Molecular Mechanisms Regulating Bacterial Two-component Signaling Systems

项目总结/摘要 金黄色葡萄球菌感染是一个主要的全球健康问题，并且仍然是一个重大的健康负担， 社会据估计，仅在美国就有超过三十万例医院相关的S。 金黄色葡萄球菌感染每年花费20亿美元。这些感染也会导致肺炎，败血症， 感染性心内膜炎、骨髓炎等疾病。S.金黄色葡萄球菌感染和相关疾病的结果 从分泌的毒力因子和细菌在广泛的环境中生存的能力 小生境，包括缺氧条件。重要的是，生长和毒力受双组分调节， TCS系统，其由膜结合的传感器组氨酸激酶（HK）和细胞质的组氨酸激酶组成。 反应调节蛋白激酶感知细胞外环境，并在适当的刺激下 通过细胞膜传递信号以诱导反应调节剂的磷酸化，导致 基因表达的变化。呼吸道反应AB（SrrAB）TCS在 缺氧条件下或在亚硝化应激的存在下，并协调毒力因子的调节， 发酵酶、一氧化氮解毒酶和生物膜形成。在本提案中，PI将 追求三个目标，旨在揭示SrrB传感器组氨酸激酶的调节机制。关于SrrB HK是一种跨膜蛋白，其含有N-末端胞外Cache结构域和胞质内Cache结构域。 含有PAS结构域的催化区（HAMP-PAS-DHpCA）。第一个目标是确定 SrrB PAS结构域以及与血红素结合如何影响SrrB功能。第二个目标是鉴定配体， 结合到该高速缓存结构域，并阐明其传感机制和在毒力中的作用。第三个目标将 使用X射线晶体学、SAXS和低温技术确定SrrB酶调节的结构基础。 在纳米级脂质盘中重构的全长SrrB的电子显微镜。成功完成 这些研究将提供SrrB感知细胞外配体和细胞内配体的分子机制。 氧化还原调节催化功能，以及破坏这种调节的生物学后果。我们的结果 将对设计靶向SrrAB TCS的新治疗策略具有重要意义， 对抗抗生素耐药的S.金黄色葡萄球菌菌株。

PROJECT SUMMARY/ABSTRACT Staphylococcus aureus infections are a major global health problem and remain a significant health burden to society. In the U.S. alone it is estimated that over three-hundred thousand cases of hospital-associated S. aureus infections occur yearly at the cost of $2 billion. These infections also contribute to pneumonia, sepsis, infective endocarditis, osteomyelitis, and other diseases. S. aureus infections and associated diseases result from secreted virulence factors and the ability of the bacterium to survive in a wide range of environmental niches, including hypoxic conditions. Importantly, growth and virulence are regulated by two-component systems (TCS), which are composed of a membrane-bound sensor histidine kinase (HK) and a cytoplasmic response regulator protein. The kinase senses the extracellular environment, and under the appropriate stimuli transmits a signal across the cell membrane to induce phosphorylation of the response regulator, resulting in changes in gene expression. The staphylococcus respiratory response AB (SrrAB) TCS is activated under hypoxic conditions or in the presence of nitrosative stress and coordinates the regulation of virulence factors, fermentation enzymes, nitric oxide detoxifying enzymes and biofilm formation. In this proposal, the PI will pursue three aims designed to reveal the regulatory mechanisms of the SrrB sensor histidine kinase. The SrrB HK is a transmembrane protein that contains an N-terminal extracellular Cache domain and a cytoplasmic catalytic region (HAMP-PAS-DHpCA) containing a PAS domain. The first aim is to determine the role of the SrrB PAS domain and how binding to heme impacts SrrB function. The second aim will identify ligands that bind to the Cache domain and elucidate its sensing mechanism and role in virulence. The third aim will determine the structural basis for SrrB enzymatic regulation using X-ray crystallography, SAXS and cryogenic electron microscopy of full-length SrrB reconstituted in nanometer-scale lipid discs. Successful completion of these studies will provide the molecular mechanism(s) by which SrrB senses extracellular ligands and cellular redox to regulate catalytic function, and the biological consequences for disrupting this regulation. Our results will have important implications for the design of novel therapeutic strategies targeting the SrrAB TCS to combat antibiotic resistant S. aureus strains.