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。人们怀疑寄生虫在这个区室内获取铁的途径发挥了重要作用，但对利什曼原虫在细胞内获取铁的具体分子机制知之甚少。通过数据库检索，我们最近发现了两个相同的利什曼原虫串联基因（LIT1），它们与IRT1（一种来自拟南芥的质膜铁转运蛋白）具有很强的相似性。我们的初步研究表明，亚马逊利什曼原虫LIT1主要由细胞内无纺丝体表达，并且该转运体在寄生虫表面的表达受铁有效性的调节。LIT1零突变体不能在巨噬细胞内生长，并且对小鼠无毒，这强烈表明通过这种转运体获取铁是利什曼原虫细胞内感染建立的关键限制步骤。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。寄生物液泡增大有利于利什曼原虫在细胞内生长，提示LYST上调是宿主细胞限制感染的先天机制。我们拟对这些新发现进行特征化和扩展，具体目的如下：1)确定LIT1是否作为亚铁转运体，并确定其在亚马逊树中表面表达的调节条件；2)评估lit1介导的铁转运在利什曼原虫细胞内存活和生长中的需求；3)阐明寄生物液泡扩张在成纤维细胞和巨噬细胞中无尾线虫细胞内存活和复制中的作用。这些研究将提供利什曼原虫克服宿主细胞防御的适应性机制的详细理解，包括寄生虫在巨噬细胞吞噬溶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发展。

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.