Mechanistic studies of rot-mediated regulation of Staphylococcus aureus virulence

描述（由申请人提供）：金黄色葡萄球菌是医院和社区环境中危及生命的细菌感染的主要原因之一。这种细菌可以定植并侵入人体几乎所有组织。S.金黄色葡萄球菌作为病原体的致病性部分是由于毒力因子库的时间表达，其由一系列双组分调节系统（TCS）和螺旋-转角-螺旋蛋白执行。遗传分析表明，在S.金黄色葡萄球菌是毒素的转录抑制因子（Rot），而S. aureus exoprotein expression two-component system（SaeRS-TCS）。使用小鼠全身感染模型的实验表明，S。缺乏rot的金黄色葡萄球菌菌株是高毒力的，而缺乏sae的菌株是完全减毒的，突出了这些菌株的重要性。 调节子S.金黄色葡萄球菌病理生理学Rot被假设为通过抑制编码细胞毒素和蛋白酶的基因的启动子并激活编码表面蛋白的基因的启动子来协调毒力因子的差异表达。相反，SaeRS-TCS参与编码大多数分泌因子（包括细胞毒素）的启动子的激活。在临床相关菌株中Rot和SaeRS调节子的比较揭示了大部分Rot抑制的基因需要SaeRS-TCS来激活。Rot调控S.金黄色葡萄球菌不被理解。类似地，Rot干扰SaeRS-TCS以改变靶基因表达的机制仍有待阐明。此外，检查rot的转录和翻译调控的研究主要是在体外进行的，不一定模拟这种转录因子对体内毒力因子调控的影响。因此，本提案的具体目标是：（i）确定Rot介导的毒力因子调节机制，以及（ii）阐明离体和体内Rot转录和翻译的调节。为了实现这些目标，将采用多学科方法，其中分子生物学、生物化学和细菌学技术将与体内和离体感染模型相结合。总的来说，本申请中提出的工作将阐明Rot如何调节所需的毒力因子的表达， S.金黄色葡萄球菌发病机制由于目前耐药沙门氏菌的流行，金黄色葡萄球菌菌株，了解S.金黄色葡萄球菌调节毒力因子的表达可能为基于该细菌致病所需的靶向调节回路开发新的抗葡萄球菌药物提供基础。

DESCRIPTION (provided by applicant): Staphylococcus aureus is one of the leading causes of life-threatening bacterial infections in both hospitals and community settings. This bacterium can colonize and invade virtually all tissues in the human body. The tremendous success of S. aureus as a pathogen is in part due to the temporal expression of an arsenal of virulence factors, which is executed by a series of two component regulatory systems (TCS) and helix-turn-helix proteins. Genetic analyses have revealed that at the center of the regulation of virulence factors in S. aureus are the transcription factors repressor of toxins (Rot) and the S. aureus exoprotein expression two-component system (SaeRS-TCS). Experiments using a murine model of systemic infection have revealed that S. aureus strains lacking rot are hypervirulent, while strains lacking sae are fully attenuated, highlighting the importance of these regulators for S. aureus pathophysiology. Rot is hypothesized to coordinate the differential expression of virulence factors by repressing the promoters of genes that code for cytotoxins and proteases, and activating the promoters of genes that code for surface proteins. In contrast, the SaeRS-TCS is involved in the activation of the promoters that code for most secreted factors, including cytotoxins. Comparison of the Rot and SaeRS regulons in clinically relevant strains has revealed that a large portion of Rot-repressed genes require the SaeRS-TCS for their activation. The precise molecular mechanism by which Rot regulates gene expression in S. aureus is not understood. Similarly, the mechanism by which Rot interferes with the SaeRS-TCS to alter the expression of target genes remains to be elucidated. Additionally, studies examining transcriptional and translational regulation of rot have primarily been done in vitro and do not necessarily mimic the impact this transcription factor has on virulence factor regulation in vivo. Thus, the Specific Aims of this proposal seek to: (i) define the mechanism of Rot-mediated regulation of virulence factors, and (ii) elucidate the regulation of rot transcriptio and translation ex vivo and in vivo. To accomplish these Aims, a multi-disciplinary approach will be employed where molecular biology, biochemistry, and bacteriology techniques will be combined with in vivo and ex vivo infection models. Collectively, the work proposed in this application will shed light into how Rot regulates the expression of virulence factors required for S. aureus pathogenesis. Due to the current epidemic of antibiotic-resistant S. aureus strains, understanding the intricacies of how S. aureus regulates the expression of virulence factors is likely to provide the foundation for the development of new anti-Staphylococcal drugs based on targeting regulatory circuits that are required for the pathogenesis of this bacterium.