Molecular determinants of intracellular survival and replication in Leishmania

描述(申请人提供)：本申请的长期目标是揭示原生动物寄生虫利什曼原虫在巨噬细胞吞噬酶体的恶劣环境中生存和复制所使用的策略。寄生虫对这一隔室内的铁的接触被怀疑起到了重要作用，但关于利什曼原虫通过细胞内获取铁的具体分子机制知之甚少。通过数据库检索，我们最近发现了两个完全相同的利什曼原虫串联基因(LIT1)，与拟南芥中一个功能齐全的质膜铁转运蛋白IRT1有很强的相似性。我们的初步研究表明，亚马逊利什曼原虫LIT1主要由细胞内的无鞭毛体表达，这种转运蛋白在寄生虫表面的表达受铁供应的调节。LIT1缺失突变体不能在巨噬细胞内生长，对小鼠是无毒的，这强烈表明通过这种转运蛋白获取铁是建立利什曼原虫细胞内感染的关键限制步骤。研究宿主细胞的反应，我们还发现感染亚马孙乳杆菌上调了Lyst的表达，Lyst是一种调节溶酶体大小的巨噬细胞胞质蛋白。寄生性空泡的扩大有利于利什曼原虫的细胞内生长，这表明Lyst上调作为宿主细胞固有的机制来限制感染。我们建议对这些新的发现进行描述和扩展，具体目的如下：1)确定LIT1是否作为铁离子转运体发挥作用，并确定其在亚马孙牛体内表达的调控条件；2)评估LIT1介导的铁转运体在利什曼原虫细胞内生存和生长中的需求；3)阐明寄生虫空泡扩张在成纤维细胞和巨噬细胞中无鞭毛体细胞内生存和复制中的作用。这些研究将提供对利什曼原虫用来克服宿主细胞防御的适应机制的详细了解，包括寄生虫在巨噬细胞吞噬酶体的敌对环境中获得铁的过程。从这个项目中获得的信息将填补我们对利什曼原虫感染的细胞生物学的了解的一个很大的空白，并将促进开发这种严重的人类疾病的新的治疗方法。

DESCRIPTION (provided by applicant): The long-term goal of this application is to uncover strategies used by the protozoan parasite Leishmania to survive and replicate within the harsh environment of macrophage phagolysosomes. Parasite access to iron within this compartment is suspected to play an important role, but little is known about the specific molecular mechanisms used by Leishmania to acquire iron intracellularly. Through database searches, we recently identified two identical Leishmania genes in tandem (LIT1) with strong similarity to IRT1, a well- characterized plasma membrane iron transporter from Arabidopsis thaliana. Our initial studies indicate that Leishmania amazonensis LIT1 is expressed predominantly by intracellular amastigotes, and that expression of this transporter on the parasite's surface is regulated by iron availability. LIT1 null mutants do not grow intracellularly in macrophages and are avirulent for mice, strongly suggesting that iron acquisition through this transporter is a critical limiting step for the establishment of intracellular infections by Leishmania. Investigating host cell responses, we also found that infection with L. amazonensis upregulates the expression of LYST, a macrophage cytosolic protein that regulates lysosomal size. Parasitophorous vacuole enlargement favors Leishmania intracellular growth, suggesting that LYST upregulation functions as a host cell innate mechanism to limit infection. We propose to characterize and extend these novel findings, with the following specific aims: 1) Determine if LIT1 functions as a ferrous iron transporter, and define the conditions regulating its surface expression in L. amazonensis; 2) Assess the requirement for LIT1-mediated iron transport in the intracellular survival and growth of Leishmania amastigotes; 3) Elucidate the role of parasitophorous vacuole expansion in the intracellular survival and replication of amastigotes in fibroblasts and macrophages. These studies will provide a detailed understanding of adaptive mechanisms utilized by Leishmania to overcome host cell defenses, including the process by which the parasites acquire iron in the hostile environment of macrophage phagolysosomes. The information derived from this project will fill a large gap in our understanding of the cell biology of Leishmania infections, and will facilitate the development of novel therapeutic approaches for this serious human disease.