A Comprehensive approach to bacterial osmotolerance

细菌渗透耐受的综合方法

• 批准号: 9925727
• 负责人: SERGEI I SUKHAREV
• 金额: $ 42.03万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-11 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9925727
• 关键词: Adaptive Behaviors; Address; Affect; Area; Bacteria; Bacterial Model; Biophysics; Cell Survival; Cell Volumes; Cell Wall; Cell membrane; Cell model; Cells; Computer Models; Coupled; Crowding; Crystallization; Cytoplasm; Data; Detergents; Disulfides; Elasticity; Electrophysiology (science); Ensure; Enterobacteriaceae; Environment; Escherichia coli; Fresh Water; Geometry; Individual; Kinetics; Link; Liquid substance; Mechanics; Mediating; Membrane; Modeling; Modernization; Molecular; Molecular Conformation; Mutation; Optics; Osmolar Concentration; Osmotic Shocks; Pathway interactions; Peptidoglycan; Permeability; Pharmacy Schools; Phase; Population; Preparation; Process; Property; Rain; Recovery; Refractive Indices; Relaxation; Rest; Role; Rupture; Shock; Soil; Speed; Stretching; Structure; Swelling; Techniques; Testing; Thermodynamics; Time; Video Microscopy; Water; Weight; Work; base; biophysical analysis; cell envelope; commensal bacteria; crosslink; density; design; enteric pathogen; experimental study; fitness; in vivo; light scattering; medical schools; metabolomics; microorganism; mutant; patch clamp; pathogen; pathogenic bacteria; physical model; response; simulation; solute; stop flow technique

Project Summary Enteric bacteria and most opportunistic pathogens transmitted through soil and fresh water show exceptional adaptability to a range of environments. Part of their adaptive potential is the ability to survive drastic osmolarity changes. Upon a sudden dilution of external medium, such as in the rain, bacteria evade mechanical rupture by engaging tension-activated channels that act as osmolyte release valves. The low-threshold MscS and high- threshold MscL, the two channel species that mediate the bulk of osmolyte exchange in E. coli, have been extensively studied in terms of their structure and gating mechanisms. Yet, despite the progress in biophysical studies of these individual mechanosensitive channels, little is known about the actual release process that takes place in the cell upon abrupt osmotic downshift. There is almost no data on the extent and rate of swelling, the kinetics of osmolyte release, the molecules that escape through specific channels, when and how the transient permeability ceases, and finally, how all these parameters are linked to osmotic fitness. Our current analysis of the mechanism strongly suggests two key aspects: (i) the channels must release osmolytes fast enough to outpace the osmotic water influx and curb cell swelling; on the other hand (ii) the massive osmolyte dissipation must be firmly terminated by inactivation of the low-threshold channel to facilitate recovery. The proposed project aims at a self-consistent kinetic/physical model of the rescuing process based on a comprehensive phenomenological description of osmotically-induced solute exchange in live cultures of E. coli, cell envelope mechanics, and on spatial and thermodynamic properties of channel gating. (1) We will determine identities and availabilities of major osmolytes leaving cells through specific channels during osmotic shock using modern metabolomics. We will study the effects of major permeable and impermeable osmolytes on MscS and MscL gating and visualize permeation and interactions which may affect state distributions in MD simulations. (2) To address several remaining questions about the mechanism of MscS opening and inactivation, we will determine crystal structures of mutants with stabilized resting and open states. The transition pathways between the states will then be reconstructed in simulations. (3) We will employ the stopped-flow technique to record the kinetics of light scattering in live cultures and assess permeabilities of the cell envelope to water and osmolytes; we will correlate the exchange rates with osmotic cell viability. Using fluidics and videomicroscopy we will to determine the elasticity of the cell wall and the amount of membrane reservoir inside the stretchable peptidoglycan. Parallel electrophysiological analysis will provide channel densities and parameters for gating and inactivation. The detailed picture of the concerted action of two non- redundant channels in the course of osmotic permeability response and a set of ‘vital’ parameters will provide grounds for a quantitative model which would predict whether a particular magnitude and speed of osmotic downshift will be tolerable or lethal.

项目摘要 通过土壤和淡水传播的大多数肠细菌和大多数机会性病原体表现出异常 适应各种环境。它们的自适应潜力的一部分是能够生存巨大的渗透压 更改。突然稀释外部培养基，例如在雨中，细菌逃避机械破裂 通过参与充当渗透释放阀的张力激活通道。低阈值MSC和高级 阈值MSCL是介导大肠杆菌中大部分Osmolyte交换的两个通道物种 从其结构和门控机制方面进行了广泛的研究。然而，阐明生物物理的进展 对这些单独的机械敏感渠道的研究，对实际释放过程知之甚少 发生在突然的渗透降档时发生在牢房中。几乎没有关于肿胀的程度和速率的数据 渗透剂释放的动力学，通过特定通道逃脱的分子，何时何时以及如何 瞬态渗透性停止，最后，所有这些参数如何与渗透性适应性链接。我们的目前 该机制的分析强烈提出了两个关键方面：（i）通道必须快速释放渗透液 足以超过渗透水影响并遏制细胞肿胀；另一方面（ii）巨大的渗透液 必须首先通过低阈值渠道灭活以促进恢复来终止耗散。这 拟议的项目的目的是基于A的自洽动力学/物理模型 渗透诱导的可溶性交换的全面现象学描述。 大肠杆菌，细胞包络力学以及通道门控的空间和热力学特性。 （1）我们会的 确定主要渗透群的身份和可用性，使细胞通过渗透期间特定通道离开细胞 使用现代代谢组学冲击。我们将研究主要的渗透和不可渗透的渗透液的影响 在MSC和MSCL门控以及可视化可能影响MD中状态分布的渗透和相互作用上 模拟。 （2）解决有关MSC开放机制的剩余问题和 失活，我们将确定具有稳定静止状态和开放状态的突变体的晶体结构。 然后将在模拟中重建状态之间的过渡途径。 （3）我们将采用 停止流量技术以记录活文化中光散射的动力学和评估的渗透率 细胞包膜到水和渗透孔；我们将将汇率与渗透细胞活力相关联。使用 流体和视频显微镜我们将确定细胞壁的弹性和膜的量 在可拉伸的胶囊内部的储层。平行电生理分析将提供通道 通信和灭活的密度和参数。两个非 - 在渗透通透性响应过程中的冗余通道和一组“重要”参数将提供 定量模型的理由，该模型可以预测特定的渗透速度和速度是否 降档将是可以忍受的或致命的。