Branched-chain fatty acids and membrane function in Listeria monocytogenes

性状（由申请方提供）：单核细胞增生李斯特菌是一种食源性细菌病原体，能够在冷藏温度下生长，因此是一种耐冷微生物。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，耐冷生物必须调整其脂肪酸组成以保持膜流动性。在正常情况下，几乎所有的L.单核细胞增生性细菌的脂肪酸是支链脂肪酸，与通常具有约40%直链脂肪酸的其它革兰氏阳性细菌相反。反异构脂肪酸C15：0在低温生长的细菌中通过脂肪酸缩短和从异脂肪酸到反异构脂肪酸的分支转换的组合而增加，并且在增加膜流动性中起关键作用。对支链脂肪酸缺陷突变体的研究表明，它们在低温生长、各种胁迫耐受性和毒性方面受到显著损害。这表明李斯特菌膜的物理结构和最佳功能取决于高含量的支链脂肪酸，这些脂肪酸的缺乏对生物体的生理和毒力有重大影响。突变体和前体喂养的研究已经揭示了存在一种新的，但不确定的脂肪酸生物合成途径，从短直链和支链羧酸前体，除了正常的途径从支链氨基酸。我们建议研究支链脂肪酸开关的机制，定义从羧酸的脂肪酸生物合成的途径，并操纵膜脂肪酸的组成，因此流动性，在一个极端的方式，并检查其对生物体的生理和发病机制的影响。脂肪酸分支转换的主要决定因素似乎在于在脂肪酸生物合成途径中进行第一缩合反应的酶ω-酮酰基载体蛋白合酶III（FabH）的温度响应性催化性质。在具体的目标我，我们将结合联合收割机诱变，功能和在silico建模，蛋白质生物化学和动力学分析，以探测的机制，温度依赖性变化的底物特异性的FabH。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、过表达由布克（丁酸激酶）和ptb（磷酸转丁酰酶）编码的蛋白质，并试图表明它们构成了生产羧酸脂肪酸前体的CoA衍生物的新途径。L.单核细胞增多症具有使用支链C6羧酸作为“非天然”偶数支链脂肪酸的前体的能力。在具体目标3中，我们将使用前体补料实验通过操纵其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的物理结构和流动性对低温耐受性的影响，细胞生理学的各个方面，以及细菌的发病机制。这些目标的实现将增加我们对低温下生长以及膜结构对生理和发病机制的影响的理解。人们希望这项工作能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：单核细胞增生李斯特菌是一种具有高致死率的食源性病原体，其在冷藏温度下在食品中生长的能力是一个关键因素。该提案的重点是支链脂肪酸在膜的物理结构，耐冷性，生理学和致病性中的作用。作为这项工作的结果，预计将开发出控制生物体生长的新方法。

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.