Contribution of Spx to Staphylococcus aureus physiology and virulence

项目概要/摘要 金黄色葡萄球菌是一种强有力的人类病原体，由于其大量的毒力因子， 细菌感染人体的任何部位。我们的长期目标是了解 S.金黄色葡萄球菌调节毒力因子的表达和活性以引起疾病。这包括揭露小说 途径有助于毒力因子的生产和对环境变化的反应。Spx蛋白 被革兰氏阳性菌用于调节基因表达以响应二硫化物和氧化应激。最 对Spx的了解来自对芽孢杆菌和链球菌的研究，只有少数研究 研究了Spx在S.金黄色。其中两项研究的解释受到阻碍， 后来发现的二级位点突变。最近，我们发表了一项研究，其中包括一个spx突变体 在USA 300谱系的临床相关MRSA菌株中。在这种背景下，Spx有助于在 二酰胺胁迫和色素产生。Spx是S.但可以通过控制 硫氧还蛋白的表达，我们采用的一种技术。尽管硫氧还蛋白的生产，spx突变体有一个 生长缺陷我们也有新的初步数据表明spx突变体改变了关键的溶血活性， 分泌的蛋白质生长缺陷和改变的毒力因子产生的原因还没有被证实。 这是本申请的重点。 基于我们的初步数据，我们假设Spx调节代谢和关键调节因子， 控制毒力因子表达的网络。为了验证这一点，提出了三个目标。Aim 1使用RNAseq ChIPseq和靶向代谢组学来定义Spx调节子和Spx对代谢的影响。Aim 2构建 我们的初步数据显示溶血素活性降低，以确定这种表型背后的机制。 这研究了关键的调控网络，据信涉及使用突变体，报告基因， 定量分析Aim 3首次检验了Spx对S.使用小鼠的金黄色葡萄球菌毒力 皮肤感染模型。此外，它使用活体动物成像来检查体内毒力因子的表达。 这些研究的完成将提供深入了解这一重要病原体的毒力因子调控。在 此外，这将是第一个定义的Spx调节子在S。金黄色葡萄球菌，并测定Spx对金黄色葡萄球菌的贡献。 金黄色葡萄球菌毒力

PROJECT SUMMARY/ABSTRACT Staphylococcus aureus is a potent human pathogen due to its large repertoire of virulence factors, allowing this bacterium to infect any part of the human body. Our long-term goal is to understand the mechanisms by which S. aureus modulates virulence factor expression and activity to cause disease. This includes uncovering novel pathways contributing to virulence factor production and responses to environmental changes. The Spx protein in used by Gram-positive bacteria to regulate gene expression in response to disulfide and oxidative stress. Most of what is known about Spx stems from studies in Bacillus and Streptococcus and only a few studies have examined the importance of Spx in S. aureus. The interpretation of two of those studies are hampered due to secondary site mutations that were later discovered. Recently, we published a study that included an spx mutant in a clinically relevant MRSA strain of the USA300 lineage. In this background, Spx contributes to survival under diamide stress and pigment production. Spx is essential in S. aureus but can be circumvented by controlled expression of thioredoxin, a technique we employ. Despite thioredoxin production, the spx mutants have a growth defect. We also have new preliminary data that the spx mutant has altered activity of key hemolytic secreted proteins. The reason for the growth deficiency and altered virulence factor production has not been elucidated and is the focus of this application. Based on our preliminary data, we hypothesize that Spx modulates metabolism and key regulatory networks that control virulence factor expression. To test this, three aims are proposed. Aim 1 uses RNAseq ChIPseq, and targeted metabolomics to define the Spx regulon and impact of Spx on metabolism. Aim 2 builds on our preliminary data showing reduced hemolysin activity to identify the mechanism behind this phenotype. This examines key regulatory networks believed to be involved using a combination of mutants, reporters, and quantitative assays. Aim 3 tests for the first time the contribution of Spx to S. aureus virulence using a murine skin infection model. Furthermore, it uses live animal imaging to examine virulence factor expression in vivo. Completion of these studies will provide insight into virulence factor regulation in this important pathogen. In addition, it will be the first to define the Spx regulon in S. aureus and determine the contribution of Spx to S. aureus virulence.