Characterization of CCR5-like Molecules in Leishmania

描述(申请人提供)：利什曼原虫感染可引起人类广泛的利什曼病。虽然众所周知，宿主和寄生虫衍生的因子对疾病结局有贡献，但我们对它们的分子相互作用的了解仍然相对有限。这项应用的长期目标是确定宿主对亚马孙乳杆菌(La)感染的易感性机制(S)。与La感染相关的皮肤利什曼病的一个特征是对这种寄生虫的细胞免疫严重不足。Th1细胞因子和炎性CC-趋化因子的产生不足与小鼠La感染的疾病进展有关。我们基于抗体和配体的研究表明，皮损和培养的La和L.miciana寄生虫可以表达CCR5样分子。这些发现导致了一种假设，即利什曼原虫表达一个或多个模仿并干扰宿主CCR5系统的分子，从而促进寄生虫归巢到其靶细胞和/或在原位调节趋化因子反应。这一假设将同时在两个具体目标上得到检验。目的1证实CCR5类分子在不同种/株利什曼原虫中的表达，并研究其表达在宿主-寄生虫相互作用中的生物学相关性。我们的努力将主要放在利用小鼠和人CCR5配体以及不与CCR5结合的配体进行功能研究(钙内流、趋化和配体结合/内化试验)。目的2是鉴定寄生虫来源的CCR5编码基因，并确定这些蛋白的性质。编码CCR5类分子的基因将用于与它们的哺乳动物或病毒同行进行序列比较。表达LaCCR5和针对利什曼CCR5样蛋白的抗体的稳定细胞系将被用于功能分析的重新评估。 这项新的研究具有重要意义，因为虽然有确凿的证据表明，包括DNA病毒、弓形虫和曼氏血吸虫在内的许多病原体对宿主细胞因子/趋化因子及其受体进行了分子模拟，但尚未在锥虫中发现这种模拟。试剂到位的事实，与PI在免疫学和分子生物学方面的经验相联系，确保了这一探索性赠款的可行性。如果被证明是正确的，这项研究将首次证明在利什曼原虫中存在功能性CCR5同源物(S)。这一应用将极大地扩展我们目前对利什曼原虫生物学的理解，并为进一步深入研究宿主与寄生虫的相互作用奠定基础。

DESCRIPTION (provided by applicant): Leishmania infection can cause a broad spectrum of leishmaniases in humans. Although it is well known that host- and parasite-derived factors contribute to disease outcome, our knowledge about their molecular interactions is still relatively limited. The long-term goal of this application is to define the mechanism(s) underlying the host susceptibility to L. amazonensis (La) infection. A hallmark of cutaneous leishmaniasis associated with La infection is the profound deficiency in cellular immunity to the parasite. An insufficient production of Th1 cytokines and inflammatory CC-chemokines is correlated with disease progression in murine La infection. Our antibody- and ligand-based studies have revealed that lesional and cultured La and L. mexicana parasites can express CCR5-like molecules. These findings have led to the hypothesis that Leishmania parasites express one or more molecules that mimic and interfere with the host CCR5 system, thus promoting homing of parasites to their target cells and/or modulating chemokine responses in situ. This hypothesis will be tested concurrently in two Specific Aims. Aim 1 is to confirm the expression of CCR5-like molecules in different species/strains of Leishmania and to examine the biological relevance of their expression in host-parasite interaction. Our efforts will mostly be placed on conducting functional studies (Ca++ influx, chemotaxis and ligand binding/internalization assays) using murine and human CCR5 ligands, as well as ligands that do not bind to CCR5. Aim 2 is to characterize the gene encoding parasite-derived CCR5 and define the nature of these proteins. The gene encoding CCR5-like molecules will be used for sequence comparison with their mammalian or viral counterparts. Stable cell lines expressing LaCCR5 and antibodies specific to leishmanial CCR5-like proteins will be generated for re-evaluation in functional assays. This proposed study is novel and highly significant because, while solid evidence exists for molecular mimicry of host cytokines/chemokines and their receptors by many pathogens, including DNA viruses, Toxoplasma gondii, and Schistosoma mansoni, such mimicry has not been identified for trypanosomatids. The fact that reagents are in place, linked with the PI's experience in immunology and molecular biology, ensures the feasibility of this exploratory grant. If proven correct, this study would, for the first time, demonstrate the presence of a functional CCR5 homologue(s) in Leishmania parasites. This application would greatly extend our current understanding of Leishmania biology and lay the foundation for additional in-depth investigations of host-parasite interactions.