Analysis of PASTA kinase function in Enterococcus faecalis

项目摘要 几乎所有的革兰氏阳性细菌合成一种跨膜的Ser/Thr激酶，其含有3至5个氨基酸残基。 细胞外PASTA结构域（即“PASTA激酶”），其控制关键过程，包括 抗生素抗性、毒素产生、毒力或细胞分裂;在某些细菌中，PASTA激酶 对生存能力至关重要因此，PASTA激酶代表了新疗法的有吸引力的靶标。 然而，对该家族中激酶的功能机制的基本理解， 在体内感知环境刺激并处理该信息以协调适应性生物学 缺乏回应。这些信息对于开发新的治疗药物至关重要。 接近。本文提出的研究旨在通过阐明基本的 该家族中代表性激酶的功能方面，粪肠球菌中的IreK激酶， 我们已经证明这是E的固有电阻所必需的。粪便细胞壁活性 抗微生物剂和胆汁中存在的去污剂，如胆酸盐。这项研究利用遗传学和 生物化学方法结合最先进的质谱分析策略，以克服关键的 通过定义体内磷酸化对IreK的程度和功能影响， 以及鉴定IreK磷酸化的下游底物。完成这些研究 将使我们在理解IreK在E.粪细胞 鉴于PASTA激酶家族中保守的结构域结构， 也会转化为其他PASTA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.