Molecular Mechanisms Regulating Bacterial Two-component Signaling Systems

项目摘要/摘要 金黄色葡萄球菌感染是一个主要的全球健康问题，仍然是严重的健康负担。 社会。仅在美国，估计就有超过30万例与医院相关的S。 金黄色葡萄球菌感染每年造成20亿美元的损失。这些感染还会导致肺炎、败血症、 感染性心内膜炎、骨髓炎等疾病。金黄色葡萄球菌感染及相关疾病 从分泌的毒力因子和细菌在广泛的环境中生存的能力 利基，包括低氧条件。重要的是，生长和毒力由两种成分调节。 系统(TCS)，它由膜结合的传感器组氨酸激酶(HK)和细胞质 反应调节蛋白。该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来诱导反应调节器的磷酸化，从而导致 基因表达的变化。葡萄球菌呼吸反应AB(SrrAB)TCS在以下条件下激活 在低氧条件下或在亚硝化胁迫存在的情况下，协调毒力因子的调节， 发酵酶、一氧化氮解毒酶和生物膜的形成。在这项提案中，私人投资公司将 追求三个目标，旨在揭示SrrB传感器组氨酸激酶的调节机制。高级公务员制度委员会 HK是一种跨膜蛋白，含有N端的胞外缓存区和细胞质 含有PAS结构域的催化区(HAMP-PAS-DHpCA)。第一个目标是确定 SrB传递结构域以及与血红素结合如何影响srB功能。第二个目标将确定 结合到Cache域，并阐明其感知机制和在毒力中的作用。第三个目标将是 用X射线结晶学、SAXS和低温确定SrrB酶调控的结构基础 纳米级脂盘中重组的全长SrrB的电子显微镜。成功完成 这些研究将提供srB感知细胞外配体和细胞的分子机制(S) 氧化还原来调节催化功能，以及破坏这种调节的生物学后果。我们的结果 将对设计针对SrrAB TCS的新治疗策略具有重要意义 对抗抗药性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.