SRP RNA level as a determinant of leishmanial parasitism of macrophages

SRP RNA 水平作为利什曼原虫巨噬细胞寄生的决定因素

• 批准号: 7628080
• 负责人: GAUTAM CHAUDHURI
• 金额: $ 18.31万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2010-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7628080
• 关键词: 7SL RNA; Age; Animal Model; Antibodies; Biological; Biological Models; Bone Marrow; Bone Marrow Cells; Bone Marrow Stem Cell; Bone Marrow Stem Cell Transplantation; Bone Marrow Transplantation; Cathepsins; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Collaborations; Consultations; Data; Dendritic Cells; Development; Down-Regulation; Endoplasmic Reticulum; Environment; Enzymes; Evaluation; Event; Exposure to; Figs - dietary; Financial compensation; Gamma Rays; Genes; Genetic Transcription; Human; Hydrolase; Immune response; Immunocompromised Host; Immunofluorescence Microscopy; Immunomodulators; Inbred BALB C Mice; Infection; Leishmania; Leishmaniasis; Lesion; Literature; Location; Lysosomes; LytA enzyme; Measures; Mediating; Medicine; Membrane; Membrane Proteins; Microarray Analysis; Modeling; Molecular; Mus; Mutate; Natural Immunity; Parasites; Peritoneal Macrophages; Peritoneum; Phagolysosome; Protein Secretion; Proteins; Protozoa; RNA; RNA chemical synthesis; Refractory; Research; Resistance; Scientist; Signal Recognition Particle; Small Interfering RNA; Sorting - Cell Movement; Staging; Stem Cell Development; Subfamily lentivirinae; Surface; Testing; Time; Tissues; Transcript; Translations; Transplantation; Transport Process; U937 Cells; Universities; Vesicular Protein Transport; Western Blotting; Work; base; chemokine; college; combat; cytokine; design; extracellular; foot; genetic manipulation; killings; knock-down; macrophage; microbicide; mouse model; mutant; overexpression; parasite invasion; parasitism; pathogen; prevent; protein distribution; public health relevance; receptor; scavenger receptor; signal recognition particle receptor

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.

描述（由申请方提供）：利什曼原虫是一组寄生原生动物，感染人类巨噬细胞并在这些细胞吞噬溶酶体的恶劣环境中茁壮成长。拟议研究的长期目标是确定在利什曼原虫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。利什曼原虫长期以来一直被认为可以“修复”巨噬细胞的分子环境，以便在其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。暴露于利什曼原虫后在巨噬细胞中下调的基因转录物包括7SL RNA，信号识别颗粒（SRP）的RNA组分。由于巨噬细胞的杀微生物功能深刻地依赖于囊泡蛋白转运过程，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J774 G8或U937细胞中过表达7SL RNA赋予对利什曼原虫感染的抗性。这些结果证明了下调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，假设是利什曼病诱导的巨噬细胞中7SL RNA水平的下调部分地有利于小鼠模型中利什曼病的发展。因此，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具有抗性。（1）通过骨髓干细胞的遗传操作和移植，建立了巨噬细胞中7SL RNA过表达的BALB/c小鼠衍生物。骨髓来源的巨噬细胞中7SL RNA的过表达将使用已经开发的慢病毒构建体进行，所述慢病毒构建体将允许表达在巨噬细胞谱系的细胞内发生。基因操作的骨髓干细胞将被移植到受辐射的BALB/c小鼠，这是利什曼原虫的易感宿主。(2)评价利什曼原虫前鞭毛体在小鼠巨噬细胞中过度表达7 SL RNA而产生足垫病变的能力。(3)评估从移植小鼠腹膜分离的巨噬细胞暴露于利什曼原虫对吞噬溶酶体蛋白、表面膜受体水平和从这些细胞分泌的蛋白水平的影响。我们将测量溶酶体中的组织蛋白酶水平、清道夫受体和从巨噬细胞暴露不同时间段（0-12 h）后的移植小鼠中分离的腹腔巨噬细胞的细胞表面上的CSF-1 R。这些评价将通过免疫荧光显微镜和Western印迹分析进行。将通过细胞因子抗体微阵列分析评价暴露或未暴露利什曼原虫的巨噬细胞分泌的细胞因子和趋化因子水平。公共卫生关系：这项研究将揭示寄生原虫利什曼原虫在巨噬细胞中建立感染的独特修复机制。在其巨噬细胞中过度表达7SL RNA的小鼠模型的开发可用于测试巨噬细胞囊泡蛋白转运是否也是巨噬细胞被其他病原体寄生的关键。了解宿主-寄生虫相互作用机制之间的相互作用巨噬细胞和利什曼原虫的分子细节将有助于我们制定对抗这种致命的人类病原体的战斗措施。