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，以更好地了解疾病进入和建立慢性阶段所涉及的因素。慢性结核（约95%的感染）的分子发病机制还远未被理解(3)，其中分枝杆菌杆菌尽管代谢活跃，但仍处于表型休眠状态。之前，我们利用我们团队开发的一种技术（体内微阵列分析，IVMA）来鉴定结核分枝杆菌（M. tb）基因，这些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在慢性结核病的发病中起着突出的作用。rv0990c株等基因突变体的进一步微阵列分析rv0990c)表明rv0990c可以调控大量基因的表达。对rv0990c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大家族的成员。基因特异性转录谱进一步证实rv0990c的转录本在暴露于热休克和长期缺氧后被诱导。在动物感染之后，？与亲本（H37Rv）相比，rv0990c毒株在BALB/c小鼠中的毒性减弱。rv0990c:: rv0990c)压力。我们假设rv0990c基因产物参与调控大量参与热休克反应的蛋白，这些蛋白是分枝杆菌在感染期间存活所必需的。本项目主要探讨rv0990c在结核分枝杆菌发病机制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）分析来鉴定受rv0990c基因影响的应激反应基因，使用2种体外热休克模型和多种应激源。在第二个目标中，我们将使用抗体下拉试验和酵母2-杂交系统来识别Rv0990c在热休克反应中的结合伙伴。最后，我们将监测rv0990c突变体在小鼠骨髓源性巨噬细胞中的存活和吞噬体成熟。我们相信，在这个建议中所利用的方法将研究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认识，并将为更详细地研究rv0990c在结核分枝杆菌毒力中的作用提供必要的基础。

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.