The electrophysiology of facultative pathogens

描述（由申请人提供）：许多疾病的传播取决于病原体的环境稳定性。虽然微生物细胞内相容的渗透剂的积累是众所周知的，但实际上没有关于渗透剂外排系统赋予对突然的环境变化的抗性的信息。也有一个缺乏实用的方法来监测膜扰动与亲脂性药物的渗透，这限制了我们的了解渗透机制，并阻碍了新的药物开发的过程。生物物理学和电生理学的方法至关重要地帮助我们破译膜的机制，参与调节和检测渗透亲脂性物质的界面效应。完整的细菌对于电生理学来说太小;然而，巨大的原生质球制备提供了直接进入细菌细胞质膜的途径。这种方法，包括用抗生素诱导丝状体，然后消化细胞壁，是为大肠杆菌开发的。大肠杆菌，并导致几个机械敏感（MS）通道介导的膨压调节渗透压下降休克。小电导和大电导的机械敏感性通道MscS和MscL占主导地位，并直接由膜张力门控。这些通道也对由亲脂性物质插入到周围双层中引起的侧向压力变化敏感。掌握了大量关于E.大肠杆菌通道，在这个探索性的项目中，我们提出了两个常见的兼性病原体，霍乱弧菌和铜绿假单胞菌的电生理平台的扩展，目的是更好地了解他们的膜，渗透液运输和亲脂性药物渗透的机电响应。两种兼性病原体的基因组都含有几种E. coliMS通道。我们将优化巨原生质球的制备过程，并对这两种病原体中的机械敏感通道进行详细的研究。这将包括在不同压力刺激下的原位电生理表征、克隆、同源和异源（E.大肠杆菌）表达的通道，测定其电导，张力依赖性和渗透性的离子和主要兼容的渗透剂。它还将包括在天然膜和在E的机械-电气特性的比较。杆菌这项研究将为我们提供有关这些病原体渗透渗透反应机制的第一个分子信息，这是一种关键的环境防御。此外，了解MS通道如何作为侧压力传感器发挥作用将为优化生物活性化合物（如抗生素，自诱导物及其合成类似物）提供机会，以通过促进渗透通过其细胞质膜来靶向特定病原体。

DESCRIPTION (provided by applicant): The spread of many diseases depends on the environmental stability of pathogens. While the accumulation of compatible osmolytes inside microbial cells is well understood, there is practically no information about osmolyte efflux systems imparting resistance to abrupt environmental changes. There is also a lack of practical approaches for monitoring membrane perturbations associated with the permeation of lipophilic drugs, which limits our understanding of permeation mechanisms and hampers the process of new drug development. Biophysical and electrophysiological approaches critically help us to decipher membrane mechanisms involved in osmoregulation and to detect interfacial effects of permeant lipophilic substances. Intact bacteria are too small for electrophysiology; however, giant spheroplast preparation provides direct access to the bacterial cytoplasmic membrane. This procedure, involving the induction of filamentous forms by antibiotics followed by cell wall digestion, was developed for E. coli and led to the identification of several mechanosensitive (MS) channels mediating turgor pressure adjustment during osmotic downshock. The mechanosensitive channels of small and large conductance, MscS and MscL, are dominant and gated directly by membrane tension. These channels are also sensitive to lateral pressure changes induced by insertion of lipophilic substances into the surrounding bilayer. Having a substantial amount of information about E. coli channels, in this exploratory project we propose extension of this electrophysiological platform to two common facultative pathogens, Vibrio cholerae and Pseudomonas aeruginosa with the aim of better understanding the mechanoelectrical responses of their membranes, osmolyte transport and lipophilic drug permeation. The genomes of both facultative pathogens contain orthologs of several E. coli MS channels. We will optimize the procedure of giant spheroplast preparation and conduct a detailed study of the mechanosensitive channels in both pathogens. This will include in situ electrophysiological characterization under different pressure stimuli, cloning, homologous and heterologous (E. coli) expression of the channels, determination of their conductances, tension dependences and permeabilities for ions and major compatible osmolytes. It will also include a comparison of the mechano-electrical characteristics in the native membranes and in E. coli. The proposed study will provide us with the first molecular information about the mechanism of an osmotic permeability response in these pathogens, which is a critical environmental defense. Furthermore, understanding how the MS channels function as lateral pressure sensors will open up opportunities for optimizing biologically active compounds such as antibiotics, autoinducers and their synthetic analogs to target specific pathogens by favoring permeation through their cytoplasmic membranes.