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Branched-chain fatty acids and membrane function in Listeria monocytogenes

单核细胞增生李斯特菌中的支链脂肪酸和膜功能

基本信息

批准号：
8289070
负责人：
BRIAN JAMES WILKINSON
金额：
$ 43.2万
依托单位：
ILLINOIS STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8289070
关键词：
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)Acyl Coenzyme A Anabolism Bacteria Branched-Chain Amino Acids Butyrates Carboxylic Acids Cell physiology Cells Characteristics Coenzyme A Complement Computer Simulation Disease Enzymes Fatty Acids Food Gene Cluster Genes Gram-Positive Bacteria Growth Hydrocarbons Infection Isoleucine Kinetics Knowledge Leucine Life Listeria Listeria monocytogenes Listeriosis Membrane Membrane Fluidity Methods Modeling Modification Molecular Mutagenesis Organism Pathogenesis Pathogenicity Pathway interactions Phosphate Acetyltransferase Phosphotransferases Physical Function Physical condensation Physiology Play Population Sizes Principal Investigator Production Property Protein Biochemistry Proteins Reaction Refrigeration Role Source Stress Structure Substrate Specificity Temperature Valine Variant Virulence Work branched chain fatty acid cold temperature fatty acid biosynthesis feeding fluidity foodborne foodborne pathogen holo-(acyl-carrier-protein) synthase insight isobutyryl-coenzyme A isovaleryl-coenzyme A mutant novel overexpression pathogen physical property programs research study tool

项目摘要

DESCRIPTION (provided by applicant): Listeria monocytogenes is a foodborne bacterial pathogen with the ability to grow at refrigeration temperatures, and is thus a psychrotolerant organism. It is an intracellular pathogen and its molecular mechanisms of pathogenesis have been studied extensively. In order to grow at low temperatures psychrotolerant organisms must adjust their fatty acid compositions in order to maintain membrane fluidity. Under normal conditions almost the entire complement (more than 90%) of L. monocytogenes fatty acids are branched-chain fatty acids, in contrast to other Gram-positive bacteria that typically have about 40% straight-chain fatty acids. Fatty acid anteiso C15:0 increases in low-temperature grown bacteria by a combination of fatty acid shortening and branching switching from iso to anteiso fatty acids, and plays a key role in increasing membrane fluidity. Study of branched-chain fatty acid-deficient mutants has shown they are significantly impaired in low temperature growth, tolerance of various stresses, and virulence. This indicates that the physical structure and optimum function of the Listeria membrane is dependent on a high content of branched-chain fatty acids, and deficiency in these has major impacts on the physiology and virulence of the organism. Mutant and precursor feeding studies have revealed the existence of a novel but undefined pathway of fatty acid biosynthesis from short straight- and branched-chain carboxylic acid precursors, in addition to the normal pathway from branched-chain amino acids. We propose to study the mechanism of branched-chain fatty acid switching, define the pathway of fatty acid biosynthesis from carboxylic acids, and manipulate the membrane fatty acid composition, and hence fluidity, in an extreme fashion and examine its impact on the physiology and pathogenesis of the organism. The major determinant of fatty acid branching switching appears to reside in the temperature- responsive catalytic properties of the enzyme ¿-keto acyl carrier protein synthase III (FabH) that carries out the first condensation reaction in the fatty aid biosynthesis pathway. In specific aim I we will combine mutagenesis, functional and in silico modeling, protein biochemistry and kinetic analyses to probe the mechanisms underlying temperature-dependent variation of substrate specificities of FabH. Short- and branched-chain carboxylic acids by pass the normal fatty acid biosynthetic pathway in both branched-chain fatty acid-deficient mutants and wild-type organisms. In specific aim 2 we will clone, overexpress the proteins encoded by buk, butyrate kinase, and ptb, phosphotransbutyrylase, and attempt to show that they constitute a novel pathway for production of the CoA derivatives of carboxylic acid fatty acid precursors. L. monocytogenes has the ability of use branched-chain C6 carboxylic acid as precursors of "unnatural" even-numbered branched-chain fatty acids. In specific aim 3 we will use precursor feeding experiments to produce bacteria of low, normal and high fluidity membranes through manipulation of their fatty acid composition. These cells will be used to study the impact of membrane physical structure and fluidity on low temperature tolerance, various aspects of cell physiology, and bacterial pathogenesis. Realization of these objectives will increase our understanding of growth at low temperatures and the impact of membrane structure on physiology and pathogenesis. It is hoped that novel ways of controlling the growth of Listeria at low temperatures will emanate from the work. PUBLIC HEALTH RELEVANCE: Listeria monocytogenes is a foodborne pathogen with a high fatality rate and its ability to grow in foods at refrigeration temperatures is a critical factor i this. The proposal focuses on the role of branched-chain fatty acids in membrane physical structure, psychrotolerance, physiology, and pathogenicity. Novel methods for controlling the growth of the organism are expected to be developed as a result of the work.

描述（由申请人提供）：单核细胞增生李斯特氏菌是一种食源性细菌病原体，能够在冷藏温度下生长，因此是一种耐冷生物体。它是一种细胞内病原体，其发病的分子机制已被广泛研究。为了在低温下生长，耐冷生物体必须调整其脂肪酸组成以维持膜的流动性。在正常条件下，几乎全部（超过 90%）单核细胞增生李斯特氏菌脂肪酸都是支链脂肪酸，而其他革兰氏阳性菌通常含有约 40% 的直链脂肪酸。脂肪酸反异 C15:0 通过脂肪酸缩短和从异脂肪酸到反异脂肪酸的分支转换相结合，增加了低温生长的细菌的数量，并在增加膜流动性方面发挥了关键作用。对支链脂肪酸缺陷突变体的研究表明，它们的低温生长、对各种胁迫的耐受性和毒力显着受损。这表明李斯特菌膜的物理结构和最佳功能依赖于高含量的支链脂肪酸，而支链脂肪酸的缺乏会对生物体的生理和毒力产生重大影响。突变体和前体喂养研究表明，除了来自支链氨基酸的正常途径外，还存在一种来自短直链和支链羧酸前体的新型但未定义的脂肪酸生物合成途径。我们建议研究支链脂肪酸转换的机制，定义从羧酸生物合成脂肪酸的途径，并以极端的方式控制膜脂肪酸的组成，从而控制流动性，并检查其对生物体生理学和发病机制的影响。脂肪酸支链转换的主要决定因素似乎在于β-酮酰基载体蛋白合酶III (FabH)的温度响应催化特性，该酶在脂肪助剂生物合成途径中进行第一个缩合反应。在具体目标 I 中，我们将结合诱变、功能和计算机模拟、蛋白质生物化学和动力学分析来探讨 FabH 底物特异性的温度依赖性变化的机制。在支链脂肪酸缺陷突变体和野生型生物体中，短链和支链羧酸绕过正常的脂肪酸生物合成途径。在具体目标 2 中，我们将克隆、过表达 buk（丁酸激酶）和 ptb（磷酸转丁酰酶）编码的蛋白质，并尝试证明它们构成了生产羧酸脂肪酸前体的 CoA 衍生物的新途径。单核细胞增生李斯特氏菌具有使用支链C6羧酸作为“非天然”偶数支链脂肪酸前体的能力。在具体目标 3 中，我们将使用前体喂养实验，通过操纵脂肪酸组成来产生低、正常和高流动性膜的细菌。这些细胞将用于研究膜物理结构和流动性对低温耐受性、细胞生理学各个方面和细菌发病机制的影响。这些目标的实现将增加我们对低温生长以及膜结构对生理和发病机制影响的理解。希望这项工作能够产生在低温下控制李斯特菌生长的新方法。公共卫生相关性：单增李斯特菌是一种食源性病原体，死亡率很高，其在冷藏温度下在食品中生长的能力是其中的一个关键因素。该提案重点关注支链脂肪酸在膜物理结构、耐冷性、生理学和致病性中的作用。这项工作的结果预计将开发出控制生物体生长的新方法。