Mechanistic control of metabolism and virulence by fatty acid kinase in MRSA

项目摘要/摘要 金黄色葡萄球菌是一种成功的人类病原体，原因是大量的毒力因子，包括 一种毒素，如α-溶血素，可杀死宿主细胞和裂解两者的多种分泌的蛋白酶 细菌和宿主蛋白。毒力因子生产的一个关键监管系统是SaeRS两个- 成分系统，它激活或抑制基因表达，以响应未知信号。最近， 我们鉴定了VfrB，它是所有革兰氏阳性细菌中的一种保守的假设蛋白，它调节着毒力 因子生产与SaeRS活性的变化一致。VfrB需要功能性的SAERS来控制溶血素 生产和结构性活性的SAE绕过了α-溶血素表达中对VfrB的需要，这表明 VfrB在激活SAE中起作用。此外，我们发现一个缺乏VfrB的突变体增强了 皮肤感染的小鼠模型中的毒性。我们的进一步研究表明，VfrB是一种新的脂肪酸激酶 它与两种未鉴定的脂肪酸载体蛋白FakB1和FakB2一起工作，以整合 外源脂肪酸进入细胞。该系统的破坏不仅改变了毒力和脂肪酸的摄取，而且 也改变了体外的生长动力学和醋酸酯代谢。有趣的是，FakB2对脂肪酸具有专一性 不是由这种细菌产生的，但在哺乳动物中大量存在，这表明VfrB和FakB可能构成了 主机识别系统。 根据这些信息，我们推测金黄色葡萄球菌中的VfrB引导碳流通过醋酸盐。 同时激活SAE系统来调节毒力。为了测试这一点 假设，提出了三个具体目标。AIM1使用定向和全局方法来检查 生长过程中细胞代谢的变化，重点是醋酸盐代谢。AIM 2使用诱变技术 确定功能所必需的VfrB、FakB1和FakB2的氨基酸。变种人将受到 一组检测毒力因子产生、蛋白质-蛋白质相互作用和脂肪酸变化的测试 有约束力的。为了支持这些研究，提出了解决VfrB结构的方法。最后，目标3将决定 VfrB控制SAE激活的阶段，利用突变体和SaeRS依赖的报告来检查 缺失VfrB的突变体中SaeRS的激活状态。Aim 3还试图定义VfrB是如何调节毒力的 宿主中的因素，以及这如何导致∆vfrB突变体的毒力增强。这将通过以下方式解决 一种皮肤感染模型，以确定α-溶血素和蛋白酶在感染过程中的作用 ∆vfrB突变体。最后，AUR蛋白水解酶和α-溶血素的表达将在体内使用 蛋白质组学和活体动物成像。这些研究的完成将使我们能够深入了解 我们最近发现的脂肪酸激酶复合体在所有革兰氏阳性细菌中都有发现，并将释放出新的 金黄色葡萄球菌感染毒力因子的代谢与调控。

PROJECT SUMMARY/ABSTRACT Staphylococcus aureus is a successful human pathogen due to a large repertoire of virulence factors, including a variety of toxins, such as α-hemolysin, that kill host cells and multiple secreted proteases that cleave both bacterial and host proteins. A key regulatory system for virulence factor production is the SaeRS two- component system, which activates or represses gene expression in response to an unknown signal. Recently, we identified VfrB, a conserved hypothetical protein in all gram-positive bacteria, which regulates virulence factor production consistent with altered SaeRS activity. VfrB requires a functional SaeRS to control hemolysin production and a constitutively-active SaeS bypasses the need for VfrB in α-hemolysin expression, suggesting that VfrB plays a role in activating SaeS. In addition, we found that a mutant lacking VfrB has enhanced virulence in a mouse model of skin infection. Our further studies revealed that VfrB is a novel fatty acid kinase that works in conjunction with two uncharacterized fatty acid carrier proteins, FakB1 and FakB2, to incorporate exogenous fatty acids into the cell. Disruption of this system not only alters virulence and fatty acid uptake, but also changes growth kinetics and acetate metabolism in vitro. Interestingly, FakB2 has specificity for fatty acids not produced by this bacterium but are abundant in mammals, suggesting that VfrB and FakB may constitute a host recognition system. Based on this information, we hypothesize that VfrB in S. aureus directs carbon flow through acetate metabolism while at the same time activating the Sae system to modulate virulence. To test this hypothesis, three specific aims are proposed. Aim1 utilizes directed and global approaches to examine changes in cellular metabolism during growth, with a focus on acetate metabolism. Aim 2 uses mutagenesis to identify amino acids of VfrB, FakB1 and FakB2 that are essential for function. The mutants will be subjected to a panel of tests to examine changes in virulence factor production, protein-protein interactions and fatty acid binding. To support these studies, solving the structure of VfrB is proposed. Finally, Aim 3 will determine the stage at which VfrB controls SaeS activation, utilizing mutants and SaeRS-dependent reporters to examine the activation state of SaeRS in a mutant lacking VfrB. Aim 3 also seeks to define how VfrB regulates virulence factors in the host, and how this leads to enhanced virulence in the ∆vfrB mutant. This will be addressed using a skin infection model to determine the contributions of α-hemolysin and proteases during infection by the ∆vfrB mutant. Finally, expression of the Aur protease and α-hemolysin will be monitored in vivo using proteomics and live-animal imaging. Completion of these studies will provide insight into the activity of our recently-identified fatty acid kinase complex found in all gram-positive bacteria and will shed new light on the metabolism and regulation of virulence factors contributing to S. aureus infection.