Characterization of A Novel Regulatory Protein in M. tuberculosis

描述（由申请人提供）：结核病是一个主要的健康负担，需要更多的调查，以更好地了解参与进入和建立慢性阶段的疾病的因素。慢性结核病（约95%的感染）的分子发病机制，其中分枝杆菌保持在表型休眠状态，尽管代谢活跃，远未被理解（3）。在此之前，我们利用我们小组开发的技术（体内微阵列分析，IVMA）来鉴定结核分枝杆菌（M。tb）负责进入结核病慢性阶段的基因。慢性结核病的IVMA分析表明rv 0990 c在建立慢性结核病中具有突出作用。rv 0990 c株的一个同基因突变株（？rv 0990 c）表明Rv 0990 c可以调控大量基因的表达。对rv 0990 c序列的生物信息学分析表明，它编码一个对热休克有反应的蛋白质大家族的成员。基因特异性转录谱分析进一步证实，rv 0990 c的转录本在热休克和长期缺氧后被诱导。动物感染后，？rv 0990 c株在BALB/c小鼠体内的毒力较亲本株（H37 Rv）或互补株（？）RV 0990 c：：RV 0990 c）菌株。我们推测rv 0990 c基因产物参与调节大量的蛋白质，这些蛋白质参与了在感染过程中分枝杆菌生存所必需的热休克反应。本研究的主要目的是探讨rv 0990 c在M. TB.在第一个目标中，我们将使用RNA序列（RNASeq）分析来鉴定受rv 0990 c基因影响的应激反应基因，使用2种热休克和多种应激源的体外模型。在第二个目标中，我们将使用抗体下拉分析和酵母双杂交样系统来鉴定Rv 0990 c在热休克反应期间的结合配偶体。最后，我们将监测小鼠骨髓源性巨噬细胞中rv 0990 c突变体的存活和吞噬体成熟。我们相信，在这项建议中利用的方法将检查一个新的调节蛋白在M。结核病发病机制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，并将为更详细地研究rv 0990 c在M.结核病毒力 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，这是一个全球性的人类健康问题。 该项目与NIH/NIAID的使命高度相关。

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.