Physiological roles of regulated changes in membrane phospholipid composition

说明(申请人提供)：膜的关键生物学功能取决于其磷脂组成。因此，细菌保持了几乎恒定的主要细胞膜甘油磷脂的比例。然而，环境信号可以引起阴离子和两性离子磷脂摩尔比的变化，这引发了头基组成的调节变化的生理作用的问题。这项拟议研究的具体目的是使用遗传和生化方法调查磷脂头基组成的调节变化是否影响涉及革兰氏阴性病原体肠沙门氏菌中与磷脂大分子相互作用的活动。目的1是设计具有可调节磷脂头基组成的沙门氏菌菌株。目的2是检测磷脂组成是否影响PhoQ的生化特性和传感能力，PhoQ是一种完整的膜传感器激酶，它利用配体介导的与阴离子磷脂的相互作用来检测信号，并对低胞质外镁离子、抗菌肽的存在和酸性pH做出反应。目的3是研究磷脂组成的调节变化是否介导了对Mainin 2的耐药性，Mainin 2是一种阳离子抗菌肽，可以破坏细胞膜以杀灭细菌。将采用独立和并行的方法来解决目标2和目标3：目标1中构建的菌株将用于体内实验，重组生化脂质体系统将用于体外实验。这些拟议实验的结果将表明，磷脂头基组成的调节变化是否调节了完整的膜传感器激酶的活性，或调节了对阳离子抗菌肽的耐药性。这些目标的实现将使对人类病原体磷脂头基组成变化的遗传学研究成为可能，并将阐明一种重要细胞成分的新的生理功能。 与公共卫生相关：生物膜在细胞周围形成屏障，包围隔间，并含有对生命具有必要功能的分子。这项建议旨在了解引起人类胃肠炎和伤寒的致病细菌肠道沙门氏菌内膜成分的变化是否会影响沙门氏菌适应新环境和抵抗抗菌肽杀死的能力。这项拟议研究的结果将提供对调节的细胞变化的理解，这些变化使沙门氏菌能够在不同的地点繁殖并导致疾病。

DESCRIPTION (provided by applicant): The critical biological functions of membranes depend on their phospholipid composition. Consequently, bacteria maintain a nearly constant ratio of major membrane glycerophospholipids. Nevertheless, environmental signals can induce changes in the molar ratio of anionic to zwitterionic phospholipids, raising questions about the physiological roles of regulated changes in headgroup composition. The specific aims of this proposed research investigate whether regulated changes in phospholipid headgroup composition affect activities that involve macromolecular interactions with phospholipids in the Gram-negative pathogen Salmonella enterica using genetic and biochemical approaches. Aim 1 is to engineer Salmonella strains with adjustable phospholipid headgroup compositions. Aim 2 is to examine whether phospholipid composition affects the biochemical properties and sensing capabilities of PhoQ, an integral membrane sensor kinase that utilizes ligand-mediated interactions with anionic phospholipids to detect signals and to respond to low extracytoplasmic Mg2+, the presence of antimicrobial peptides, and acidic pH. Aim 3 is to investigate whether regulated changes in phospholipid composition mediate resistance to magainin 2, a cationic antimicrobial peptide that disrupts membranes to kill bacteria. Independent and parallel approaches will be adopted to address Aims 2 and 3: strains constructed in Aim 1 will be utilized for in vivo experiments, and reconstituted biochemical liposome systems will be utilized for in vitro experiments. Results from these proposed experiments will indicate whether regulated changes in phospholipid headgroup composition modulate the activities of an integral membrane sensor kinase or mediate resistance to a cationic antimicrobial peptide. Accomplishment of these aims will enable genetic investigations of alterations in phospholipid headgroup composition in a human pathogen and will illuminate novel physiological functions for an essential cellular component. PUBLIC HEALTH RELEVANCE: Biological membranes form barriers around cells, enclose compartments, and contain molecules that have essential functions for life. This proposal aims to understand whether changes in the composition of the inner membrane of the pathogenic bacterium Salmonella enterica, which causes gastroenteritis and typhoid fever in humans, affect the ability of Salmonella to adapt to new environments and to resist killing by antimicrobial peptides. Results from this proposed research will provide an understanding of the regulated cellular changes that enable Salmonella to prosper in different locales and to cause disease.