Analysis of PASTA kinase function in Enterococcus faecalis

粪肠球菌PASTA激酶功能分析

• 批准号: 9451474
• 负责人: CHRISTOPHER J KRISTICH
• 金额: $ 24.24万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9451474
• 关键词: Address; Adopted; Antibiotic Resistance; Antimicrobial Resistance; Architecture; Bacteria; Bile fluid; Biochemical; Biological; Biological Adaptation; Biological Process; Cell Extracts; Cell Wall; Cell division; Cells; Cholates; Collection; Coupled; Cytoplasm; Data; Detergents; Development; Enterococcus; Enterococcus faecalis; Family; Foundations; Funding Mechanisms; Future; Genetic; Gram-Positive Bacteria; In Vitro; Incidence; Infection; Intervention; Knowledge; Mass Spectrum Analysis; Mediating; Membrane; Molecular; Multi-Drug Resistance; Mutagenesis; Nosocomial Infections; Outcome; Pathway interactions; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Process; Production; Proteome; Regulation; Research; Resistance; Role; Site; Stimulus; Stress; Therapeutic; Toxin; Translating; Virulence; Work; antimicrobial; antimicrobial drug; base; design; extracellular; in vitro activity; in vivo; innovation; insight; member; new therapeutic target; novel; novel therapeutic intervention; novel therapeutics; pathogen; pathogenic bacteria; phosphoproteomics; prevent; response; targeted treatment; trait

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.

项目概要 几乎所有革兰氏阳性菌都会合成含有 3 至 5 个氨基酸的跨膜 Ser/Thr 激酶 细胞外 PASTA 结构域（即“PASTA 激酶”）控制关键过程，包括 抗生素耐药性、毒素产生、毒力或细胞分裂；在某些细菌中，PASTA 激酶 对于生存能力至关重要。因此，PASTA 激酶代表了新疗法的有吸引力的靶标。 然而，对该家族中激酶发挥作用的机制的基本了解 感知体内环境刺激并处理该信息以协调适应性生物 缺乏回应。这些信息对于新疗法的开发至关重要 接近。这里提出的研究旨在通过阐明基本原理来帮助解决这一差距 该家族中代表性激酶（粪肠球菌中的 IreK 激酶）的功能方面， 我们已经证明这是粪肠球菌对细胞壁活性的内在抵抗力所必需的 抗菌剂和胆汁中存在的去污剂，例如胆酸盐​​。这项研究利用遗传和 生化方法与最先进的质谱策略相结合，以克服关键问题 通过定义体内 IreK 磷酸化的程度和功能影响来解决进展的障碍， 并通过识别 IreK 磷酸化的下游底物。完成这些研究 将使我们在了解 IreK 如何在粪肠球菌细胞中发挥作用方面迈出重要的一步。 鉴于 PASTA 激酶家族中的保守结构域结构，见解很可能 这项工作的成果也将转化为其他 PASTA 激酶。