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。虽然相容性渗透液在微生物细胞内的积累已被很好地理解，但实际上没有关于渗透液外排系统对环境突变的抵抗力的信息。也缺乏监测与亲脂药物渗透相关的膜扰动的实用方法，这限制了我们对渗透机制的理解，阻碍了新药开发的进程。生物物理和电生理方法有助于我们破译涉及渗透调节的膜机制，并检测渗透亲脂物质的界面效应。完整的细菌对于电生理来说太小了；然而，巨球质体的制备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式，然后是细胞壁消化，是为大肠杆菌开发的，并导致了几个机械敏感（MS）通道的鉴定，这些通道在渗透下行冲击期间介导膨胀压力调节。小电导和大电导的机械敏感通道，MscS和MscL，是主导和直接由膜张力门控的。这些通道对由亲脂物质插入周围双分子层引起的侧压变化也很敏感。有了大量关于大肠杆菌通道的信息，在这个探索性项目中，我们提出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响应，渗透物运输和亲脂性药物渗透。这两种兼性病原体的基因组都含有几个大肠杆菌质谱通道的同源物。我们将优化巨球质体的制备工艺，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、克隆、通道的同源和异源（大肠杆菌）表达、它们的电导率、张力依赖性和离子和主要相容渗透物的渗透性的测定。它还将包括对天然膜和大肠杆菌的机电特性的比较。该研究将为我们提供关于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，了解质谱通道如何作为侧压传感器发挥作用，将为优化生物活性化合物（如抗生素、自诱导剂及其合成类似物）提供机会，使其通过细胞质膜渗透以靶向特定病原体。

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.