The Role of the Kharon Complex in Leishmania Virulence

 描述(申请人提供)：相关性：寄生原生动物，如利什曼原虫和锥虫，感染全世界数百万人，并导致毁灭性和致命性疾病。这一应用将阐明这些寄生虫成功入侵人类细胞并导致疾病的一个重要机制。总结。最近的发现表明，参与这些单细胞寄生虫运动的鞭毛在疾病发生阶段起着关键作用，利什曼原虫的无鞭毛体和血液形成非洲锥虫。具体地说，我们的实验室发现了一种新的蛋白质KHARON1，它参与将完整的膜蛋白靶向墨西哥利什曼原虫的鞭毛膜。重要的是，KHARON1对于致病墨西哥乳杆菌的无鞭毛体在人巨噬细胞的吞噬酶体囊泡内生存是必不可少的。虽然Δkharon1缺失突变体在培养基中以无菌无鞭毛体的形式复制，但一旦它们进入宿主巨噬细胞，它们就不会复制，而是在这些宿主细胞内死亡。这些结果表明KHARON1在巨噬细胞吞噬酶体内无鞭毛体的存活中起着特殊的作用。最近的实验也表明，Δkharon1零突变体在感染Balb/C小鼠后是无毒的。其他初步数据证实，KHARON1是被称为Kharon复合体的高分子量多蛋白质复合体的一部分。这项应用的总体目标是剖析Kharon复合体在致病无鞭毛体中的关键功能，从而阐明无鞭毛体鞭毛在寄生虫毒力中的作用。KHARON1蛋白定位于昆虫阶段前鞭毛体的鞭毛轴突基底部，通过高分辨荧光显微镜和电子显微镜将其定位于无鞭毛体。KHARON1介导膜蛋白运输到无鞭毛体鞭毛的能力将被证明，以证实该蛋白参与鞭毛 无鞭毛体和前鞭毛体中的膜靶向性。最近发表的实验表明，无鞭毛体鞭毛的尖端与巨噬细胞的吞噬体膜形成了“突触”，这表明这些突触在寄生虫/巨噬细胞的相互作用中可能是重要的。Δkharon1缺失突变体可能无法形成这种潜在的关键突触的可能性将通过电子显微镜进行测试。此外，还将研究Δkharon1突变体不能将蛋白质从鞭毛输送到巨噬细胞胞浆的可能性。这些实验可以为Δkharon1缺失突变体的无毒提供一个机制解释。最后，初步研究已经利用生物素邻近标记确定了Kharon复合体的几个候选成分。实验将解决这些候选亚基是否存在于细胞内无鞭毛体中与KHARON1相同的高分子量复合体中。总体而言，这一应用将阐明哈龙复合体在寄生虫毒力中的关键作用。

 DESCRIPTION (provided by applicant): Relevance: Parasitic protozoa such as Leishmania and Trypanosoma infect millions of people worldwide and cause devastating and fatal diseases. This application will elucidate an important mechanism whereby these parasites successfully invade human cells and cause disease. Summary. Recent discoveries have revealed that the whip-like flagellum that mediates motility of these single-cell parasites plays a critical role in he disease causing stages, the amastigote of Leishmania and the bloodstream form African trypanosomes. Specifically, our laboratory has discovered a novel protein, KHARON1, that is involved in targeting integral membrane proteins to the flagellar membrane in Leishmania mexicana. Importantly, KHARON1 is essential for disease-causing amastigotes of L. mexicana to survive inside the phagolysosomal vesicles of human macrophages. Although Δkharon1 null mutants replicate as axenic amastigotes in culture medium, once they enter host macrophages they do not replicate but rather die inside these host cells. These results suggest a role for KHARON1 specifically in survival of amastigotes inside the macrophage phagolysosome. Recent experiments also indicate that Δkharon1null mutants are avirulent following infection of Balb/C mice. Other preliminary data establish that KHARON1 is part of a high molecular weight multi-protein complex designated the KHARON Complex. The overall objective of this application is to dissect the critical function of the KHARON Complex in disease causing amastigotes and to thus illuminate the role of the amastigote flagellum in parasite virulence. The KHARON1 protein, which localizes to the base of the flagellar axoneme in insect stage promastigotes, will be localized in amastigotes by high-resolution fluorescence microscopy and electron microscopy. The ability of KHARON1 to mediate trafficking of a membrane protein to the amastigote flagellum will be demonstrated to confirm that this protein is involved in flagellar membrane targeting in amastigotes as well as promastigotes. Recently published experiments have demonstrated that the tips of the amastigote flagella form `synapses' with the phagolysomal membrane of the macrophage, suggesting that these synapses could be important in parasite/macrophage interactions. The possibility that Δkharon1 null mutants may fail to form such potentially critical synapses will be tested by electron microscopy. Additionally the possibility that Δkharon1 mutants fail to deliver a protein from the flagellum to the macrophage cytosol will also be examined. These experiments could provide a mechanistic explanation for the avirulence of Δkharon1 null mutants. Finally, preliminary studies have identified several candidate components of the KHARON Complex using biotin proximity labeling. Experiments will address whether these candidate subunits exist in the same high molecular weight complex as KHARON1 in intracellular amastigotes. Overall, this application will elucidate the critical role of the KHARON Complex in parasite virulence.