Molecular determinants of intracellular survival and replication in Leishmania

利什曼原虫细胞内存活和复制的分子决定因素

基本信息

批准号：
7304302
负责人：
Norma Windsor Andrews
金额：
$ 41.21万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-06-01 至 2011-05-31
项目状态：
已结题

项目摘要

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.

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。寄生虫在该隔室内获取铁被怀疑发挥着重要作用，但人们对利什曼原虫在细胞内获取铁的具体分子机制知之甚少。通过数据库搜索，我们最近鉴定了两个相同的串联利什曼原虫基因 (LIT1)，与拟南芥质膜铁转运蛋白 IRT1 高度相似。我们的初步研究表明，亚马逊利什曼原虫 LIT1 主要由细胞内无鞭毛体表达，并且该转运蛋白在寄生虫表面的表达受到铁可用性的调节。 LIT1无效突变体不能在巨噬细胞内生长，并且对小鼠无毒，这强烈表明通过该转运蛋白获取铁是利什曼原虫建立细胞内感染的关键限制步骤。在研究宿主细胞反应时，我们还发现亚马逊乳杆菌感染会上调 LYST 的表达，LYST 是一种调节溶酶体大小的巨噬细胞胞质蛋白。寄生泡增大有利于利什曼原虫细胞内生长，表明 LYST 上调是宿主细胞限制感染的先天机制。我们建议表征和扩展这些新发现，具体目标如下：1）确定 LIT1 是否作为亚铁转运蛋白发挥作用，并定义调节其在亚马逊 L. amazonensis 中表面表达的条件； 2) 评估利什曼原虫无鞭毛体细胞内存活和生长对 LIT1 介导的铁转运的需求； 3）阐明寄生液泡扩张在成纤维细胞和巨噬细胞中无鞭毛体的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性机制，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。该项目获得的信息将填补我们对利什曼原虫感染细胞生物学理解的巨大空白，并将促进针对这种严重人类疾病的新治疗方法的开发。