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

• 批准号: 8114402
• 负责人: ADEL M TALAAT
• 金额: $ 21.89万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-15 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8114402
• 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; stressor

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（由申请人提供）：结核病代表了一个重大的健康负担，需要进行更多的调查，以更好地了解该疾病的慢性阶段所涉及的因素。慢性结核病的分子发病机理（大约95％的感染），其中分枝杆菌杆菌尽管具有代谢性活性，但仍未被了解（3）。以前，我们利用了我们的小组开发的技术（体内微阵列分析，IVMA）来鉴定结核分枝杆菌（M. TB）基因，负责进入结核病的慢性阶段。慢性结核病的IVMA分析表明，RV0990C在建立慢性结核病中起着重要作用。对RV0990C菌株（？rv0990c）的同基因突变体的进一步微阵列分析表明，RV0990C可以调节大量基因的表达。 RV0990C序列的生物信息学分析表明，它编码了响应热休克的大型蛋白质家族的成员。基因特异性转录分析进一步证实，暴露于热休克和长期缺氧后，诱导了RV0990C的转录本。在动物感染之后，与其母体（H37RV）或补充（？RV0990C :: RV0990C）菌株相比，BALB/C小鼠中的？RV0990C菌株被减弱。我们假设RV0990C基因产物参与调节感染过程中分生不清生存所需的大量蛋白质参与热休克反应。该项目的主要重点是探索RV0990C在M. TB发病机理中的作用和潜在功能。在第一个目标中，我们将使用RNA序列（RNASEQ）分析来识别使用2种体外模型用于热休克和多种应激源的压力响应基因。在第二个目标中，我们将使用抗体下拉测定法和酵母2杂交系统类似系统来识别热冲击反应期间RV0990C的结合伴侣。最后，我们将监测鼠骨髓来源的巨噬细胞中RV0990C突变体的生存和吞噬体成熟。我们认为，该提案中利用的方法将检查新的调节蛋白在M. TB发病机理中的作用。拟议的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RV0990C在M. TB毒力中的作用方面提供更详细和机械的研究提供必要的基础。 公共卫生相关性：该项目着重于更好地理解结核病的分子基础，这是全球健康问题。 该项目与NIH/NIAID的任务相对高。