Phagolysosome Biogenesis Research Articles

ARL5b inhibits human rhinovirus 16 propagation and impairs macrophage-mediated bacterial clearance

Human rhinovirus is the most frequently isolated virus during severe exacerbations of chronic respiratory diseases, like chronic obstructive pulmonary disease. In this disease, alveolar macrophages display significantly diminished phagocytic functions that could be associated with bacterial superinfections. However, how human rhinovirus affects the functions of macrophages is largely unknown. Macrophages treated with HRV16 demonstrate deficient bacteria-killing activity, impaired phagolysosome biogenesis, and altered intracellular compartments. Using RNA sequencing, we identify the small GTPase ARL5b to be upregulated by the virus in primary human macrophages. Importantly, depletion of ARL5b rescues bacterial clearance and localization of endosomal markers in macrophages upon HRV16 exposure. In permissive cells, depletion of ARL5b increases the secretion of HRV16 virions. Thus, we identify ARL5b as a novel regulator of intracellular trafficking dynamics and phagolysosomal biogenesis in macrophages and as a restriction factor of HRV16 in permissive cells.

Propulsive cell entry diverts pathogens from immune degradation by remodeling the phagocytic synapse.

Phagocytosis is a critical immune function for infection control and tissue homeostasis. During phagocytosis, pathogens are internalized and degraded in phagolysosomes. For pathogens that evade immune degradation, the prevailing view is that virulence factors are required to disrupt the biogenesis of phagolysosomes. In contrast, we present here that physical forces from motile pathogens during cell entry divert them away from the canonical degradative pathway. This altered fate begins with the force-induced remodeling of the phagocytic synapse formation. We used the parasite Toxoplasma gondii as a model because live Toxoplasma actively invades host cells using gliding motility. To differentiate the effects of physical forces from virulence factors in phagocytosis, we employed magnetic forces to induce propulsive entry of inactivated Toxoplasma into macrophages. Experiments and computer simulations show that large propulsive forces hinder productive activation of receptors by preventing their spatial segregation from phosphatases at the phagocytic synapse. Consequently, the inactivated parasites are engulfed into vacuoles that fail to mature into degradative units, similar to the live motile parasite's intracellular pathway. Using yeast cells and opsonized beads, we confirmed that this mechanism is general, not specific to the parasite used. These results reveal new aspects of immune evasion by demonstrating how physical forces during active cell entry, independent of virulence factors, enable pathogens to circumvent phagolysosomal degradation.

High content quantitative imaging of Mycobacterium tuberculosis responses to acidic microenvironments within human macrophages.

Intracellular pathogens such as Mycobacterium tuberculosis (Mtb) have evolved diverse strategies to counteract macrophage defence mechanisms including phagolysosomal biogenesis. Within macrophages, Mtb initially resides inside membrane-bound phagosomes that interact with lysosomes and become acidified. The ability of Mtb to control and subvert the fusion between phagosomes and lysosomes plays a key role in the pathogenesis of tuberculosis. Therefore, understanding how pathogens interact with the endolysosomal network and cope with intracellular acidification is important to better understand the disease. Here, we describe in detail the use of fluorescence microscopy-based approaches to investigate Mtb responses to acidic environments in cellulo. We report high-content imaging modalities to probe Mtb sensing of external pH or visualise in real-time Mtb intrabacterial pH within infected human macrophages. We discuss various methodologies with step-by-step analyses that enable robust image-based quantifications. Finally, we highlight the advantages and limitations of these different approaches and discuss potential alternatives that can be applied to further investigate Mtb-host cell interactions. These methods can be adapted to study host-pathogen interactions in different biological systems and experimental settings. Altogether, these approaches represent a valuable tool to further broaden our understanding of the cellular and molecular mechanisms underlying intracellular pathogen survival.

Leishmania donovani infection induce Extracellular signal-regulated kinase ½ (ERK½) mediated lipid droplet generation in macrophages

Recently unfolded mechanisms showed lipid droplet helps in pathogen survival and paralyzes host immune response. In the present study, we showed the extent of lipid droplet(LD) generation in Leishmania donovani infection, the signaling involved, and their function concerning pathogenicity. RAW 264.7 and J774A.1 cells were used to infect with L. donovani and then flow cytometry and confocal microscopy were used to detect lipid droplet generation and subsequent assays. In this study, we showed that L. donovani AG83 (AG83/MHOM/1983) triggers lipid droplet formation in macrophages in a time-dependent manner. We provide novel insight into the signaling molecules which is responsible for LD accumulation. Interestingly, LPG deficient attenuated Leishmania strain UR6 (UR6/MHOM/1978) failed to fuel LD generation. But inhibition of phagosome maturation drastically stimulates LD accumulation in UR6 infected MΦs. Aspirin treatment in AG83 infected MΦs does not only lower LD load but also favors phagolysosome biogenesis and corrects cytokine balance. Employing strategies to circumvent halt in phagosome maturation using drugs that manipulate lipid droplet generation could be used as a therapeutic tool to resist parasite growth in the early hour of infection.

COMMD10 is critical for Kupffer cell survival and controls Ly6Chi monocyte differentiation and inflammation in the injured liver.

Liver-resident macrophages Kupffer cells (KCs) and infiltrating Ly6Chi monocytes both contribute to liver tissue regeneration in various pathologies but also to disease progression upon disruption of orderly consecutive regeneration cascades. Little is known about molecular pathways that regulate their differentiation, maintenance, or inflammatory behavior during injury. Here, we show that copper metabolism MURR1 domain (COMMD)10-deficient KCs adopt liver-specific identity. Strikingly, COMMD10 deficiency in KCs and in other tissue-resident macrophages impedes their homeostatic survival, leading to their continuous replacement by Ly6Chi monocytes. While COMMD10 deficiency in KCs mildly worsens acetaminophen-induced liver injury (AILI), its deficiency in Ly6Chi monocytes results in exacerbated and sustained hepatic damage. Monocytes display unleashed inflammasome activation and a reduced type I interferon response and acquire "neutrophil-like" and lipid-associated macrophage differentiation fates. Collectively, COMMD10 appears indispensable for KC and other tissue-resident macrophage survival and is an important regulator of Ly6Chi monocyte fate decisions and reparative behavior in the diseased liver.

ZKSCAN3 in severe bacterial lung infection and sepsis-induced immunosuppression

The mortality rates among patients who initially survive sepsis are, in part, associated with a high risk of secondary lung infections and respiratory failure. Given that phagolysosomes are important for intracellular killing of pathogenic microbes, we investigated how severe lung infections associated with post-sepsis immunosuppression affect phagolysosome biogenesis. In mice with P. aeruginosa-induced pneumonia, we found a depletion of both phagosomes and lysosomes, as evidenced by decreased amounts of microtubule associated protein light chain 3-II (LC3-II) and lysosomal-associated membrane protein (LAMP1). We also found a loss of transcription factor E3 (TFE3) and transcription factor EB (TFEB), which are important activators for transcription of genes encoding autophagy and lysosomal proteins. These events were associated with increased expression of ZKSCAN3, a repressor for transcription of genes encoding autophagy and lysosomal proteins. Zkscan3−/− mice had increased expression of genes involved in the autophagy-lysosomal pathway along with enhanced killing of P. aeruginosa in the lungs, as compared to wild-type mice. These findings highlight the involvement of ZKSCAN3 in response to severe lung infection, including susceptibility to secondary bacterial infections due to immunosuppression.P. aeruginosa dependent pneumonia decreased lysosomal proteins, transcription factor E3 (TFE3) and transcription factor EB (TFEB); and increased transcription repressor Zinc finger protein with KRAB and SCAN domains 3 (ZKSCAN3). ZKSCAN3 knockout mice exhibited enhanced bacterial killing.

Better Together: Current Insights Into Phagosome-Lysosome Fusion.

Following phagocytosis, the nascent phagosome undergoes maturation to become a phagolysosome with an acidic, hydrolytic, and often oxidative lumen that can efficiently kill and digest engulfed microbes, cells, and debris. The fusion of phagosomes with lysosomes is a principal driver of phagosomal maturation and is targeted by several adapted intracellular pathogens. Impairment of this process has significant consequences for microbial infection, tissue inflammation, the onset of adaptive immunity, and disease. Given the importance of phagosome-lysosome fusion to phagocyte function and the many virulence factors that target it, it is unsurprising that multiple molecular pathways have evolved to mediate this essential process. While the full range of these pathways has yet to be fully characterized, several pathways involving proteins such as members of the Rab GTPases, tethering factors and SNAREs have been identified. Here, we summarize the current state of knowledge to clarify the ambiguities in the field and construct a more comprehensive phagolysosome formation model. Lastly, we discuss how other cellular pathways help support phagolysosome biogenesis and, consequently, phagocyte function.

Vacuolins and myosin VII are required for phagocytic uptake and phagosomal membrane recycling in Dictyostelium discoideum.

Flotillins are lipid raft residents involved in membrane trafficking and recycling of plasma membrane proteins. Dictyostelium discoideum uses phagocytosis to kill, digest and feed on bacteria. It possesses three flotillin-like vacuolins that are strongly associated with membranes and that gradually accumulate on maturing phagosomes. Absence of vacuolins reduced adhesion and particle recognition resulting in a drastic reduction in the uptake of various types of particles. This was caused by a block in the recycling of plasma membrane components and the absence of their specific cortex-associated proteins. In addition, absence of vacuolins also impaired phagolysosome biogenesis, without significantly impacting killing and digestion of a range of bacteria. Strikingly, both absence and overexpression of vacuolins induced a strong downregulation of myosin VII (also known as MyoI) expression, as well as its binding partner talin A. Episomal expression of myosin VII fully rescued defects in uptake and adhesion but not in phagosome maturation. These results suggest a dual role for vacuolins: a novel mechanism involving membrane microdomains and myosin VII-talin A in clustering phagosomal receptors and adhesion molecules at the plasma membrane, and a role in phagolysosomal biogenesis.

Leishmania braziliensis: Strain-Specific Modulation of Phagosome Maturation.

Leishmania (Viannia) braziliensis is responsible for the largest number of American tegumentary leishmaniasis (ATL) in Brazil. ATL can present several clinical forms including typical (TL) and atypical (AL) cutaneous and mucocutaneous (ML) lesions. To identify parasite and host factors potentially associated with these diverse clinical manifestations, we first surveyed the expression of two virulence-associated glycoconjugates, lipophosphoglycan (LPG) and the metalloprotease GP63 by a panel of promastigotes of Leishmania braziliensis (L. braziliensis) strains isolated from patients with different clinical manifestations of ATL and from the sand fly vector. We observed a diversity of expression patterns for both LPG and GP63, which may be related to strain-specific polymorphisms. Interestingly, we noted that GP63 activity varies from strain to strain, including the ability to cleave host cell molecules. We next evaluated the ability of promastigotes from these L. braziliensis strains to modulate phagolysosome biogenesis in bone marrow-derived macrophages (BMM), by assessing phagosomal recruitment of the lysosome-associated membrane protein 1 (LAMP-1) and intraphagosomal acidification. Whereas, three out of six L. braziliensis strains impaired the phagosomal recruitment of LAMP-1, only the ML strain inhibited phagosome acidification to the same extent as the L. donovani strain that was used as a positive control. While decreased phagosomal recruitment of LAMP-1 correlated with higher LPG levels, decreased phagosomal acidification correlated with higher GP63 levels. Finally, we observed that the ability to infect and replicate within host cells did not fully correlate with the inhibition of phagosome maturation. Collectively, our results revealed a diversity of strain-specific phenotypes among L. braziliensis isolates, consistent with the high genetic diversity within Leishmania populations.

COMMD10-Guided Phagolysosomal Maturation Promotes Clearance of Staphylococcus aureus in Macrophages

SummaryStaphylococcus aureus is a major cause of infectious disease. Liver Kupffer cells (KCs) are responsible for sequestering and destroying S. aureus through the phagolysosomal pathway. Proteins belonging to the COMMD family emerge as key intracellular regulators of protein trafficking, but the role of COMMD10 in macrophage-mediated S. aureus eradication is unknown. Here we report that COMMD10 in macrophages was necessary for its timely elimination, as demonstrated with two different S. aureus subspecies. In vivo, COMMD10-deficient liver KCs exhibited impaired clearance of systemic S. aureus infection. S. aureus-infected COMMD10-deficient macrophages exhibited impaired activation of the transcription factor EB, resulting in reduced lysosomal biogenesis. Moreover, S. aureus-initiated phagolysosomal maturation and function were significantly attenuated in COMMD10-deficient macrophages. Finally, expression of COMMD/CCDC22/CCDC93 complex, linked to phagolysosomal maturation, was reduced by COMMD10 deficiency. Collectively, these results support an important role for COMMD10 in instructing macrophage phagolysosomal biogenesis and maturation during S. aureus infection.

Mycobacterium tuberculosis with different virulence reside within intact phagosomes and inhibit phagolysosomal biogenesis in alveolar macrophages of patients with pulmonary tuberculosis

Tuberculosis (TB) is a dangerous airborne disease caused by Mycobacterium tuberculosis (Mtb) and characterized by a tight interplay between pathogen and host cells, mainly alveolar macrophages. Studies of the mechanisms of Mtb survival within human cells during TB disease are extremely important for the development of new strategies and drugs for TB treatment. We have used the ex vivo cultures of alveolar macrophages and histological sections obtained from the resected lungs of patients with pulmonary TB to establish the unique features of Mtb lifestyle in host cells. Our data indicate that Mtb with different virulence, as single and in colonies, with or without cording morphology, are exclusively intravacuolar pathogens with intact phagosomal membranes in viable host cells of TB patients and Mtb-infected guinea pig. Mycobacteria were detected in the cytoplasm and/or damaged vacuoles only in alveolar macrophages with morphological signs of cell death after prolonged ex vivo culture, however Mtb were found inside phagosomes in viable alveolar macrophages or cells with apoptotic/necrotic morphology in the same ex vivo cell culture. The Mtb phagosomes interacted with human different endocytic pathways, but inhibited phagolysosomal biogenesis, while intracellular vesicles containing Mtb products were fused with lysosomes in the same host cells.

Functions of the Dictyostelium LIMP-2 and CD36 homologues in bacteria uptake, phagolysosome biogenesis and host cell defence.

Phagocytic cells take up, kill and digest microbes by a process called phagocytosis. To this end, these cells bind the particle, rearrange their actin cytoskeleton, and orchestrate transport of digestive factors to the particle-containing phagosome. The mammalian lysosomal membrane protein LIMP-2 (also known as SCARB2) and CD36, members of the class B of scavenger receptors, play a crucial role in lysosomal enzyme trafficking and uptake of mycobacteria, respectively, and generally in host cell defences against intracellular pathogens. Here, we show that the Dictyostelium discoideum LIMP-2 homologue LmpA regulates phagocytosis and phagolysosome biogenesis. The lmpA knockdown mutant is highly affected in actin-dependent processes, such as particle uptake, cellular spreading and motility. Additionally, the cells are severely impaired in phagosomal acidification and proteolysis, likely explaining the higher susceptibility to infection with the pathogenic bacterium Mycobacterium marinum, a close cousin of the human pathogen Mycobacterium tuberculosis Furthermore, we bring evidence that LmpB is a functional homologue of CD36 and specifically mediates uptake of mycobacteria. Altogether, these data indicate a role for LmpA and LmpB, ancestors of the family of which LIMP-2 and CD36 are members, in lysosome biogenesis and host cell defence.

CD82 hypomethylation is essential for tuberculosis pathogenesis via regulation of RUNX1-Rab5/22

The tumor suppressor gene CD82/KAI1 is a member of the tetraspanin superfamily and organizes various membrane-based processes. Mycobacterium tuberculosis (MTB) persists in host macrophages by interfering with phagolysosome biogenesis and inflammatory responses, but the role of CD82 in controlling the intracellular survival of pathogenic mycobacteria within macrophages remains poorly understood. In this study, we demonstrated that the virulent MTB strain H37Rv (MTB Rv) induced CD82 promoter hypomethylation, resulting in CD82 expression. Targeting of the runt-related transcription factor 1 (RUNX1) by CD82 is essential for phagosome arrest via interacting with Rab5/22. This arrest is required for the intracellular growth of MTB in vitro and in vivo, but not for that of MTB H37Ra (MTB Ra) in macrophages. In addition, knockdown or knockout of CD82 or RUNX1 increased antibacterial host defense via phagolysosome biogenesis, inflammatory cytokine production, and subsequent antimicrobial activity both in vitro and in vivo. Notably, the levels of CD82 and RUNX1 in granulomas were elevated in tuberculosis (TB) patients, indicating that CD82 and RUNX1 have clinical significance in human TB. Our findings identify a previously unrecognized role of CD82 hypomethylation in the regulation of phagosome maturation, enhanced intracellular survival, and the innate host immune response to MTB. Thus, the CD82–RUNX1–Rab5/22 axis may be a previously unrecognized virulence mechanism of MTB pathogenesis.

COMMD10-Guided Phagolysosomal Maturation Promotes Clearance of <i>Staphylococcus Aureus</i> in Macrophages

Staphylococcus aureus (S. aureus) is a major cause of infectious disease. Macrophages, and especially liver Kupffer cells (KCs), are responsible for sequestering and destroying S. aureus bacteria through the phagolysosomal pathway. Proteins belonging to the COMMD family emerge as key intracellular regulators of protein trafficking, but the role of COMMD10 in macrophage-mediated eradication of S. aureus is unknown. Here we report that COMMD10-deficiency in macrophages did not affect S. aureus internalization, but was necessary for its timely elimination, as demonstrated with two different S. aureus subspecies. In vivo, COMMD10-deficient liver KCs exhibited impaired clearance of systemic S. aureus infection. S. aureus-infected COMMD10-deficient macrophages exhibited impaired activation of the transcription factor EB, resulting in reduced lysosomal biogenesis. Moreover, S. aureus-initiated phagolysosomal maturation and function was significantly attenuated in the COMMD10-deficient macrophages. Collectively, these results support an important role for COMMD10 in instructing macrophage phagolysosomal biogenesis and maturation during S. aureus infection.

Moesin and myosin IIA modulate phagolysosomal biogenesis in macrophages

Biogenesis of phagolysosomes is central to the elimination of pathogens by macrophages. We previously showed that Src homology region 2 domain-containing phosphatase 1 (SHP-1) participates in the regulation of phagosome maturation. Through proteomics, we identified moesin and the non-muscle myosin-IIA as proteins interacting with SHP-1 during phagocytosis. Silencing of either moesin or myosin IIA with small interfering RNA inhibited phagosomal acidification and recruitment of LAMP-1. Moreover, the intraphagosomal oxidative burst was impaired in the absence of either SHP-1 or myosin IIA but not moesin. Finally, absence of either SHP-1, moesin, or myosin IIA ablated the capacity of macrophages to clear bacterial infection. Collectively, these results implicate both moesin and myosin IIA in the regulation of phagolysosome biogenesis and in host defense against infections.

The Macrophage: A Disputed Fortress in the Battle against Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the etiological agent of human tuberculosis (TB), has plagued humans for thousands of years. TB still remains a major public health problem in our era, causing more than 4,400 deaths worldwide every day and killing more people than HIV. After inhaling Mtb-contaminated aerosols, TB primo-infection starts in the terminal lung airways, where Mtb is taken up by alveolar macrophages. Although macrophages are known as professional killers for pathogens, Mtb has adopted remarkable strategies to circumvent host defenses, building suitable conditions to survive and proliferate. Within macrophages, Mtb initially resides inside phagosomes, where its survival mostly depends on its ability to take control of phagosomal processing, through inhibition of phagolysosome biogenesis and acidification processes, and by progressively getting access to the cytosol. Bacterial access to the cytosolic space is determinant for specific immune responses and cell death programs, both required for the replication and the dissemination of Mtb. Comprehension of the molecular events governing Mtb survival within macrophages is fundamental for the improvement of vaccine-based and therapeutic strategies in order to help the host to better defend itself in the battle against the fierce invader Mtb. In this mini-review, we discuss recent research exploring how Mtb conquers and transforms the macrophage into a strategic base for its survival and dissemination as well as the associated defense strategies mounted by host.

Maturation of phagosomes containing different erythrophagocytic particles in primary macrophages.

Erythrophagocytosis is a physiological process that aims to remove damaged red blood cells from the circulation in order to avoid hemolysis and uncontrolled liberation of iron. Many efforts have been made to understand heme trafficking inside macrophages, but little is known about the maturation of phagosomes containing different types of erythrophagocytic particles with different signals at their surfaces. Therefore, we performed a comparative study on the maturation of phagosomes containing three different models of red blood cells (RBC): aged/senescent, complement‐opsonized, and IgG‐opsonized. We also used two types of professional phagocytes: bone marrow‐derived and peritoneal macrophages. By comparing markers from different stages of phagosomal maturation, we found that phagosomes carrying aged RBC reach lysosomes with a delay compared to those containing IgG‐ or complement‐opsonized RBC, in both types of macrophages. These findings contribute to understanding the importance of the different signals at the RBC surface in phagolysosome biogenesis, as well as in the dynamics of RBC removal.

Involvement of the p62/NRF2 signal transduction pathway on erythrophagocytosis

Erythrophagocytosis, the phagocytic removal of damaged red blood cells (RBC), and subsequent phagolysosome biogenesis are important processes in iron/heme metabolism and homeostasis. Phagolysosome biogenesis implies the interaction of nascent phagosomes with endocytic compartments and also autophagy effectors. Here, we report that besides recruitment of microtubule-associated protein-1-light chain 3 (LC3), additional autophagy machinery such as sequestosome 1 (p62) is also acquired by single-membrane phagosomes at very early stages of the phagocytic process and that its acquisition is very important to the outcome of the process. In bone marrow-derived macrophages (BMDM) silenced for p62, RBC degradation is inhibited. P62, is also required for nuclear translocation and activation of the transcription factor Nuclear factor E2-related Factor 2 (NRF2) during erythrophagocytosis. Deletion of the Nrf2 allele reduces p62 expression and compromises RBC degradation. In conclusion, we reveal that erythrophagocytosis relies on an interplay between p62 and NRF2, potentially acting as protective mechanism to maintain reactive oxygen species at basal levels and preserve macrophage homeostasis.

The Monomeric GTPase Rab35 Regulates Phagocytic Cup Formation and Phagosomal Maturation in Entamoeba histolytica

One of the hallmarks of amoebic colitis is the detection of Entamoeba histolytica (Eh) trophozoites with ingested erythrocytes. Therefore, erythrophagocytosis is traditionally considered as one of the most important criteria to identify the pathogenic behavior of the amoebic trophozoites. Phagocytosis is an essential process for the proliferation and virulence of this parasite. Phagocytic cargo, upon internalization, follows a defined trafficking route to amoebic lysosomal degradation machinery. Here, we demonstrated the role of EhRab35 in the early and late phases of erythrophagocytosis by the amoeba. EhRab35 showed large vacuolar as well as punctate vesicular localization. The spatiotemporal dynamics of vacuolar EhRab35 and its exchange with soluble cytosolic pool were monitored by fluorescence recovery after photobleaching experiments. Using extensive microscopy and biochemical methods, we demonstrated that upon incubation with RBCs EhRab35 is recruited to the site of phagocytic cups as well as to the nascent phagosomes that harbor Gal/GalNAc lectin and actin. Overexpression of a dominant negative mutant of EhRab35 reduced phagocytic cup formation and thereby reduced RBC internalization, suggesting a potential role of the Rab GTPase in the cup formation. Furthermore, we also performed a phagosomal maturation assay and observed that the activated form of EhRab35 significantly increased the rate of RBC degradation. Interestingly, this mutant also significantly enhanced the number of acidic compartments in the trophozoites. Taken together, our results suggest that EhRab35 is involved in the initial stage of phagocytosis as well as in the phagolysosomal biogenesis in E. histolytica and thus contributes to the pathogenicity of the parasite.

Leishmania major Promastigotes Evade LC3-Associated Phagocytosis through the Action of GP63.

The protozoan Leishmania parasitizes macrophages and evades the microbicidal consequences of phagocytosis through the inhibition of phagolysosome biogenesis. In this study, we investigated the impact of this parasite on LC3-associated phagocytosis, a non-canonical autophagic process that enhances phagosome maturation and functions. We show that whereas internalization of L. major promastigotes by macrophages promoted LC3 lipidation, recruitment of LC3 to phagosomes was inhibited through the action of the parasite surface metalloprotease GP63. Reactive oxygen species generated by the NOX2 NADPH oxidase are necessary for LC3-associated phagocytosis. We found that L. major promastigotes prevented, in a GP63-dependent manner, the recruitment of NOX2 to phagosomes through a mechanism that does not involve NOX2 cleavage. Moreover, we found that the SNARE protein VAMP8, which regulates phagosomal assembly of the NADPH oxidase NOX2, was down-modulated by GP63. In the absence of VAMP8, recruitment of LC3 to phagosomes containing GP63-deficient parasites was inhibited, indicating that VAMP8 is involved in the phagosomal recruitment of LC3. These findings reveal a role for VAMP8 in LC3-associated phagocytosis and highlight a novel mechanism exploited by L. major promastigotes to interfere with the host antimicrobial machinery.