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

Abstract

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.

Exploitation of the Host Cell Membrane Fusion Machinery by Leishmania Is Part of the Infection Process

“…Several roles have been attributed to LAP, including increased phagosomal microbicidal activity and enhanced antigen presentation on MHC II molecules [17]. By cleaving VAMP8 and preventing phagosomal recruitment of NOX2, GP63 allows Leishmania major promastigotes to evade LAP [18], possibly contributing to the impairment of phagosome maturation and further inhibiting antigen presentation to T cells. Consistent with the role of Syt XI as a negative regulator of cytokine secretion, cleavage of this endosomal protein by GP63 from L .…”

The WD and linker domains of ATG16L1 required for non-canonical autophagy limit lethal respiratory infection by influenza A virus at epithelial surfaces

“…In a reciprocal experiment the linker and WD domains of ATG16L1 were deleted specifically from myeloid cells. These mice, which lack non-canonical autophagy in The observation that virulence factors such as the GP63 metalloprotease of Leishmania major and melanin of Aspergillus fumigatus prevent recruitment of NOX2 to phagosomes to prevent LAP [13][14][15] further suggest that non-canonical autophagy in phagocytes should provide a defence against infection. One reason for the discrepancy may be that the 5 studies cited above have focused on in vitro experiments using microbes with a tropism for macrophages, rather than in vivo studies where pathogens encounter epithelial barriers.…”

“…At present a role for non-canonical autophagy in host defence has been implied from in vitro studies of LAP in phagocytes and microbes with a tropism for macrophages such as Listeria monocytogenes 11 , Legionella dumoffii 12 , Leishmania major and Aspergillus fumigatus [13][14][15] . Critically, these studies have rarely been extended to model organisms with intact epithelial barriers and complex immune systems.…”

Mannose receptor (MR) and Toll‐like receptor 2 (TLR2) influence phagosome maturation during Leishmania infection

“…An additional mechanism that may contribute to phagosome formation is the role of actin at the nascent phagosome. While certainly not the only mechanism, as prevention of several fusion partners including VAMP8 and the direct effects of LPG inserted into the membrane disrupting the lipid microdomains influence the process, actin likely plays an early role in phagosome maturation . Previously, actin loss has correlated with Leishmania ‐containing phagosome acquisition of LAMP1 .…”

“…There are several different glycoconjugates present on the surface of Leishmania with potential MR‐binding properties, including gp63, glycosylinositolphospholipids and LPG. Additionally, gp63 has been implicated in cleavage of phagosome fusion proteins and modulation of host cell lipid rafts that may influence parasite survival and phagosome maturation . LPG coats the outside of Leishmania metacyclic promastigotes engaging multiple receptors on the host cell surface.…”