Analysis of PASTA kinase function in Enterococcus faecalis

项目总结 几乎所有的革兰氏阳性细菌都会合成一种跨膜的丝氨酸/苏氨酸激酶，它含有3到5个 控制关键过程的胞外通心区(即‘通心粉激酶’)，包括 抗生素抗药性、毒素产生、毒力或细胞分裂；在某些细菌中，意大利面酶 对于生存来说是必不可少的。因此，意大利面激酶是新疗法的诱人靶点。 然而，对这个家族中的激酶作用于 在体内感知环境刺激并处理该信息以协调适应生物 目前还缺乏回应。这些信息对开发新的治疗方法至关重要 接近了。这里提出的研究试图通过阐明基本原理来帮助解决这一差距 该家族中具有代表性的一种激酶，粪肠球菌中的Irek激酶的功能， 我们已经证明，这是粪肠球菌对细胞壁活性产生内在抗性所必需的 抗菌剂和胆汁中存在的洗涤剂，如胆酸盐。这项研究使用了遗传和 生化方法与最先进的质谱学策略相结合，以克服关键 通过确定体内Irek的磷酸化程度和功能影响，阻碍了进展， 以及通过鉴定用于Irek磷酸化的下游底物。完成这些研究 这将使我们在了解Irek如何在粪肠球菌细胞中发挥作用方面向前迈出重要的一步。 考虑到意大利面蛋白激酶家族中保守的结构域结构，很可能有这样的见解 这项工作也将转化为其他意大利面蛋白激酶。

PROJECT SUMMARY Nearly all Gram-positive bacteria synthesize a transmembrane, Ser/Thr kinase containing 3 to 5 extracellular PASTA-domains (i.e. a ‘PASTA kinase’) that controls critical processes including antibiotic resistance, toxin production, virulence, or cell division; in some bacteria the PASTA kinase is essential for viability. As such, PASTA kinases represent attractive targets for new therapeutics. However, a basic understanding of the mechanisms by which kinases in this family function to perceive environmental stimuli in vivo and process that information to coordinate adaptive biological responses is lacking. Such information is critical to inform development of new therapeutic approaches. The research proposed here seeks to help address this gap by elucidating fundamental aspects of function for a representative kinase in this family, the IreK kinase in Enterococcus faecalis, which we have shown is required for intrinsic resistance of E. faecalis to cell-wall-active antimicrobials and to detergents present in bile, such as cholate. This research uses genetic and biochemical approaches coupled with state-of-the-art mass spectrometry strategies to overcome key roadblocks to progress by defining the extent and functional impact of phosphorylation on IreK in vivo, and by identifying downstream substrates for phosphorylation by IreK. Completion of these studies will enable us to take important steps forward in understanding how IreK functions in E. faecalis cells. Given the conserved domain architecture among the family of PASTA kinases, it is likely that insights from this work will translate to other PASTA kinases as well.