SRP RNA level as a determinant of leishmanial parasitism of macrophages

描述(申请人提供)：利什曼原虫是一组寄生原虫，感染人类巨噬细胞，并在这些细胞的吞噬溶酶体的恶劣环境中茁壮成长。这项拟议研究的长期目标是确定在利什曼病与巨噬细胞相互作用的早期阶段必须发生的分子事件，从而建立成功的寄生。众所周知，利什曼原虫可以“更新”巨噬细胞的分子环境，以便在巨噬细胞的吞噬酶体内建立感染。利什曼原虫在感染的早期阶段专门操纵宿主巨噬细胞基因的表达。暴露在利什曼原虫下的巨噬细胞下调的基因转录本包括7SL RNA，信号识别颗粒(SRP)的RNA成分。由于巨噬细胞的杀菌功能在很大程度上依赖于囊泡蛋白的运输过程，7SL RNA的下调可能在利什曼原虫感染的建立中具有重要意义。更重要的是，在J774G8或U937细胞中过表达7SL RNA可增强对利什曼原虫感染的抵抗力。这些结果表明，下调7SL RNA合成在利什曼原虫感染的建立中具有生物学意义。基于这些发现，假设利什曼原虫诱导的巨噬细胞中7SL RNA水平的下调在一定程度上有利于利什曼病在小鼠模型中的发展。因此，巨噬细胞中7SL RNA的外源补偿将使这些小鼠对利什曼原虫感染产生抵抗力。具体目的如下：(1)通过骨髓干细胞的遗传操作和移植，建立巨噬细胞高表达7SL RNA的BALB/c小鼠。7SL RNA在骨髓来源的巨噬细胞中的过度表达将使用已经开发的慢病毒结构来完成，这种结构将允许表达发生在巨噬细胞系的细胞内。基因操作的骨髓干细胞将被移植到受辐射的BALB/c小鼠体内，这些小鼠是利什曼原虫的易感宿主。(2)评价利什曼原虫前鞭毛体在其巨噬细胞中过度表达7SL RNA，在小鼠体内产生足垫损伤的能力。(3)评价利什曼原虫暴露对移植小鼠腹膜巨噬细胞吞噬酶蛋白、表膜受体和分泌蛋白水平的影响。我们将从移植小鼠分离的巨噬细胞暴露不同时间(0-12h)后，检测溶酶体中组织蛋白酶和细胞表面清道夫受体和CSF-1R的水平。这些评估将通过免疫荧光显微镜和Western blotting分析进行。巨噬细胞分泌的细胞因子和趋化因子水平将通过细胞因子抗体微阵列分析进行评估。公共卫生相关性：拟议的研究将揭示寄生原生动物利什曼原虫用来建立巨噬细胞感染的独特更新机制。建立小鼠巨噬细胞7SL RNA高表达模型，可用于检测巨噬细胞泡蛋白转运是否也是其他病原体寄生巨噬细胞的关键。在分子细节上了解巨噬细胞和利什曼原虫之间相互作用的宿主-寄生虫相互作用机制将有助于我们制定对抗这种往往致命的人类病原体的措施。

DESCRIPTION (provided by applicant): Leishmania is a group of parasitic protozoa that infect human macrophages and thrive inside the hostile environment of the phagolysosomes of these cells. The long-term objective of the proposed research is to identify the molecular events that must occur at the early stage of leishmanial interactions with the macrophages leading to the establishment of successful parasitism. Leishmania has long been known to 'renovate' the molecular environment of macrophages in order to establish infection inside their phagolysosomes. Leishmania specifically manipulates the expression of host macrophage genes in the early stages of their infection. Gene transcripts that are down regulated in macrophages upon exposure to Leishmania include 7SL RNA, the RNA component of the signal recognition particle (SRP). Since the microbicidal functions of macrophages profoundly count on vesicular protein transport processes, down regulation of 7SL RNA may be significant in the establishment of infection by Leishmania. More importantly, over expression of 7SL RNA in J774G8 or U937 cells confers resistance to Leishmania infection. These results demonstrate the biological significance of down-regulating 7SL RNA synthesis in the establishment of infection by Leishmania. Based on these findings, the hypothesis is that Leishmania-induced down regulation of the level of 7SL RNA in the macrophages favors in part the development of leishmaniasis in the mouse model. Exogenous compensation of 7SL RNA in their macrophages will thus make these mice resistant to Leishmania infection. Specific aims to test the hypothesis are the following: (1) Development of BALB/c mice derivatives with over expression of 7SL RNA in their macrophages by genetic manipulation of the bone marrow stem cells and transplantation. Over expression of 7SL RNA in the bone marrow derived macrophages will be done using already developed lentiviral constructs that will allow the expression to occur inside the cells of macrophage lineage. Genetically manipulated bone marrow stem cells will be transplanted into irradiated BALB/c mice, which are susceptible hosts for Leishmania. (2) Evaluation of the ability of Leishmania promastigotes to produce footpad lesions in mice over expressing 7SL RNA in their macrophages. (3) Evaluation of the effects of Leishmania exposure of macrophages isolated from the peritoneum of the transplanted mice on the levels of phagolysosomal proteins, surface membrane receptors and the levels of proteins secreted from these cells. We will measure the levels of cathepsins in lysosomes, and scavenger receptor, and CSF-1R on the cell surfaces of peritoneal macrophages isolated from transplanted mice after exposure of the macrophages for various time periods (0-12 h). These evaluations will be done by immunofluorescence microscopy and Western blotting analysis. Levels of cytokines and chemokines secreted from the macrophages with or without Leishmania exposure will be evaluated by cytokine antibody microarray analysis. PUBLIC HEALTH RELEVANCE: The proposed study will reveal a unique renovating mechanism employed by the parasitic protozoan Leishmania to establish infection in the macrophages. Development of the mouse model with over expression of 7SL RNA in its macrophages may be used to test whether macrophage vesicular protein transport is also critical for the parasitism of macrophages by other pathogens. Understanding host-parasite interaction mechanisms interplayed between macrophages and Leishmania in molecular details will help us in developing combat measures against this often deadly human pathogen.