Characterization of M. tuberculosis kinases PknA & PknB

说明(申请人提供)：尽管努力广泛实施有效的治疗战略，但全球结核病负担仍然极高，许多国家耐多药结核病的发病率正在上升。深入了解结核分枝杆菌在感染期间适应宿主环境的机制，可能为治疗或预防结核病，包括耐多药结核病提供新的机会。结核分枝杆菌基因组序列的一个惊人发现是存在编码几种真核细胞样丝氨酸-苏氨酸激酶的基因。除了两个外，所有这些似乎都是受体类型的激酶，可能与细胞外环境相互作用，通过细胞内底物的可逆磷酸化传递信号，导致细菌生理的变化。其中的两个基因，pnuA和pnuB对结核分枝杆菌的生存是必不可少的，并且连接在一个操纵子中，该操纵子还包括参与细胞壁合成和细胞形状控制的基因的同源基因。我们假设，这些基因编码的蛋白质介导了调节分枝杆菌细胞分裂的信号通路。本研究的目的是研究结核分枝杆菌中PKNOA和PKNOB的功能和信号转导途径。为了实现这一目标，我们提出了四个具体的目标：1)完成pKnA和pKnB的最佳底物识别基序的鉴定；2)鉴定pKnA和pKnB的体内磷酸化靶标；3)研究pKnA和pKnB在分枝杆菌细胞生理学中的表达和功能；4)开始研究与pKnA和pKnB胞外结构域相互作用的分子。

DESCRIPTION (PROVIDED BY APPLICANT): Despite efforts to broadly implement effective treatment strategies, the global burden of tuberculosis remains extremely high, and the incidence of multidrug-resistant tuberculosis is increasing in many countries. Insight into mechanisms by which M. tuberculosis adapts to the environment of the host during infection may provide new opportunities for the treatment or prevention or tuberculosis, including multidrug-resistant tuberculosis. A striking finding of the M. tuberculosis genome sequence was the presence of genes encoding several eukaryotic-like serine-threonine kinases. All but two of these appear to be receptor type kinases that likely interact with the extracellular environment and transmit signals by reversible phosphorylation of intracellular substrates, resulting in changes in bacterial physiology. Two of these genes, pknA and pknB are essential for M. tuberculosis viability, and are linked in an operon that also includes orthologues of genes involved in cell wall synthesis and cell shape control. We hypothesize that the proteins encoded by these genes mediate signaling pathways that regulate cell division in mycobacteria. The goal of this research is to characterize the function and signaling pathways of PknA and PknB in M. tuberculosis. To achieve this goal, we propose four specific aims: 1) To complete the characterization of the optimal substrate recognition motif of PknA and PknB, 2) To identify the in vivo phosphorylation targets of PknA and PknB, 3) To investigate the expression and function of PknA and PknB in mycobacterial cell physiology, and 4) To begin to investigate the molecules that interact with the extracellular domains of PknA and PknB.