Redefining fatty acid degradation by Staphylococcus aureus

重新定义金黄色葡萄球菌的脂肪酸降解

• 批准号: 10040249
• 负责人: Jeffrey Lee Bose
• 金额: $ 19.13万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10040249
• 关键词: Affect; Animal Model; Bacteria; Bioinformatics; Biological Assay; Biological Models; Biological Process; Bypass; Cadmium; Carbon; Carrier Proteins; Cell physiology; Complement; Data; Degradation Pathway; Energy-Generating Resources; Enoyl-CoA Hydratase; Ensure; Environment; Enzymes; Escherichia coli; Fatty Acids; Future; Generations; Genes; Genetic; Genetic Transcription; Gram-Positive Bacteria; Growth; Knowledge; Label; Laboratories; Length; Lipids; Literature; Mass Spectrum Analysis; Membrane; Metabolic; Metabolism; Modeling; Nomenclature; Northern Blotting; Open Reading Frames; Operon; Organism; Pathogenesis; Pathway interactions; Phospholipids; Phosphotransferases; Proteins; Radio; Radioactivity; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Role; Site; Source; Staphylococcus aureus; Structural Models; Substrate Specificity; System; Testing; Time; Transcript; Virulence; antimicrobial drug; base; drug development; enzyme activity; fatty acid biosynthesis; fatty acid metabolism; fatty acid oxidation; genetic architecture; human disease; inhibitor/antagonist; methicillin resistant Staphylococcus aureus; mutant; overexpression; oxidation; predictive modeling; preference; promoter; protein degradation; stem; transcriptome sequencing; transcriptomics

PROJECT SUMMARY/ABSTRACT Bacteria require fatty acids for a variety of biological functions, including the construction of phospholipids. Staphylococcus aureus, like other bacteria, synthesize fatty acids using a fatty acid biosynthesis pathway referred to as FASII, but can also scavenge fatty acids from the environment using the recently described Fak pathway. In addition to fatty acid biosynthesis and acquisition, it is common for bacteria to degrade fatty acids using fatty acid degradation (Fad) enzymes. In some cases, this is to adjust chain length but also occurs for -oxidation of fatty acids for energy generation. S. aureus does not perform -oxidation of fatty acids and has been thought to not possess the capacity to degrade fatty acids. This is due, in part, to the absence of a key crotonase enzyme. This is surprising considering that S. aureus is annotated to encode all the other necessary Fad enzymes, though their function has not been confirmed. During RNAseq studies of the Fak pathway, we observed an ~17-fold increased expression of the group of genes annotated to encode Fad proteins. Indeed, no crotonase enzyme was apparent, though all the other Fad functionalities were annotated. Using bioinformatics, we identified a putative crotonase enzyme in S. aureus that we now call FadB based on nomenclature in other systems. When expressed with S. aureus FabA, the S. aureus FadB can functionally complement an E. coli fadAB mutant on minimal media with fatty acid as a sole carbon source. This demonstrates that 1) S. aureus FadB can substitute for the E. coli crotonase activity-containing enzyme, and 2) S. aureus does possess a complete Fad pathway and likely can degrade fatty acids. We seek to characterize this pathway using two complementary Specific Aims. Aim 1 uses the E. coli model to confirm the identity of the other S. aureus Fad proteins and to define the activity of S. aureus FadB, including the substrate range in a well-described system with the benefit of minimal media. We found that under rich media conditions (there is no minimal media for S. aureus) that the S. aureus Fad pathway is lowly expressed and it is unknown if these genes are co-transcribed. Aim 2 determines the genetic context of the Fad-encoding genes. In addition, Aim 2 examines the ability for S. aureus to degrade fatty acids using controlled expression of the Fad genes by a combination of radio-labelled fatty acids and mass spectrometry approaches. The Fad-encoding genes have been identified in a variety of transcriptomic studies, but remain unstudied likely due to the known dogma in the field that S. aureus does not possess a complete Fad pathway and cannot degrade fatty acids. We anticipate that the completion of this application will redefine fatty acid metabolism in S. aureus and determine for the first time that S. aureus can degrade fatty acids. This will change how the field understands S. aureus metabolism and will set the stage for future applications examining the role of this pathway in cell physiology and pathogenesis.

项目总结/摘要 细菌需要脂肪酸用于各种生物功能，包括构建 磷脂金黄色葡萄球菌，像其他细菌一样，使用脂肪酸生物合成来合成脂肪酸 途径称为FASII，但也可以从环境中吸收脂肪酸， 描述了Fak途径。除了脂肪酸的生物合成和获取，细菌降解也很常见 使用脂肪酸降解（Fad）酶测定脂肪酸。在某些情况下，这是为了调整链长，但也 发生脂肪酸的反式氧化以产生能量。S.金黄色葡萄球菌不进行脂肪酸的反式氧化 并且被认为不具有降解脂肪酸的能力。这部分是由于缺乏 一种关键的巴豆酸酶这是令人惊讶的考虑到S。aureus被注释为编码所有其他 必需的Fad酶，尽管它们的功能尚未得到证实。在Fak的RNAseq研究期间， 在Fad通路中，我们观察到注释为编码Fad的基因组的表达增加了~17倍 proteins.事实上，没有巴豆酸酶是明显的，尽管所有其他Fad功能都被注释了。 利用生物信息学，我们确定了一个假定的巴豆酸酶在S。我们现在称之为FadB， 其他系统中的命名。当用S表示时。aureus FabA、S.金黄色葡萄球菌FadB可以在功能上 补充一个E。coli fadAB突变体在基本培养基上以脂肪酸作为唯一碳源。这表明 1）S。aureus FadB可替代E. coli巴豆酸酶活性的酶; 2）S.金黄色葡萄球菌 拥有完整的Fad途径，可能可以降解脂肪酸。我们试图描述这一途径， 两个互补的具体目标。 Aim 1使用E. coli模型进行鉴定。金黄色葡萄球菌Fad蛋白，并定义 S.的活动。金黄色葡萄球菌FadB，包括在一个良好描述的系统中的底物范围， 媒体我们发现，在丰富的媒体条件下（没有最低限度的媒体为S。aureus），S.金黄色 Fad途径是低表达的，并且不知道这些基因是否共转录。目标2确定了 Fad编码基因的遗传背景。此外，目标2考察了S.金黄色葡萄球菌降解脂肪 通过放射性标记的脂肪酸和质谱的组合， 光谱方法。 Fad编码基因已在多种转录组学研究中被鉴定，但仍未被研究 可能是由于该领域中已知的教条，S.金黄色葡萄球菌不具有完整的Fad途径， 降解脂肪酸。我们预计，这一应用的完成将重新定义脂肪酸代谢的S。 金黄色葡萄球菌，并首次确定了S.金黄色葡萄球菌可以降解脂肪酸。这将改变这个领域 理解S。金黄色葡萄球菌代谢，并将为未来研究其作用的应用奠定基础 细胞生理学和发病机制的途径。