The electrophysiology of facultative pathogens

描述(申请人提供)：许多疾病的传播取决于病原体的环境稳定性。虽然在微生物细胞内相容的渗透物质的积累是众所周知的，但几乎没有关于渗透物质外排系统赋予对突然的环境变化的抵抗力的信息。目前还缺乏实用的方法来监测与亲脂药物渗透相关的膜扰动，这限制了我们对渗透机制的了解，阻碍了新药开发的进程。生物物理学和电生理学的方法有助于我们破译渗透调节的膜机制，并检测亲脂性物质的界面效应。完整的细菌太小，不适合电生理学；然而，巨大的球体制剂提供了直接进入细菌细胞质膜的途径。这一过程包括抗生素诱导丝状形态，然后细胞壁消化，这是为大肠杆菌开发的，并导致鉴定出几个机械敏感(MS)通道，在渗透压下激波期间介导膨压调节。小电导和大电导的机械敏感通道MSCs和MSCL占主导地位，并直接受膜张力门控。这些通道对亲脂性物质插入周围双分子层所引起的侧向压力变化也很敏感。有了大量的关于大肠杆菌通道的信息，在这个探索性的项目中，我们建议将这个电生理平台扩展到两种常见的兼性病原体，霍乱弧菌和铜绿假单胞菌，目的是更好地了解它们的膜的机电反应，渗透物质的运输和亲脂药物的渗透。这两种兼性病原体的基因组都含有几个E.ColiMS通道的同源基因。我们将优化巨球形体的制备程序，并对这两种病原体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，通道的克隆，同源和异源(E.Coli)表达，通道电导的测定，离子和主要兼容渗透压的张力依赖性和渗透性的测定。它还将包括对天然膜和大肠杆菌中的机电特性的比较。这项拟议的研究将为我们提供关于这些病原体渗透反应机制的第一个分子信息，这是一个关键的环境防御。此外，了解MS通道作为侧压传感器的作用将为优化生物活性化合物(如抗生素、自身诱导剂及其合成类似物)提供机会，通过促进其细胞膜的渗透来靶向特定的病原体。

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.