Characterization of ??S in the stress & virulence responses of S. aureus

描述（由申请人提供）：金黄色葡萄球菌是一种高毒力和广泛成功的病原体，被推测是人类疾病的最常见原因。随着耐多药菌株的不断出现，确定新的靶点对于我们防止回到前抗生素时代至关重要。金黄色葡萄球菌作为一种病原体的成功很大程度上是由于其大量的毒力决定因素，或者更具体地说，是调节这些和其他遗传成分的调控元件的复杂网络。从发病机制的角度来看，这个控制网络的存在可能是为了微调宿主环境中生物体的需求。我们最近发现了这个调控网络的一个新组成部分：一个替代西格玛因子（CS），它不仅是金黄色葡萄球菌适应和生存所必需的，而且对其毒力也至关重要。使用小鼠脓毒性关节炎模型，我们发现感染sigS突变体的小鼠表现出明显的体重减轻、死亡率、感染严重程度、全身传播和免疫反应增加。因此，我们的具体假设是，CS代表了金黄色葡萄球菌毒力调控网络的主要缺失部分。我们认为，在本应用中提出的对其功能和控制的深入分析将最终使我们能够更好地了解该生物体的发病机制，并可能有助于合理开发新的治疗方法。为此，我们建议：分析金黄色葡萄球菌诱导sigS所需的机制。我们将使用启动子定位、DNA-蛋白下拉试验和高通量报告基因融合技术，结合生长操作来确定在金黄色葡萄球菌中诱导CS活性的确切机制。2. 定义CS在金黄色葡萄球菌应激和毒力反应中的作用。我们将使用微阵列分析来确定哪些基因是由CS控制的；和表型微阵列，探讨CS对金黄色葡萄球菌生物学的代谢和生理贡献。3. 研究控制金黄色葡萄球菌CS活性的具体途径。ECF-sigma因子通常在翻译后通过复杂的级联蛋白相互作用调节，由抑制和蛋白水解介导。我们将利用基因转录技术、特异性蛋白分析、酵母-2杂交研究和诱变筛选来探索金黄色葡萄球菌CS调控网络的特异性成分。随着金黄色葡萄球菌引起的感染在全球范围内的上升，尽一切努力了解其发病机制是至关重要的。这里提出的研究将为我们提供一个独特的和特定的洞察金黄色葡萄球菌疾病的分子机制，通过绘制一个主要的新的毒力调节因子的功能和控制。所获得的知识也可能有助于合理设计新的抗葡萄球菌疗法。最后，考虑到葡萄球菌中CS的独立进化起源，我们的研究可能会提供有关原核调控网络的新信息。

DESCRIPTION (provided by applicant): Staphylococcus aureus is a highly virulent and widely successful pathogen that is speculated to be the most common cause of human disease. With the continued emergence of multi-drug resistant isolates, the identification of novel targets is crucial in our fight against a return to the pre-antibiotic era. The success of S. aureus as a pathogen is largely due to its vast array of virulence determinants, or more specifically, the complex network of regulatory elements that modulate these and other genetic components. From the perspective of pathogenesis, this network of control presumably exists to fine-tune the requirements of the organism within the host environment. We have recently identified a novel component of this regulatory network: an alternative sigma factor (CS) which is required not only for S. aureus adaptation and survival, but is also vital for its virulence. Using a murine model of septic arthritis we found that mice infected with a sigS mutant displayed significantly decreased weight-loss, mortality, severity of infection, systemic dissemination and mounted immune responses. Thus our specific hypothesis is that CS represents a major missing component of the S. aureus virulence regulatory network. We contend that the thorough analysis of its function and control proposed in this application will ultimately enable us to better understand the pathogenesis of this organism, and may aid in the rational development of novel therapeutic treatments. To this end we propose to: 1. Analyze the mechanisms required for sigS induction in S. aureus. We will use promoter mapping, DNA- protein pulldown assays, and high-throughput reporter gene fusion technologies coupled with growth manipulation to determine the exact mechanisms responsible for the induction of CS activity in S. aureus. 2. Define the function of CS in the S. aureus stress and virulence responses. We will use microarray analysis to determine exactly which genes are controlled by CS; and phenotypic microarrays to probe the metabolic and physiological contributions made by CS to S. aureus biology. 3. Investigate the specific pathways that control CS activity in S. aureus. ECF-sigma factors are commonly regulated in a post- translational manner via a complex cascade of protein interaction, mediated by inhibition and proteolysis. We will probe S. aureus specific components of the CS regulatory network using gene-transcription technologies, specific protein analysis, yeast-2-hybrid studies and mutagenic screens. With infections caused by S. aureus on the rise globally it is vital that every effort is exerted to understand the mechanisms involved in its pathogenesis. The investigations proposed here will provide us with a unique and specific insight into the molecular mechanisms of S. aureus disease, by mapping the function and control of a major new virulence regulator. The knowledge derived may also aid in the rational design of novel anti-staphylococcal therapies. Finally, given the independent evolutionary origins of CS in the staphylococci, our investigations will likely deliver new information regarding prokaryotic regulatory networks. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus is a highly virulent and widely successful pathogen that is speculated to be the most common cause of human infection. With the continued emergence of multi-drug resistant isolates of S. aureus (such as MRSA), there is an urgent need to understand the mechanisms by which this deadly pathogen causes disease. This proposal seeks to investigate and understand how a novel regulator of virulence, CS, contributes to disease causation in S. aureus.