Osmolarity-dependent control of cellular c-di-AMP levels and characterization of osmolyte transporters in Listeria monocytogenes

环二磷酸腺苷是一种信号核苷酸，由许多革兰氏阳性细菌产生，包括人类病原体单核细胞增生李斯特菌。C-di-AMP分别由特定的二腺苷酸环化酶和磷酸二酯酶合成和降解。对于单核增生乳杆菌和系统发育相关的细菌，已经证明c-二- amp是生长所必需的。在过去的几年里，已经确定了几个与c-di-AMP结合的靶标。到目前为止，已知的c-二- amp靶标都不是细菌生存所必需的，而细菌的生存依赖于信号核苷酸。最近，我们发现CdaR蛋白负调控L.单核细胞增生二腺苷酸环化酶CdaA的活性。观察到这两种蛋白质都位于细胞包膜，表明c-二- amp代谢与细胞壁稳态有关。事实上，据报道，对于许多细菌，细胞c-二- amp水平的改变会影响保护性细胞包膜的完整性。然而，c-二- amp在细胞壁代谢中的作用尚不清楚。在拟议的项目中，我们希望确定CdaR接收的信号以及CdaA产生的c-di-AMP如何影响细菌细胞包膜的完整性。通过应用遗传学和生物化学方法，我们旨在阐明CdaA及其调控因子CdaR的相互作用网络。此外，我们希望进行结构分析，以研究蛋白质复合物的分子细节。我们还想确定抑制CdaA的物质，因为必需的二腺苷酸环化酶是新型抗生素的极好靶点。

Cyclic di-AMP is a signalling nucleotide that is produced by many Gram-positive bacteria, including the human pathogen Listeria monocytogenes. C-di-AMP is synthesized and degraded by specific diadenylate cyclases and phosphodiesterases, respectively. For L. monocytogenes and phylogenetically related bacteria it has been shown that c-di-AMP is essential for growth. Several targets have been identified in the past years that bind to c-di-AMP. As yet, none of the known targets of c-di-AMP is essential in the bacteria whose viability, however depends on the signalling nucleotide. Recently, we have shown that the CdaR protein negatively regulates the activity of the L. monocytogenes diadenylate cyclase CdaA. The observation that both proteins are located at the cell envelope, suggests that c-di-AMP metabolism is linked to cell wall homeostasis. Indeed, for many bacteria it has been reported that alterations of the cellular c-di-AMP levels affect integrity of the protective cell envelope. However, the role of c-di-AMP in cell wall metabolism is unknown. In the proposed project we want to identify the signals that are received by CdaR and how c-di-AMP produced by CdaA affects the integrity of the bacterial cell envelope. By applying genetic as well as biochemical approaches we aim to elucidate the interaction network of CdaA and its regulator CdaR. Moreover, we want to perform structural analyses to investigate the molecular details of the protein complexes. We also want to identify substances that inhibit CdaA because the essential diadenylate cyclase is an excellent target for novel antibiotics.