ROLE OF RHOMBOID HOMOLOGUES IN MYCOBACTERIA SIGNALING

描述（由申请人提供）：结核分枝杆菌（MTB）是结核病（TB）的病原体，感染全球17亿人。每年有300万人死于结核病。这使得结核病成为全球与传染病相关的主要死亡原因。参与MTB许多表型调节的因素，如毒力、潜伏期、抗生素耐药性和在恶劣条件下生存的能力还不清楚。细菌细胞密度依赖性信号传导（群体感应）已被证明在控制细菌的毒力、孢子形成、细胞分裂、生物膜发育和耐药性中起主要作用。关于群体感应在MTB复合物表型调节中的作用的数据是有限的。然而，最近的两项研究已经证明了真核生物和原核生物中细胞外信号传导的保守机制。该信号系统由膜内信号前体组成，其被膜内蛋白酶切割以释放活性细胞外肽信号。在果蝇中，Rhomboid是膜内丝氨酸蛋白酶，其催化表皮生长因子受体（EGFr）配体的释放。EGFr信号是动物正常细胞分化、生长和发育所必需的。在果蝇中，EGFr信号已被证明对复眼和翅静脉的正确发育至关重要。在斯氏普罗威登斯菌（Providencia stuartii）中，细菌需要菱形样蛋白AarA来产生细胞外肽样信号。在结核分枝杆菌和分枝杆菌基因组中发现了两个菱形样序列。恶臭为了深入了解细胞间信号传导在结核分枝杆菌中的作用，将使用M.作为一个模特。具体目的包括：（1）研究分枝杆菌Rhomboid信号通路的组成成分。这将通过确定分枝杆菌中的菱形同源物是否可以取代斯氏毕赤酵母中的菱形同源物（AarA）来实现。(2)利用遗传学技术研究菱形样蛋白在分枝杆菌生理学中的作用。这将通过在M中构建缺失突变来解决。smegalciumRhomboid同源物，并确定这如何影响信号产生，形态，生存和分枝杆菌的其他表型。(3)鉴定分枝杆菌群体感应调节基因融合体。这将通过在M中构建随机lacZ报告转录基因融合体来实现。耻垢染色体在高细胞密度下其活性变化的融合将被识别和映射为受群体感应调节的融合。这项研究将提供信息的分枝杆菌的功能，调节细胞间的通讯。这将扩大我们对分枝杆菌生物学的理解，并可能提供新的候选疫苗和药物靶点。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB) infects 1.7 billion people worldwide. Three million people die of TB annually. This makes TB the leading cause of death associated with infectious diseases globally. Factors involved in the regulation of many phenotypes of MTB such as virulence, latency, antibiotic resistance and ability to survive under harsh conditions are not well understood. Bacterial cell density dependent signaling (quorum sensing) has been shown to play a major role in the control of virulence, sporulation, cell division, biofilm development and drug resistance in bacteria. Data on the role of quorum sensing in the regulation of MTB complex phenotypes are limited. Two recent studies, however, have demonstrated a conserved mechanism of extracellular signaling in eukaryotes and prokaryotes. This signaling system consists of an intramembrane signal precursor that is cleaved by an intramembrane protease to release an active extracellular peptide signal. In Drosophila, Rhomboid is the intramembrane serine protease that catalyses the release of epidermal growth factor receptor (EGFr) ligands. The EGFr signals are required for proper cell differentiation, growth and development in animals. In Drosophila, the EGFr signaling has been shown to be crucial for the correct development of the compound eye and wing veination. In Providencia stuartii, a bacterium, a Rhomboid-like protein, AarA, is required for production of an extracellular peptide-like signal. Two Rhomboid-like sequences have been demonstrated in the MTB genome and M. smegmatis. To gain insight into the role of cell-to-cell signaling in MTB, the role of the Rhomboid-like proteins in mycobacteria will be investigated using M. smegmatis as a model. The specific objectives include: (1) To investigate the components of the Rhomboid signaling pathway in mycobacteria. This will be accomplished by determining whether the Rhomboid homologues in mycobacteria can substitute the Rhomboid homologue (AarA) in P. stuartii. (2) To use genetic techniques to examine the role of the Rhomboid-like proteins in the physiology of mycobacteria. This will be addressed by construction of deletion mutation in M. smegmatis Rhomboid homologues and determining how this affects signal production, morphology, survival and other phenotypes of mycobacteria. (3) To identify quorum sensing regulated gene fusions in mycobacteria. This will be achieved through construction of random lacZ reporter transcriptional gene fusions in M. smegmatis chromosome. Fusions whose activity changes at high cell density will be identified and mapped as those regulated by quorum sensing. This study will provide information on mycobacterial functions that are regulated by cell-to-cell communication. This will expand our understanding of mycobacterial biology and may provide novel candidate vaccines and drug targets.