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Characterization of A Novel Regulatory Protein in M. tuberculosis

结核分枝杆菌中新型调节蛋白的表征

基本信息

批准号：
8220779
负责人：
ADEL M TALAAT
金额：
$ 18.17万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-02-15 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8220779
关键词：
Animals Antibodies Attenuated Bacillus (bacterium)Binding Bioinformatics Biological Assay Bone Marrow Chronic Complement Disease Exposure to Foundations Gene Expression Profile Genes Health Heat-Shock Response Hybrids Hypoxia Inbred BALB C Mice Infection Investigation Microarray Analysis Mission Modeling Molecular Molecular Chaperones Monitor Mus Mycobacterium Infections Mycobacterium tuberculosis National Institute of Allergy and Infectious Disease Nature Parents Pathogenesis Phagosomes Play Protein Family Proteins RNA Sequences Regulon Relative (related person)Role Sequence Analysis Staging Stress System Technology Transcript Tuberculosis United States National Institutes of Health Virulence Yeasts base genetic regulatory protein improved in vitro Model in vivo macrophage member mutant mycobacterial novel pathogen protein folding public health relevance stressor

项目摘要

DESCRIPTION (provided by applicant): Tuberculosis represents a major health burden that requires more investigation to better understand the factors involved in the entry and establishment of chronic stage of the disease. The molecular pathogenesis of chronic tuberculosis (approximately 95% of infections), where mycobacterial bacilli stay in a phenotypically dormant state despite being metabolically active, is far from being understood (3). Previously, we utilized a technology developed by our group (In Vivo Microarrays Analysis, IVMA) to identify Mycobacterium tuberculosis (M. tb) genes responsible for entering into the chronic stage of tuberculosis. The IVMA analysis of chronic tuberculosis suggested a prominent role for rv0990c in establishing the chronic tuberculosis. Further microarrays analysis of an isogenic mutant of the rv0990c strain (?rv0990c) suggested that Rv0990c could regulate the expression of a large number of genes. Bioinformatics analysis of the rv0990c sequence indicated it encodes a member of a large family of proteins responsive to heat shock. Gene specific transcriptional profiling further confirmed that transcripts of rv0990c were induced following exposure to heat shock and long-term hypoxia. Following animal infections, the ?rv0990c strain was attenuated in BALB/c mice compared to its parent (H37Rv) or complemented (?rv0990c::rv0990c) strains. We hypothesize that the rv0990c gene product participates in regulating a large number of proteins involved in heat shock responses necessary for mycobacterial survival during infection. The main focus of this project is to explore the role and potential function(s) of rv0990c in the pathogenesis of M. tb. In the first aim, we will use RNA Sequence (RNASeq) profiling to identify stress responsive genes that are impacted by the rv0990c gene using 2 in vitro models for heat shock and multiple stressors. In the second aim, we will use antibody pull-down assays and yeast 2-hybrid like system to identify binding partners for Rv0990c during heat shock responses. Finally, we will monitor the survival and phagosome maturation of the rv0990c mutant in murine bone marrow-derived macrophages. We believe that the approaches exploited in this proposal will examine the role of a novel regulatory protein in M. tb pathogenesis. The proposed studies will further improve our understanding of the nature of the host-pathogen interactions and will provide the necessary foundation for more detailed and mechanistic studies on the role of rv0990c in M. tb virulence. PUBLIC HEALTH RELEVANCE: This project focuses on better understanding of the molecular basis of tuberculosis, a worldwide health problem to mankind. This project is highly relative to the mission of the NIH/NIAID.

描述(申请人提供)：结核病是一种主要的健康负担，需要进行更多的调查，以更好地了解疾病进入和确立慢性病阶段所涉及的因素。慢性结核病(约95%的感染)的分子发病机制尚不清楚，尽管分枝杆菌在代谢方面处于活跃状态，但仍处于表型休眠状态。此前，我们利用我们团队开发的一项技术(在体内微阵列分析中，IVMA)来识别导致结核病进入慢性阶段的结核分枝杆菌(M.TB)基因。对慢性结核病的IVMA分析表明，rv0990c在慢性结核病的诊断中起着重要作用。对rv0990c株的一个同基因突变株(Rv0990c)的进一步芯片分析表明，rv0990c可以调节大量基因的表达。对rv0990c序列的生物信息学分析表明，它编码热休克反应蛋白大家族的成员。基因特异性转录谱进一步证实，rv0990c的转录本是在热休克和长期低氧条件下诱导的。动物感染后，在BALB/c小鼠中，rv0990c株与其亲本(H37Rv)相比减弱或补充(？rv0990c：：rv0990c)株。我们假设rv0990c基因产物参与调节大量参与热休克反应的蛋白质，这些蛋白质是分枝杆菌在感染期间生存所必需的。本项目的主要目的是探讨rv0990c在结核分枝杆菌发病机制中的作用和潜在功能(S)。在第一个目标中，我们将使用RNA序列(RNAseq)分析来识别受rv0990c基因影响的应激反应基因，使用两个热休克和多应激源的体外模型。在第二个目标中，我们将使用抗体下拉试验和酵母双杂交系统来确定Rv0990c在热休克反应中的结合伙伴。最后，我们将监测rv0990c突变体在小鼠骨髓来源的巨噬细胞中的存活和吞噬小体成熟情况。我们相信，这项提案中采用的方法将检验一种新的调节蛋白在结核分枝杆菌发病机制中的作用。这些研究将进一步提高我们对宿主-病原体相互作用性质的理解，并将为更详细和更详细地研究rv0990c在结核分枝杆菌毒力中的作用提供必要的基础。公共卫生相关性：该项目的重点是更好地了解结核病的分子基础，结核病是人类面临的一个世界性健康问题。该项目与NIH/NIAID的任务高度相关。