Laboratory of Host-Parasite Interaction and Epidemiology, Gonçalo Moniz Institute.
Laboratory of Host-Parasite Interaction and Epidemiology, Gonçalo Moniz Institute; National Institute of Science and Technology of Tropical Diseases - National Council for Scientific Research and Development (CNPq);
J Vis Exp. 2021 Jul 29(173). doi: 10.3791/62459.
Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3). Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.
吞噬作用是一个协调的过程，涉及多个步骤：识别、结合和内化。专业的吞噬细胞通过吞噬作用摄取利什曼原虫寄生虫，包括通过多种宿主细胞受体识别寄生虫表面的配体。利什曼原虫与巨噬细胞膜的结合通过补体受体 1（CR1）和补体受体 3（CR3）和模式识别受体发生。脂磷壁酸（LPG）和 63 kDa 糖蛋白（gp63）是参与巨噬细胞-利什曼原虫相互作用的主要配体。在宿主细胞受体对寄生虫配体的初始识别之后，寄生虫被内化，在吞噬小泡内存活和繁殖。利什曼原虫诱导的空泡的成熟过程涉及从细胞内囊泡中获取分子，包括单体 G 蛋白 Rab5 和 Rab7、溶酶体相关膜蛋白 1（LAMP-1）、溶酶体相关膜蛋白 2（LAMP-2）和微管相关蛋白 1A/1B-轻链 3（LC3）。在这里，我们描述了使用共聚焦显微镜评估利什曼原虫与宿主细胞相互作用过程中早期事件的方法，包括（i）结合、（ii）内化和（iii）吞噬体成熟。通过增加对这些感染结果决定因素的了解，我们希望提高对利什曼原虫感染发病机制的理解，并支持最终寻找新的化学治疗靶点。
