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.

项目摘要/摘要 金黄色葡萄球菌是一种成功的人类病原体，这是由于病毒因素的大量曲目，包括 杀死宿主细胞和多种分泌蛋白酶的多种毒素，例如α-羟蛋白酶素 细菌和宿主蛋白。病毒因子生产的关键调节系统是saers二 组件系统，该系统响应未知信号而激活或反映基因表达。最近， 我们鉴定了VFRB，这是所有革兰氏阳性细菌中构成假设蛋白的构成，它调节病毒 因子产生与Saers活性改变一致。 VFRB需要功能性的saer来控制血素蛋白 生产和组成性活跃的SAE绕过了对α-蛋白酶表达中VFRB的需求，这表明 该VFRB在激活SAE中起着作用。此外，我们发现缺乏VFRB的突变体增强了 皮肤感染的小鼠模型中的毒力。我们的进一步研究表明，VFRB是一种新颖的脂肪酸激酶 与两个未表征的脂肪酸载体蛋白FAKB1和FAKB2一起使用 外源脂肪酸进入细胞。该系统的破坏不仅改变了病毒和脂肪酸的摄取，而且会改变 还会改变动力学和乙酸代谢体外。有趣的是，FAKB2对脂肪酸具有特异性 该细菌不是在哺乳动物中产生的，但表明VFRB和FAKB可能构成一个 主机识别系统。 基于这些信息，我们假设金黄色葡萄球菌中的VFRB引导碳流经乙酸 代谢同时激活SAE系统调节病毒。测试这个 假设提出了三个具体目标。 AIM1利用有指导和全球方法来检查 生长过程中细胞代谢的变化，重点是乙酸代谢。 AIM 2使用诱变 鉴定对功能必不可少的VFRB，FAKB1和FAKB2的氨基酸。突变体将受到 一系列测试，以检查病毒因子产生，蛋白质 - 蛋白质相互作用和脂肪酸的变化 结合。为了支持这些研究，提出了解决VFRB的结构。最后，AIM 3将确定 VFRB控制SAE激活的阶段，利用突变体和依赖SAER的记者检查 缺乏VFRB的突变体中的sa族激活状态。 AIM 3还试图定义VFRB如何调节病毒 宿主中的因素，以及这如何导致ΔVFRB突变体中的病毒增强。这将使用 皮肤感染模型，以确定α-蛋白酶和蛋白酶在感染过程中的贡献 ∆VFRB突变体。最后，将使用体内监测AUR蛋白和α-脱糖蛋白的表达 蛋白质组学和活动画成像。这些研究的完成将洞悉 我们最近识别的脂肪酸激酶复合物在所有革兰氏阳性细菌中发现，并将散发出新的 关于导致金黄色葡萄球菌感染的病毒因子的代谢和调节的光明。