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.原生动物利什曼原虫（Leishmania）可寄生在巨噬细胞内，并通过抑制吞噬溶酶体生成（phagolysosome biogenesis）来逃避吞噬作用带来的杀菌后果。本研究探讨了该寄生虫对LC3相关吞噬作用（LC3-associated phagocytosis）的影响——这是一类能够增强吞噬体成熟与功能的非经典自噬过程。研究结果显示，尽管巨噬细胞对硕大利什曼原虫（L. major）前鞭毛体的内化作用可促进LC3脂酰化（LC3 lipidation），但寄生虫表面金属蛋白酶GP63的介导作用会抑制LC3向吞噬体的募集。由NOX2型NADPH氧化酶（NOX2 NADPH oxidase）产生的活性氧（reactive oxygen species，ROS）是LC3相关吞噬作用顺利进行的必要条件。我们发现，硕大利什曼原虫前鞭毛体可通过不涉及NOX2裂解的机制，以GP63依赖的方式阻碍NOX2向吞噬体的募集。此外，我们还发现，调控NADPH氧化酶NOX2吞噬体组装的SNARE蛋白（SNARE protein）VAMP8可被GP63下调。在缺失VAMP8的情况下，携带GP63缺陷型寄生虫的吞噬体对LC3的募集会受到抑制，这表明VAMP8参与了LC3向吞噬体的募集过程。本研究结果揭示了VAMP8在LC3相关吞噬作用中的功能，并阐明了硕大利什曼原虫前鞭毛体用以干扰宿主抗菌机制的全新途径。