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

MRSA 脂肪酸激酶代谢和毒力的机制控制

• 批准号: 9176725
• 负责人: Jeffrey Lee Bose
• 金额: $ 39.49万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-24 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9176725
• 关键词: Acetates; Address; Amino Acids; Antibiotics; Attenuated; Bacteria; Binding; Biological Assay; Bypass; Carbon; Carrier Proteins; Cell physiology; Cells; Citric Acid Cycle; Cleaved cell; Complement; Complex; Cytolysis; Data; Development; Disease; Electron Transport; Environment; Enzymes; Essential Amino Acids; Fatty Acids; Gene Expression; Genes; Glucose; Goals; Gram-Positive Bacteria; Growth; Hemolysin; Human; In Vitro; Infection; Infectious Skin Diseases; Kinetics; Lead; Life; Light; Link; Mammals; Membrane Lipids; Membrane Potentials; Metabolism; Modeling; Monitor; Mus; Mutagenesis; Mutation; PAWR protein; Pathogenicity; Peptide Hydrolases; Phenotype; Phosphotransferases; Play; Production; Proteins; Proteomics; Regulation; Reporter; Resources; Role; Signal Transduction; Source; Specificity; Staging; Staphylococcus aureus; Streptococcus; Structure; Structure-Activity Relationship; Surface; System; Testing; Time; Toxin; Virulence; Virulence Factors; Work; animal imaging; base; combat; commensal microbes; cytotoxic; design; fatty acid-binding proteins; genetic regulatory protein; human disease; in vivo; insight; killings; member; membrane synthesis; metabolomics; methicillin resistant Staphylococcus aureus; mouse model; mutant; novel; novel strategies; novel therapeutic intervention; pathogen; protein function; protein protein interaction; response; tool; transcriptome sequencing; uptake

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.

项目总结/摘要 金黄色葡萄球菌是一种成功的人类病原体，由于大量的毒力因子，包括 一种杀死宿主细胞的毒素，如α-溶血素和多种分泌的蛋白酶， 细菌和宿主蛋白质。毒力因子产生的一个关键调控系统是SaeRS两个- 组成系统，其响应于未知信号而激活或抑制基因表达。最近， 我们鉴定了VfrB，一种存在于所有革兰氏阳性菌中的保守假设蛋白，它调节着致病力 因子生产与改变的SaeRS活性一致。VfrB需要功能性SaeRS来控制溶血素 生产和组成型活性SaeS绕过了α-溶血素表达中对VfrB的需要，这表明 VfrB在激活SaeS中起作用。此外，我们发现缺乏VfrB的突变体增强了 在小鼠皮肤感染模型中的毒力。我们的进一步研究表明VfrB是一种新的脂肪酸激酶 它与两种未知的脂肪酸载体蛋白FakB 1和FakB 2结合， 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摄取， 也改变了体外生长动力学和乙酸盐代谢。有趣的是，FakB 2对脂肪酸具有特异性， 不是由这种细菌产生的，但在哺乳动物中很丰富，这表明VfrB和FakB可能构成了一种新的基因。 主机识别系统 基于这些信息，我们假设S. aureus引导碳流通过醋酸盐 代谢，同时激活Sae系统以调节毒力。为了验证这一 假设，提出了三个具体目标。AIM 1利用定向和全局方法来检查 生长过程中细胞代谢的变化，重点是乙酸盐代谢。Aim 2使用诱变来 鉴定VfrB、FakB 1和FakB 2的功能必需氨基酸。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 约束力为了支持这些研究，解决VfrB的结构。最后，目标3将确定 VfrB控制SaeS激活的阶段，利用突变体和SaeRS依赖性报告基因来检查 缺乏VfrB的突变体中SaeRS的激活状态。目标3还试图确定VfrB如何调节毒力 因子在宿主中，以及这如何导致增强的毒力在p53 vfrB突变体。这将通过使用 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。 HPVfrB突变体。最后，Aur蛋白酶和α-溶血素的表达将使用 蛋白质组学和活体动物成像。这些研究的完成将使人们深入了解 我们最近发现的脂肪酸激酶复合物存在于所有革兰氏阳性细菌中， 阐明了导致S.金黄色葡萄球菌感染。