Molecular determinants of intracellular survival and replication in Leishmania

描述（由申请人提供）：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溶酶体的恶劣环境中生存和复制的策略。寄生虫获得铁在这个车厢内被怀疑发挥重要作用，但鲜为人知的是利什曼原虫细胞内获得铁的具体分子机制。通过数据库搜索，我们最近确定了两个相同的利什曼原虫基因串联（LIT 1）与IRT 1，一个充分表征的质膜铁转运蛋白拟南芥的高度相似性。我们的初步研究表明，亚马逊利什曼原虫LIT 1主要由细胞内无鞭毛体表达，这种转运蛋白在寄生虫表面的表达受铁的可用性调节。LIT 1无效突变体不生长在巨噬细胞内，对小鼠无毒，强烈表明通过这种转运蛋白获得铁是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L。amazonensis上调LYST的表达，LYST是一种调节溶酶体大小的巨噬细胞胞质蛋白。寄生虫空泡扩大有利于利什曼原虫细胞内生长，表明LYST上调作为宿主细胞先天机制发挥作用以限制感染。我们建议表征和扩展这些新的发现，具有以下具体目标：1）确定LIT 1是否作为亚铁转运蛋白，并定义其在L.亚马逊河流域; 2）评估LIT 1介导的铁转运在利什曼原虫无鞭毛体细胞内存活和生长中的需求; 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存活和复制中的作用。这些研究将详细了解利什曼原虫用来克服宿主细胞防御的适应机制，包括寄生虫在巨噬细胞吞噬溶酶体的恶劣环境中获取铁的过程。来自该项目的信息将填补我们对利什曼原虫感染的细胞生物学理解的巨大空白，并将促进这种严重人类疾病的新治疗方法的开发。

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.