MOLECULAR BASIS OF PKNB ESSENTIALITY IN MYCOBACTERIA

分枝杆菌是一类用途广泛的微生物，包括医学上重要的病原菌和环境细菌。分枝杆菌已经发展出不同的机制，使它们能够在恶劣的条件下长期生存，并适应广泛的环境生态位。结核分枝杆菌(Mtb)是一种生长缓慢的细菌，具有复杂的富含脂质的细胞壁，使用多种信号机制来协调细胞成分的分裂和生物合成，是结核病的病原体。11个丝氨酸/苏氨酸蛋白激酶对结核分枝杆菌细胞过程的调节尤为重要。丝氨酸/苏氨酸蛋白激酶B(PounB)对分枝杆菌的生长是必不可少的，尽管其原因尚不清楚。以前试图耗尽或过度生产PKnB会导致分枝杆菌迅速死亡。我们最近发现了支持不产生PuncB的分枝杆菌生长的特殊条件，并比较了产生和缺失PuncB的分枝杆菌中的磷酸化蛋白。这一新的方法有助于鉴定被PKnB磷酸化的蛋白质，这是建立PKnB重要性的分子机制的第一步。我们的发现和以前发表的结果表明，PKnB磷酸化了参与细菌细胞壁的主要成分肽聚糖的生物合成和重塑的酶，然而这些蛋白的确切磷酸化作用仍然不清楚。在这个项目中，我们建议通过测量磷酸化和非磷酸化的酶的活性，它们在细胞中的定位和输出到细胞表面，以及磷酸化在酶与其他蛋白质相互作用中的作用来研究PKnB介导的肽聚糖生物合成的调节机制。我们将研究两种遗传相关的生物，Mtb和耻垢分枝杆菌，并产生缺失PnuB底物或表达其不能被PnuB磷酸化的改变形式的突变体。将评估这些突变体在各种介质中的生长情况。我们还将使用我们建立的方法来寻找与PnuB磷酸化蛋白相互作用的伙伴，并阐明分枝杆菌中肽聚糖生物合成的机制细节以及PnuB对其调控。在单独的实验中，我们将研究PnuB磷酸化如何有助于休眠分枝杆菌的形成和它们的复苏。该项目的预期结果将提高我们对分枝杆菌基本细胞过程的理解，并将刺激针对不同生理状态下的结核分枝杆菌的新方法的发展。我们的结果也将有助于破译原核生物中丝氨酸/苏氨酸蛋白激酶的功能。

Mycobacteria are a group of versatile microorganisms which include medically important pathogens and environmental bacteria. Mycobacteria have developed distinct mechanisms enabling their prolonged survival in hostile conditions and adaptation to a wide range of environmental niches. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a slow-growing bacterium with a complex lipid-rich cell wall that uses multiple signaling mechanisms to coordinate division with the biosynthesis of cellular components. Eleven serine/threonine protein kinases are particularly important for regulation of cellular processes in Mtb. The serine/threonine protein kinase B (PknB) is essential for mycobacterial growth, although the reasons for this are unknown. Previous attempts to deplete or over-produce PknB resulted in rapid death of mycobacteria. We have recently discovered special conditions, which support growth of mycobacteria that do not produce PknB, and compared phosphorylated proteins in mycobacteria producing and missing PknB. This novel approach has aided the identification of proteins that are phosphorylated by PknB, which is the first step in establishing the molecular mechanisms of PknB essentiality. Our findings and previously published results suggest that PknB phosphorylates enzymes involved in biosynthesis and remodelling of peptidoglycan, the major component of bacterial cell wall, however the precise role of phosphorylation of these proteins remains elusive. Within this project we propose to investigate the mechanisms of PknB-mediated regulation of peptidoglycan biosynthesis by measuring the activities of phosphorylated and non-phosphorylated enzymes, their localization in cells and export onto the cell surface, and the role of phosphorylation in the interaction of enzymes with other proteins. We will work on two genetically related organisms, Mtb and Mycobacterium smegmatis, and generate mutants missing the PknB substrates or expressing their altered forms which cannot be phosphorylated by PknB. The growth of these mutants in various media will be assessed. We will also use methods established by us to identify partners interacting with PknB-phosphorylated proteins and to elucidate the mechanistic details of peptidoglycan biosynthesis in mycobacteria and its regulation by PknB. In separate experiments we will study how PknB phosphorylation contributes to the formation of dormant mycobacteria and their resuscitation. The expected outcomes of this project will improve our understanding of fundamental cellular processes in mycobacteria and will stimulate the development of novel approaches to target Mtb in different physiological states. Our results will also contribute to deciphering the function of serine/threonine protein kinases in prokaryotes.