophagy pathway similar to LAP targets T. gondii-containing vacuoles in IFN-γ-stimulated cells. It does, however, not result in enhanced fusion with lysosomes but induces recruitment of IRGs, which induce membrane rupture of the vacuole and cytosolic death of the parasite.
decipher the relative roles of LAP and canonical autophagy in immunity to C. albicans. Moreover, a study using a different strain of C. albicans did not find any increased susceptibility of mice with ATG7 deficiency in myeloid cells [53]. Therefore, targeting of C. albicans by the autophagic machinery may be strain-specific. Whether this is due to strainspecific virulence factors that limit targeting and killing of C. albicans by autophagy and/or LAP remains elusive.
that leads to recruitment of Rubicon and Nox2 to the phagosome and, thereby, LAP. Melanin is used also by Rhizopus oryzae, filamentous fungi causing mucormycosis, to prolong intracellular survival in macrophages [49]. Melanin-expressing R. oryzae largely prevent targeting by LAP and arrest phagosome maturation. Removal of melanin enhances targeting by LAP and phagosome maturation, resulting in elimination of R. oryzae by macrophages in vivo. The role of LAP in immunity to Candida albicans is less clear [50]. C. albicans are targeted by LAP [9,10] and survive more often in LC3ßdeficient macrophages in vitro [9]. Mice deficient for ATG5 [51] or ATG7 [52] in myeloid cells are more susceptible to C. albicans infection but this is at least to some degree due to impaired canonical autophagy [52]. Thus, an unambiguous role for LAP in killing of C. albicans remains to be demonstrated, and additional experiments are required to
4.3. LAP vs. parasites The promastigotes of Leishmania major are targeted by LAP [54] (Fig. 5). Whether this results in enhanced killing of the parasite remains to be investigated, though. In particular, because L. major limit targeting by LAP via their surface metalloprotease gp63. Mechanistically, gp63 directly cleaves the SNARE protein VAMP8 [55] resulting in 6
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impaired recruitment of Nox2 to L. major-containing phagosomes and, thereby, LAP [54]. Thus, viable L. major promastigotes actively limit targeting by LAP. By contrast, apoptotic-like L. major promastigotes, which are important for disease development, readily enter LAPsomes [56]. Since they mimic apoptotic host cells by exposing phosphatidylserine on their surface, their LAP reduces cytokine secretion and T cell activation by macrophages resulting in enhanced survival of viable L. major promastigotes present in the inoculum. Hence, L. major on the one hand avoid targeting by LAP and on the other hand subvert LAP to impair immunity. It will be interesting to see the relative contributions of these two pathways to immunity in vivo. In IFN-γ-stimulated cells, vacuoles containing the tachyzoites of Toxoplasma gondii are targeted by a non-canonical autophagy pathway that shares many features with LAP yet is different in others (Fig. 5). It requires the LC3 conjugation complex but not components of the ULK complex or Atg14 [57]. However, decoration of T. gondii-containing vacuoles with LC3 does not result in fusion with lysosomes but in recruitment of immunity-related GTPases (IRGs) [57–59]. These IRGs induce disruption of the vacuole and subsequent death of the parasite in the cytosol. Thus, although this pathway also promotes killing of the pathogen, it is different from LAP. Plasmodium berghei are targeted and killed by a non-canonical form of autophagy that does require ATG5 but not FIP200 in HeLa cells [60]. Similarly, Plasmodium vivax are targeted and killed by a non-canonical form of autophagy that does require ATG5 but not ULK1 in IFN-γ-stimulated hepatocytes [61]. Whether similar pathways are triggered in professional phagocytes and whether they have more in common with LAP than the independence from the ULK complex remains to be investigated, though.
macrophages not target all L. monocytogenes via LAP to ensure complete elimination? As expected for such a highly microbicidal pathway, pathogens have evolved strategies to evade targeting by LAP. Evasion of LAP is known for a number of pathogens, knowledge on microbial evasion strategies for LAP on molecular level, however, is scarce. Interestingly, all molecularly understood strategies of pathogens to evade LAP (i.e. those of M. tuberculosis, A. fumigatus and L. major) rely on prevention of ROS production by Nox2 highlighting its crucial role during LAP. Of course, inhibiting Nox2 may be particularly advantageous for the pathogen since ROS can also have antimicrobial functions beyond LAP [40]. Notably, to date, it only is clear that Nox2-derived ROS are crucial for LAP, their function on the molecular level, however, remains elusive. Moreover, while there is little doubt that LAP promotes fusion with lysosomes, how this is achieved on the molecular level is completely unknown. LC3 family proteins may play a crucial role but also other, yet to be identified, factors may play a role. In conclusion, while there has been a lot of progress in characterization of the molecular mechanisms and functions of LAP, also a lot of open questions remain to be solved.
5. Concluding remarks
References
With the discovery of LAP in 2007 [4], a highly microbicidal pathway used by phagocytes for elimination of invading microbes has been identified more than ten years ago. However, most of the molecular characteristics discriminating LAP from canonical autophagy have been identified only relatively recently [5]. With this at hand, research on LAP now has gained considerable momentum and a number of novel functions of LAP have already been identified [26,27,62,63]. With respect to microbial infections, the list of pathogens targeted by LAP and the knowledge about microbial evasion strategies for LAP certainly will grow. Particularly, as for some pathogens it is not yet clear whether they really are targeted by bona fide LAP or rather by other non-canonical autophagy pathways. Moreover, some of the antimicrobial functions currently attributed to canonical autophagy most probably will turn out to be rather carried out by LAP. Similarly, with L. major, at least one pathogen exploiting LAP in its favor has been identified [56], and of the pathogens currently thought to exploit canonical autophagy [64] some may turn out to in fact rather exploit LAP. On the other hand, some functions prematurely attributed to LAP may turn out to be rather executed by other non-canonical autophagy pathways. Of note, a noncanonical autophagy pathway very much resembling LAP operates in non-phagocytic cells during entosis, micropinocytosis, virus infection or lysosomotropic drug treatment [19,65–68]. Therefore, which pathogens are really targeted and killed by LAP and which not, remains a very interesting question. The recently introduced mouse line lacking the WD domain of ATG16L1 that seems to be deficient specifically for LAP [20] may be very instrumental for answering this question. Even for pathogens that are definitely targeted and killed by LAP, the question remains why usually only a subpopulation of phagosomes containing bacteria becomes decorated with LC3. For example, L. monocytogenes manage to escape from conventional phagosomes but are killed in LAPosomes, yet only about 20–25% of L. monocytogenes-containing phagosomes become decorated with LC3 [12]. In consequence, at least some ingested L. monocytogenes manage to escape into the cytosol and proliferate even in LAP-proficient macrophages. So, why do
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Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declarations of interest None.
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LC3-associated phagocytosis - The highway to hell for phagocytosed microbes Marc Herb, Alexander Gluschko, Michael Schramm
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Institute for Medical Microbiology, Immunology and Hygiene, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: LC3-associated phagocytosis Non-canonical autophagy Immunity Macrophages
Phagocytes ingest, kill and degrade invading microbes in a process called phagocytosis. LC3-associated phagocytosis (LAP) combines the molecular machinery of phagocytosis with that of autophagy, the cellular pathway for ingestion of cytoplasmic components, resulting in the eponymous association of ‘microtubule-associated proteins 1 A/1B light chain 3’ (LC3) with the phagosomal membrane. The LC3-decorated phagosomes, or LAPosomes, show enhanced fusion with lysosomes resulting in enhanced killing and degradation of contained pathogens. Thus, LAP is a particularly microbicidal pathway. In this review, we discuss the molecular mechanisms involved in induction and execution of LAP and its crucial role in antimicrobial immunity against bacteria, fungi and parasites. As LAP has only recently been defined, we also point out the key open questions that remain to be answered.
1. Introduction Phagocytosis, the engulfment of extracellular particles into vesicles called phagosomes (Fig. 1), is one of the key components of cellular antimicrobial immunity [1]. Specialized immune cells, phagocytes, such as granulocytes, dendritic cells, monocytes and macrophages use phagocytosis to ingest, kill and degrade invading microbes. Of paramount importance for killing and subsequent degradation of phagocytosed microbes is maturation of the phagosome into a phagolysosome by fusion with lysosomes as this results in transfer of microbicidal acid hydrolases from lysosomes into the phagosome. Remarkably, a number of microbes have evolved strategies to avoid being killed in phagolysosomes [1]. Some pathogens such as Staphylococcus aureus or Yersinia spp. avoid being phagocytosed at all. Protozoans such as Leishmania and bacteria such as Mycobacteria, Legionella or Salmonella remodel phagosomes in their favor, usually by halting phagosome maturation into phagolysosomes. Other bacterial pathogens such as Listeria monocytogenes, Shigella flexneri or Burkholderia pseudomallei, destroy the phagosome before it fuses with lysosomes and escape into the cytosol. Analogous to the ingestion of extracellular particles by phagocytosis, cells have evolved a pathway for ingestion of intracellular structures: macroautophagy (although macroautophagy is only one of three subtypes of autophagy, the term ‘autophagy’ usually is used synonymously for macroautophagy; we follow this convention throughout this
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manuscript) [2]. Like phagocytosis, autophagy results in sequestration of the target structure in a vesicle, the autophagosome (Fig. 1). Autophagosomes, however, are surrounded not by a single membrane but by two (or multiple) membranes. This characteristic double-membrane is a consequence of the process leading to autophagosome formation: a cup-shaped structure called phagophore becomes elongated until it completely surrounds the target. Like phagosomes, autophagosomes mature and finally fuse with lysosomes into autolysosomes, resulting in degradation of their cargo. Basically, any component of the cytoplasm can be targeted by autophagy. In the context of microbial infection, cytosolic microbes as well as altered or damaged pathogen-containing vacuoles can be targeted by autophagy. In this case, i.e. when autophagy targets structures of non-self origin, it is also referred to as xenophagy (nonetheless, for the sake of simplicity, the term ‘autophagy’ is used instead of ‘xenophagy’ throughout this manuscript). As being targeted by autophagy usually means being killed and degraded, a number of pathogens with an intracellular lifestyle have evolved strategies to avoid being sequestered in autophagosomes [3]. For example, Listeria monocytogenes prevent being recognized as targets for autophagy, Salmonella block autophagy induction, Legionella proteolytically inactivate components of the autophagic machinery and Mycobacteria prevent autophagosome-lysosome fusion. In recent years, a third pathway for ingestion of particulate structures has been discovered: LC3-associated phagocytosis (LAP). During LAP, components of the autophagic machinery are recruited to
Corresponding author. E-mail address: [email protected] (M. Schramm).
https://doi.org/10.1016/j.semcdb.2019.04.016 Received 17 January 2019; Received in revised form 18 April 2019; Accepted 23 April 2019 1084-9521/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Marc Herb, Alexander Gluschko and Michael Schramm, Seminars in Cell and Developmental Biology, https://doi.org/10.1016/j.semcdb.2019.04.016
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Fig. 1. Graphical overview of phagocytosis, canonical autophagy and LC3-associated phagocytosis. During phagocytosis, extracellular particles such as microbes are recognized by specific surface receptors and ingested into vesicles called phagosomes. During canonical autophagy, a membrane structure called phagophore surrounds cytosolic components such as altered or damaged pathogen-containing vacuoles or microbes, giving rise to a double-membrane vesicle called autophagosome. Originally, the protein LC3 had been considered to be specifically recruited to autophagosomal membranes. However, by a non-canonical autophagy pathway called LC3-associated phagocytosis, LC3 can also be recruited to phagosomes that then are referred to as LAPosomes. Phagocytosis, canonical autophagy and LAP are initiated via distinct pathways and phagosomes, autophagosomes and LAPosomes form and mature by different mechanisms. The terminal step for all of them, however, is the fusion with lysosomes by which they acquire the lysosomal acid hydrolases that are essential for killing and degradation of phagocytosed microbes.
phagosomes leading to the eponymous association of ‘microtubule-associated proteins 1 A/1B light chain 3’ (LC3) with the phagosomal membrane (Fig. 1). Of note, ‘LC3’ is a collective term for the mammalian orthologs of yeast Atg8, i.e. LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2/GATE-16. To discriminate such LC3-decorated phagosomes from conventional phagosomes, they have been named LAPosomes. As LAPosome formation requires many, yet not all (!), components of the molecular machinery required for generation of canonical, double-membrane autophagosomes but uses them for conjugation of LC3 to single-membrane phagosomes, LAP is considered a non-canonical form of autophagy. Nonetheless, LAP also shares many features of conventional phagocytosis such as the initiation by surface receptors. In this review, we will delineate the molecular mechanisms of LAP and discuss its distinct function in antimicrobial immunity as well as how some pathogens manage to evade LAP. Moreover, we also point out the key open questions that remain to be answered about this relatively recently identified process.
membrane via one of three class III PI(3)K complexes (PI3KC3) [15]. All three PI3KC3 contain the core components VPS34, VPS15 and Beclin-1 but contain different Beclin-1-binding proteins that define specificity and function. The two different PI3KC3s that are involved in canonical autophagy contain either ATG14 L (for PI3KC3-C1) or UVRAG (for PI3KC3-C2). The PI3KC3 formed during LAP contains not only UVRAG but, importantly, also Rubicon [5]. While this Rubicon-containing PI3KC3 is essential for PI(3)P formation during LAP [5], Rubicon negatively regulates canonical autophagy at multiple steps [16,17]. How this differential effect is achieved is incompletely understood [18]. In the case of autophagy, a complex consisting of WIPI proteins and ATG2 binds to the PI(3)P generated in the target membrane. By binding to both PI(3)P and ATG16L1, the WIPI-ATG2 complex recruits the LC3 conjugation complex consisting of the ATG12 and the LC3 conjugation systems, resulting in attachment of LC3 to the target membrane. Also for LAP, ATG16L1 recruitment to the PI(3)P-containing target membrane is essential. However, the factor directly binding to both PI(3)P and ATG16L1 during LAP is still unknown. WIPI2, at least, is not involved [5]. Interestingly, a different domain of ATG16L1 seems to be responsible for its recruitment to PI(3)P-containing membranes during LAP than during autophagy [19,20] suggesting that a completely different factor than WIPI proteins may recruit ATG16L1 to the PI(3)Pcontaining membrane during LAP. The second fundamental difference in induction of LAP and autophagy is that generation of reactive oxygen species (ROS) by the phagocyte oxidase Nox2 is essential for LAP but dispensable for autophagy (redox regulation of canonical autophagy rather occurs via mitochondria-derived ROS [21]) [5,13]. For activation of ROS production by Nox2, the cytosolic subunits p67phox, p47phox, p40phox and Rac1/2 have to be recruited to gp91phox and p22phox, the integral membrane subunits that form the catalytic center. Rubicon regulates Nox2 activity in two ways: i) because Rubicon stabilizes p22phox (and hence also gp91phox) via direct binding [22] and ii) because p40phox binding to PI(3)P generated by the Rubicon-containing PI3PKC3 is required for full Nox2 activation [5]. Notably, why Nox2-derived ROS are so crucial for LAP is not understood at all. As mentioned above, the LC3 conjugation complex consisting of the ATG12 and the LC3 conjugation systems are the same for autophagy and LAP. Hence, only the mechanisms directing the LC3 conjugation complex to the target membrane differ between autophagy and LAP. This target membrane is the phagophore or the omegasome in the case of autophagy [2] and the phagosome in the case of LAP [4]. In summary, the defining characteristics of LAP are i) induction by surface receptors, ii) independence of ULK complex components, iii) involvement of the Rubicon-containing PI3PKC3 instead of PI3PKC3-C1 or -C2, iv) strict dependence on Nox2-derived ROS and v) the
2. Mechanisms of LAP induction The molecular mechanisms of LAPosome formation are by far not completely understood. What is known so far is that LAP shares many components of the molecular machinery responsible for LC3 conjugation to the target membrane with canonical autophagy (Fig. 2). For example, the two ubiquitin-like conjugation systems responsible for the conjugation of LC3 to the membrane lipid phosphatidylethanolamine, i.e. for the attachment of LC3 to the target membrane, are the same for LAP and canonical autophagy [4,5]. In both cases, the ATG12 conjugation system consists of ATG7 (E1-like) and ATG10 (E2-like), the LC3 conjugation system of ATG7 (E1-like), ATG3 (E2-like) and a complex of ATG16L1, ATG5 and ATG12 (E3-like as a complex). Yet, there also are some clear differences in the molecular machineries of LAP and canonical autophagy (Fig. 2). The most fundamental difference is the activation mechanism. Canonical autophagy is induced by the kinases mTORC1 and/or AMPK via a pre-initiation complex composed of ULK1 and/or ULK2, FIP200, ATG13 and ATG101 [6]. By contrast, induction of LAP is completely independent of this pre-initiation complex [5] and, instead, is triggered by surface receptors. Among those are pattern recognition receptors such as TLRs [4,7], Dectin-1 [8–10], Dectin-2 [11] and Mac-1/CR3/ integrin αmβ2 [12], IgG receptors such as FcγR [13] and receptors recognizing dead cells such as Tim4 [14]. Pre-initiation complex activation (during autophagy) or surface receptor activation (during LAP) then induces generation of the membrane lipid phosphatidylinositol-3-phosphate (PI(3)P) in the target 2
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Fig. 2. Molecular machineries leading to induction of LAP and canonical autophagy. The molecular machineries of LAP and canonical autophagy differ in some important aspects. While canonical autophagy is induced via a pre-initiation complex that is also referred to as ULK complex, LAP does not require any ULK complex components but is induced by specific surface receptors. Ulk complex or surface receptor activation then lead to formation of PI(3)P in the target membrane by class III PI(3)K complexes that are specific for either canonical autophagy or LAP. The two canonical autophagy-specific class III PI(3)K complexes contain either ATG14 L or UVRAG whereas the LAP-specific class III PI(3)K complex contains UVRAG and, importantly, Rubicon. During canonical autophagy, a complex of WIPI proteins (mainly WIPI2) and ATG2 binds to PI(3)P in the target membrane and. This complex recruits the LC3 conjugation system that mediates the conjugation of LC3 to the target membrane. LC3 conjugation to the target membrane during LAP is mediated by the same LC3 conjugation system. However, LC3 conjugation system recruitment to the target membrane during LAP does not require WIPI2. Instead, ROS generated by the Nox2 complex are absolutely crucial.
of factors such as membrane lipid composition, recruitment of a molecular machinery including Rab7, RILP, PLEKHM1 and the HOPS complex and SNARE complex formation [29]. To date, no data exist on specific membrane lipid composition of LAPosomes. Interestingly, LC3 proteins, and in particular the LC3 proteins of the GABARAP family [30], have been shown to directly regulate fusion with lysosomes by binding and recruiting PLEKHM1 [31], an adapter protein required for Rab7 and HOPS complex recruitment, to autophagosomes. It will be interesting to see if promotion of phagolysosomal fusion by LAP also is due to LC3-mediated recruitment of PLEKHM1. Another possibility is that LAP promotes phagolysosomal fusion via formation of a specific SNARE complex. The SNARE complex mediating fusion of autophagosomes with lysosomes consists of STX17 and SNAP29 or VTI1b on autophagosomal side and VAMP8 on lysosomal side [32,33]. The SNAREs mediating fusion of conventional phagosomes with lysosomes are not well defined, but include STX7, STX8, VTI1b, SNAP23 and VAMP7 [34]. On which SNAREs (autophagosomal, phagosomal or completely distinct) fusion of LAPosomes with lysosomes relies remains elusive, though. In summary, how LAP enhances phagolysosomal fusion on the molecular level remains one of the most interesting open questions.
recruitment of LC3 to single-membrane phagosomes. 3. Functions of LAP With some notable exceptions, the main function of LAP is to promote fusion of phagosomes with lysosomes resulting in enhanced degradation of the cargo. For example, fusion of phagosomes containing microbe-like particles such as TLR ligand-coated latex beads and zymosan [4] or live microbes such as Aspergillus fumigatus [5], Legionella dumoffii [23] and Listeria monocytogenes [12] with lysosomes is substantially enhanced by LAP. Furthermore, fusion with lysosomes of phagosomes containing dead cells is promoted by LAP [14]. Some stimuli such as Fcγ receptor-engaging particles may be an exception, as LAP did not further enhance lysosomal fusion of phagosomes containing IgG-opsonized zymosan or sheep red blood cells [24]. Moreover, LAP can even delay phagosome maturation resulting in prolonged antigen presentation by MHCII [25]. Indeed, LAP-deficient dendritic cells show impaired antigen processing [8,19,25]. in vivo, LAP does not seem to be required for tissue homeostasis as LAP-deficient mice do not show any respective defects [20]. With age, however, defective clearance of dead cells in LAP-deficient mice results in increased serum levels of pro-inflammatory cytokines, development of autoantibodies and of a systemic lupus erythematosus-like syndrome [26]. Moreover, LAP actually is detrimental for tumor control as it enhances immune tolerance [27]. For anti-microbial immunity, however, LAP is crucial [5,12,28]. The main reason why LAP is crucial for anti-microbial immunity is the promotion of phago-lysosomal fusion [12,28]. The molecular mechanisms how LAP enhances fusion with lysosomes are not understood at all, though. Fusion of vesicles with lysosomes depends on a number
4. LAP in anti-microbial immunity As elaborated above, engagement of LAP usually promotes phagolysosomal fusion resulting in enhanced microbial killing. However, a number of pathogens have evolved strategies to avoid being targeted and killed by LAP. Among these pathogens are bacteria, fungi and parasites. 3
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containing phagosomes promotes fusion with lysosomes and subsequent killing of L. monocytogenes [12]. Thus, sufficient production of ROS by Nox2 seems to be crucial to fully retrieve the anti-listerial potential of LAP. Importantly, Nox2-derived ROS do not seem to be listericidal by themselves but enhance anti-listerial activity of macrophages by inducing LAP [12]. As Nox2-derived ROS are not only crucial for LAP but also can have direct antimicrobial and signaling functions [40] it often is difficult to discriminate between these alternatives. Yet, inhibition of lysosomal acid hydrolase activity under conditions that preserve Nox2 activity as well as LC3 recruitment to L. monocytogenescontaining phagosomes strongly impairs anti-listerial activity of macrophages [12]. Therefore, it indeed is enhanced fusion of LAPosomes with lysosomes that underlies enhancement of bacterial killing by LAP. Like L. monocytogenes, Mycobacterium tuberculosis specialize in using macrophages as a proliferative niche. To this end, M. tuberculosis prevent fusion of phagosomes with lysosomes and targeting by canonical autophagy [41]. M. tuberculosis also possess a virulence factor, CpsA, that prevents targeting and killing by LAP [28] (Fig. 3). In contrast to phagosomes containing wildtype M. tuberculosis, phagosomes containing CpsA-deficient M. tuberculosis become positive for Nox2 and decorated with LC3 and fuse with lysosomes [28]. Both, deficiency for Nox2 or that for components of the autophagic machinery involved in LAP, strongly impairs killing of CpsA-deficient M. tuberculosis by macrophages in vitro and in vivo. By contrast, deficiency for components of the autophagic machinery specific for canonical autophagy does not markedly impair killing of wildtype or CpsA-deficient M. tuberculosis. Thus, M. tuberculosis clearly prevent being killed by LAP via CpsA. Mechanistically, CpsA prevents recruitment of Nox2 to M. tuberculosiscontaining phagosomes resulting in impaired ROS production and, thereby, induction of LAP. In the case of M. tuberculosis, direct evidence that Nox2 contributes to antibacterial activity of macrophages predominantly by inducing LAP is missing but deficiency for Nox2 resulted in a similar degree of impairment as deficiency for components of the autophagic machinery involved in LAP [28]. Burkholderia pseudomallei also has evolved a strategy to avoid targeting by LAP. Mutations affecting the type III secretion system (T3SS) or the effector protein BopA result in substantially increased localization of B. pseudomallei in LAPsomes [42]. Much more B. pseudomalleicontaining LAPosomes acquire the late phagosomal/lysosomal marker protein LAMP1 than conventional B. pseudomallei-containing phagosomes indicating that, also in this case, LAP promotes fusion with lysosomes. However, if targeting of B. pseudomallei by LAP results in enhanced killing was not investigated. Moreover, there is at least some contribution also of canonical autophagy to killing of B. pseudomallei as autophagy induction further enhances bacterial killing [43]. Hence, direct evidence for an involvement of LAP in immunity against B. pseudomallei remains to be presented. In contrast to the more commonly known Legionella pneumophila, Legionella dumoffii are targeted and killed by LAP [23]. The majority of L. dumoffii survive in macrophages by using their type IV secretion system (T4SS) to remodel the phagosome into an endoplasmatic reticulum-like vacuole that does not fuse with lysosomes. However, a subpopulation of L. dumoffii is targeted by LAP, which results in phagosome maturation and killing of the pathogen. Of note, while targeting of L. dumoffii by LAP is induced via TLR2 activation and ROS production, it also strictly requires expression of the T4SS. T4SS mutant L. dumoffii are not targeted by LAP at all. How macrophages use TLR2 to discriminate between T4SS-expressing and T4SS-deficient L. dumoffii remains elusive, though. Furthermore, if LAP also plays a role in immunity to L. dumoffii in vivo remains to be shown. Other bacteria such as Yersinia pseudotuberculosis [44] or Shigella flexneri [45] reside in LC3-decorated single-membrane vacuoles after infection of non-myeloid cells. However, if these vacuoles are decorated with LC3 via bona fide LAP or rather via a different form of non-canonical autophagy remains to be investigated.
Fig. 3. Targeting by LAP enhances killing of ingested bacteria. L. monocytogenes specialize in escaping from conventional phagosomes. In LAPosomes, however, L. monocytogenes are killed and degraded. LAP of L. monocytogenes is initiated by the surface receptor Mac-1, which induces ROS production by Nox2 and subsequent recruitment of LC3. L. monocytogenescontaining LAPosomes show enhanced fusion with lysosomes and, thus, acquire more bactericidal acid hydrolases. In consequence, L. monocytogenes are efficiently eliminated by LAP. M. tuberculosis uses the virulence factor CpsA to prevent Nox2 recruitment to the phagosome and, thus, induction of LAP. Phagosomes containing wildtype M. tuberculosis do not mature into degradative compartments. By contrast, phagosomes containing CpsA-deficient M. tuberculosis acquire Nox2, associate with LC3 and fuse with lysosomes resulting in elimination of the pathogen by LAP.
4.1. LAP vs. bacteria An excellent, and to date also the only, example of a pathogenic bacterium that is readily targeted and killed by LAP in vivo, is L. monocytogenes [12] (Fig. 3). Initially, L. monocytogenes had been suggested to exploit LAP to establish an intracellular proliferative niche [35]. However, the exact opposite is the case as tissue macrophages rely on promotion of fusion of L. monocytogenes -containing phagosomes with lysosomes by LAP for killing of L. monocytogenes [12]. While L. monocytogenes possess virulence factors to escape from phagosomes and to avoid targeting by canonical autophagy [12,36–39], they do not seem to have virulence factors to avoid LAP as heat-killed L. monocytogenes and L. monocytogenes deficient for the transcriptional regulator of most listerial virulence factors, prfA, are targeted by LAP to the same extent as wildtype L. monocytogenes [12]. In consequence, LAP, but not canonical autophagy, promotes killing of L. monocytogenes by macrophages and enhances immunity to L. monocytogenes infection in vivo [12]. Interestingly, L. monocytogenes are properly targeted and killed by LAP only in tissue macrophages that produce sufficient amounts of ROS by Nox2. In Nox2-deficient tissue macrophages and in in vitro-differentiated macrophages, which produce significantly less ROS by Nox2 than tissue macrophages [12], LC3 recruitment to L. monocytogenescontaining phagosomes is induced via an alternative pathway by the pore-forming toxin of L. monocytogenes, Listeriolysin O (LLO), [12,35]. However, this ROS-independent LC3 recruitment to L. monocytogenescontaining phagosomes does not confer anti-listerial properties as L. monocytogenes escape from these LC3-decorated phagosomes [37]. By contrast, ROS-dependent LC3 recruitment to L. monocytogenes4
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Fig. 4. Targeting by LAP enhances killing of ingested fungi. Recognition of ingested S. cerevisiae mainly by Dectin-1 and -2 induces ROS production by Nox2 and subsequent recruitment of LC3 to S. cerevisiae-containing phagosomes. These LAPosomes show enhanced fusion with lysosomes resulting in increased exposure of S. cerevisiae to lysosomal acid hydrolases and, thus, enhanced killing and degradation of S. cerevisiae. A. fumigatus are also targeted and killed by LAP. However, their conidia limit targeting by LAP via their cell wall component melanin. Melanin sequesters Ca2+ in the phagosome, which prevents activation of a Ca2+/calmodulin signaling pathway that leads to recruitment of Rubicon and Nox2 to the phagosome and, thereby, LAP. Accordingly, A. fumigatus deficient for melanin, for example because of germination, are targeted and degraded more efficiently by LAP.
receptors for induction of LAP here are Dectin-1 and -2 [8–11], with some contribution also of TLRs [4]. That deficiency for LAP, but not that for canonical autophagy, impairs immunity to the filamentous fungi Aspergillus fumigatus [5] demonstrates that LAP contributes to anti-fungal immunity also in vivo. However, A. fumigatus manage to limit the contribution of LAP via their cell wall component melanin [47] (Fig. 4). Conidia (the spores of fungi) of A. fumigatus are efficiently targeted by LAP only when devoid of melanin, either caused by genetic deficiency, chemical removal or swelling during germination. Consequently, melanin-deficient A. fumigatus are killed in vivo much more efficiently and in a LAP-dependent manner. Mechanistically, melanin sequesters Ca2+ in the phagosome [48]. This prevents activation of a Ca2+/calmodulin signaling pathway
Similarly, LC3 recruitment to Salmonella typhimurium infecting epithelial cells [13] or to adherent and invasive Escherichia coli and Staphylococcus aureus in macrophages [46] have been shown to require Nox2-derived ROS. However, other defining characteristics of LAP have not been investigated in these studies. Thus, whether these processes represent bona fide LAP remains to be demonstrated. 4.2. LAP vs. fungi LAP obviously has a fundamental role in immunity to fungal infections. Already in their landmark paper introducing LAP, Sanjuan et al. identified LAP as a mechanism that promotes killing of ingested Saccharomyces cerevisiae in vitro [4] (Fig. 4). The most important 5
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Fig. 5. Targeting by LAP enhances killing of L. major. L. major limit targeting by LAP by using their surface metalloprotease gp63 to cleave the SNARE protein VAMP8 that is necessary for recruitment of Nox2 and, thus, induction of LAP. Moreover, a subpopulation mimicking apoptotic host cells subverts LAP to dampen the host response. A non-canonical autophagy pathway similar to LAP targets T. gondii-containing vacuoles in IFN-γ-stimulated cells. It does, however, not result in enhanced fusion with lysosomes but induces recruitment of IRGs, which induce membrane rupture of the vacuole and cytosolic death of the parasite.
decipher the relative roles of LAP and canonical autophagy in immunity to C. albicans. Moreover, a study using a different strain of C. albicans did not find any increased susceptibility of mice with ATG7 deficiency in myeloid cells [53]. Therefore, targeting of C. albicans by the autophagic machinery may be strain-specific. Whether this is due to strainspecific virulence factors that limit targeting and killing of C. albicans by autophagy and/or LAP remains elusive.
that leads to recruitment of Rubicon and Nox2 to the phagosome and, thereby, LAP. Melanin is used also by Rhizopus oryzae, filamentous fungi causing mucormycosis, to prolong intracellular survival in macrophages [49]. Melanin-expressing R. oryzae largely prevent targeting by LAP and arrest phagosome maturation. Removal of melanin enhances targeting by LAP and phagosome maturation, resulting in elimination of R. oryzae by macrophages in vivo. The role of LAP in immunity to Candida albicans is less clear [50]. C. albicans are targeted by LAP [9,10] and survive more often in LC3ßdeficient macrophages in vitro [9]. Mice deficient for ATG5 [51] or ATG7 [52] in myeloid cells are more susceptible to C. albicans infection but this is at least to some degree due to impaired canonical autophagy [52]. Thus, an unambiguous role for LAP in killing of C. albicans remains to be demonstrated, and additional experiments are required to
4.3. LAP vs. parasites The promastigotes of Leishmania major are targeted by LAP [54] (Fig. 5). Whether this results in enhanced killing of the parasite remains to be investigated, though. In particular, because L. major limit targeting by LAP via their surface metalloprotease gp63. Mechanistically, gp63 directly cleaves the SNARE protein VAMP8 [55] resulting in 6
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impaired recruitment of Nox2 to L. major-containing phagosomes and, thereby, LAP [54]. Thus, viable L. major promastigotes actively limit targeting by LAP. By contrast, apoptotic-like L. major promastigotes, which are important for disease development, readily enter LAPsomes [56]. Since they mimic apoptotic host cells by exposing phosphatidylserine on their surface, their LAP reduces cytokine secretion and T cell activation by macrophages resulting in enhanced survival of viable L. major promastigotes present in the inoculum. Hence, L. major on the one hand avoid targeting by LAP and on the other hand subvert LAP to impair immunity. It will be interesting to see the relative contributions of these two pathways to immunity in vivo. In IFN-γ-stimulated cells, vacuoles containing the tachyzoites of Toxoplasma gondii are targeted by a non-canonical autophagy pathway that shares many features with LAP yet is different in others (Fig. 5). It requires the LC3 conjugation complex but not components of the ULK complex or Atg14 [57]. However, decoration of T. gondii-containing vacuoles with LC3 does not result in fusion with lysosomes but in recruitment of immunity-related GTPases (IRGs) [57–59]. These IRGs induce disruption of the vacuole and subsequent death of the parasite in the cytosol. Thus, although this pathway also promotes killing of the pathogen, it is different from LAP. Plasmodium berghei are targeted and killed by a non-canonical form of autophagy that does require ATG5 but not FIP200 in HeLa cells [60]. Similarly, Plasmodium vivax are targeted and killed by a non-canonical form of autophagy that does require ATG5 but not ULK1 in IFN-γ-stimulated hepatocytes [61]. Whether similar pathways are triggered in professional phagocytes and whether they have more in common with LAP than the independence from the ULK complex remains to be investigated, though.
macrophages not target all L. monocytogenes via LAP to ensure complete elimination? As expected for such a highly microbicidal pathway, pathogens have evolved strategies to evade targeting by LAP. Evasion of LAP is known for a number of pathogens, knowledge on microbial evasion strategies for LAP on molecular level, however, is scarce. Interestingly, all molecularly understood strategies of pathogens to evade LAP (i.e. those of M. tuberculosis, A. fumigatus and L. major) rely on prevention of ROS production by Nox2 highlighting its crucial role during LAP. Of course, inhibiting Nox2 may be particularly advantageous for the pathogen since ROS can also have antimicrobial functions beyond LAP [40]. Notably, to date, it only is clear that Nox2-derived ROS are crucial for LAP, their function on the molecular level, however, remains elusive. Moreover, while there is little doubt that LAP promotes fusion with lysosomes, how this is achieved on the molecular level is completely unknown. LC3 family proteins may play a crucial role but also other, yet to be identified, factors may play a role. In conclusion, while there has been a lot of progress in characterization of the molecular mechanisms and functions of LAP, also a lot of open questions remain to be solved.
5. Concluding remarks
References
With the discovery of LAP in 2007 [4], a highly microbicidal pathway used by phagocytes for elimination of invading microbes has been identified more than ten years ago. However, most of the molecular characteristics discriminating LAP from canonical autophagy have been identified only relatively recently [5]. With this at hand, research on LAP now has gained considerable momentum and a number of novel functions of LAP have already been identified [26,27,62,63]. With respect to microbial infections, the list of pathogens targeted by LAP and the knowledge about microbial evasion strategies for LAP certainly will grow. Particularly, as for some pathogens it is not yet clear whether they really are targeted by bona fide LAP or rather by other non-canonical autophagy pathways. Moreover, some of the antimicrobial functions currently attributed to canonical autophagy most probably will turn out to be rather carried out by LAP. Similarly, with L. major, at least one pathogen exploiting LAP in its favor has been identified [56], and of the pathogens currently thought to exploit canonical autophagy [64] some may turn out to in fact rather exploit LAP. On the other hand, some functions prematurely attributed to LAP may turn out to be rather executed by other non-canonical autophagy pathways. Of note, a noncanonical autophagy pathway very much resembling LAP operates in non-phagocytic cells during entosis, micropinocytosis, virus infection or lysosomotropic drug treatment [19,65–68]. Therefore, which pathogens are really targeted and killed by LAP and which not, remains a very interesting question. The recently introduced mouse line lacking the WD domain of ATG16L1 that seems to be deficient specifically for LAP [20] may be very instrumental for answering this question. Even for pathogens that are definitely targeted and killed by LAP, the question remains why usually only a subpopulation of phagosomes containing bacteria becomes decorated with LC3. For example, L. monocytogenes manage to escape from conventional phagosomes but are killed in LAPosomes, yet only about 20–25% of L. monocytogenes-containing phagosomes become decorated with LC3 [12]. In consequence, at least some ingested L. monocytogenes manage to escape into the cytosol and proliferate even in LAP-proficient macrophages. So, why do
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Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declarations of interest None.
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