SRP RNA level as a determinant of leishmanial parasitism of macrophages

描述（由申请人提供）：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敌对环境中茁壮成长。拟议研究的长期目标是确定利什曼原虫与巨噬细胞相互作用早期必须发生的分子事件，从而建立成功的寄生。人们早就知道利什曼原虫能够“修复”巨噬细胞的分子环境，从而在巨噬细胞的吞噬溶酶体内建立感染。利什曼原虫在感染的早期阶段特异性地操纵宿主巨噬细胞基因的表达。巨噬细胞暴露于利什曼原虫后下调的基因转录物包括7SL RNA，这是信号识别颗粒（SRP）的RNA成分。由于巨噬细胞的杀微生物功能在很大程度上依赖于囊泡蛋白的转运过程，因此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，7SL RNA的过表达赋予了利什曼原虫感染的抗性。这些结果表明下调7SL RNA合成在利什曼原虫感染建立中的生物学意义。基于这些发现，假设利什曼病诱导巨噬细胞中7SL RNA水平的下调在一定程度上有利于利什曼病小鼠模型的发展。巨噬细胞中7SL RNA的外源性补偿将使这些小鼠对利什曼原虫感染产生抗性。验证这一假设的具体目的如下：(1)通过对骨髓干细胞的遗传操作和移植，在巨噬细胞中培养过表达7SL RNA的BALB/c小鼠衍生物。7SL RNA在骨髓源性巨噬细胞中的过表达将使用已经开发的慢病毒结构来完成，该慢病毒结构将允许在巨噬细胞谱系的细胞内表达。经过基因处理的骨髓干细胞将被移植到受辐照的BALB/c小鼠体内，这些小鼠是利什曼原虫的易感宿主。(2)巨噬细胞中过量表达7SL RNA的promatigotes利什曼原虫对小鼠足垫病变的能力评价。(3)评价移植小鼠腹膜分离巨噬细胞暴露利什曼原虫对吞噬溶酶体蛋白、表面膜受体及其分泌蛋白水平的影响。我们将测量移植小鼠腹膜巨噬细胞暴露于不同时间段（0-12小时）后，细胞表面溶酶体、清道夫受体和CSF-1R中组织蛋白酶的水平。这些评估将通过免疫荧光显微镜和免疫印迹分析进行。细胞因子抗体微阵列分析将评估有或没有利什曼原虫暴露的巨噬细胞分泌的细胞因子和趋化因子水平。公共卫生相关性：该研究将揭示寄生原生动物利什曼原虫在巨噬细胞中建立感染的独特修复机制。建立巨噬细胞中7SL RNA过表达的小鼠模型，可以用来检验巨噬细胞囊泡蛋白转运是否也是其他病原体寄生巨噬细胞的关键。从分子细节上了解巨噬细胞与利什曼原虫之间的宿主-寄生虫相互作用机制将有助于我们制定对抗这种通常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.