Control of bacterial peptidoglycan biosynthesis through protein phosphorylation and regulated proteolysis

肽聚糖是细菌细胞壁的主要成分。它围绕着细胞形成一个三维网络，承受内部的膨胀压力，对细胞形状的维持很重要。肽聚糖的生物合成、重组和降解必须与细胞生长和分裂过程中发生的大小和形状变化紧密协调，以维持细菌的完整性。我们发现了一种在单核增生李斯特菌中调控肽聚糖生物合成的新途径，该途径在其他革兰氏阳性菌中也是保守的。根据我们的研究结果，MurA是肽聚糖生物合成的第一步酶，是整个途径调控的关键控制点。我们已经证明单核增生乳杆菌MurA被ClpCP蛋白酶降解，并且这种ClpCP依赖性的MurA降解依赖于小细胞质蛋白ReoM的磷酸化状态。我们还发现ReoM磷酸化依赖于prka。PrkA是一种跨膜丝氨酸/苏氨酸蛋白激酶，具有胞外PASTA结构域，可感知肽聚糖重组和降解过程中积累的肽聚糖片段。通过这一途径，激活PrkA和磷酸化ReoM影响MurA的蛋白水解稳定性，最终控制肽聚糖的生物合成，使其适应当前的生长条件和修复需要。PrkA→ReoM→ClpCP→MurA信号级联的基本问题应该在这个项目中得到解答。单核增生L. ClpCP蛋白酶的底物应使用蛋白质组学方法进行鉴定。ReoM及其两个辅助因子ReoY和MurZ在多大程度上控制了ClpCP底物的降解，这个问题应该在随后的分析中得到回答。此外，ReoM， ReoY和MurZ作为潜在的ClpC适配器的可能功能应该在遗传和生化实验中进行测试。应该使用不同的筛选方法来确定对MurA蛋白水解降解重要的新决定因素。此外，应该确定导致体内ReoM磷酸化刺激的生长条件。最后，利用上位实验研究PrkA→ReoM→ClpCP→MurA信号通路在单核增生乳杆菌对头孢菌素的内在耐药中的作用。该项目将有助于更深入地了解革兰氏阳性菌中肽聚糖生物合成的调控及其对细胞壁损伤条件和抗生素的反应。该项目应有助于通过鉴定新的目标结构开发新的抗菌化合物。

Peptidoglycan is the main component of the bacterial cell wall. It surrounds the cell as a three-dimensional network that withstands the internal turgor pressure and is important for cell shape maintenance. Biosynthesis, reorganization and degradation of peptidoglycan must be tightly coordinated with the size and shape changes that occur during cell growth and division in order to maintain the integrity of the bacterium. We have discovered a novel pathway for regulation of peptidoglycan biosynthesis in the pathogenic bacterium Listeria monocytogenes and this pathway is also conserved in other Gram-positive bacteria. According to our results, MurA, the first committed step enzyme of peptidoglycan biosynthesis, acts as a key control point for the regulation of the entire pathway. We have demonstrated that L. monoctyogenes MurA is degraded by the ClpCP protease and that this ClpCP-dependent degradation of MurA depends on the phosphorylation state of the small cytosolic protein ReoM. We also have shown that ReoM phosphorylation is PrkA-dependent. PrkA is a transmembrane serine/threonine protein kinase with extracellular PASTA domains and senses peptidoglycan fragments that accumulate during reorganization and degradation of peptidoglycan. Using this route, activation of PrkA and phosphorylation of ReoM influence the proteolytic stability of MurA and finally control peptidoglycan biosynthesis to adjust it to the current growth conditions and repair needs.Fundamental questions regarding the PrkA→ReoM→ClpCP→MurA signaling cascade should be answered in this project. Substrates of the L. monocytogenes ClpCP protease should be identified using proteomic approaches. The question to what degree ReoM and its two auxiliary factors ReoY and MurZ control degradation of ClpCP substrates should then be answered in subsequent analyses. Furthermore, a possible function of ReoM, ReoY and MurZ as potential ClpC adaptors should be tested in genetic and biochemical experiments. Different screening approaches should be used to identify novel determinants that are important for the proteolytic degradation of MurA. Furthermore, growth conditions leading to stimulation of ReoM phosphorylation in vivo should be identified. Finally, epistasis experiments should be used to investigate the role of the PrkA→ReoM→ClpCP→MurA signaling pathway in the intrinsic resistance of L. monocytogenes against cephalosporins. This project should lead to a deeper understanding of the regulation of peptidoglycan biosynthesis in Gram positive bacteria and their response to cell wall damaging conditions and antibiotics. The project should contribute to the development of new antimicrobial compounds through the identification of novel target structures.