A Comprehensive approach to bacterial osmotolerance

细菌渗透耐受的综合方法

• 批准号: 10163120
• 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/10163120
• 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; experimental study; fitness; in vivo; light scattering; medical schools; metabolomics; microorganism; mutant; opportunistic pathogen; patch clamp; 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.

项目概要 通过土壤和淡水传播的肠道细菌和大多数机会性病原体表现出异常的特征 对各种环境的适应性。它们的部分适应潜力是能够承受剧烈的渗透压 变化。当外部介质突然稀释时，例如在雨中，细菌会逃避机械破裂 通过接合充当渗透剂释放阀的张力激活通道。低门槛的MSCS和高门槛的 阈值MscL是介导大肠杆菌中大量渗透剂交换的两种通道物种，已被 对其结构和门控机制进行了广泛的研究。然而，尽管生物物理学取得了进展 对这些单独的机械敏感通道的研究，人们对实际的释放过程知之甚少。 当渗透压突然下降时，细胞内就会发生这种情况。几乎没有关于肿胀程度和速率的数据， 渗透剂释放的动力学、通过特定通道逸出的分子、何时以及如何逸出 瞬时渗透性停止，最后，所有这些参数如何与渗透适应性相关。我们目前的 对机制的分析强烈表明两个关键方面：（i）通道必须快速释放渗透剂 足以超过渗透水的流入并抑制细胞肿胀；另一方面 (ii) 大量渗透剂 必须通过低阈值通道的失活来牢固终止耗散，以促进恢复。这 拟议项目旨在建立一个基于救援过程的自洽动力学/物理模型 对大肠杆菌活培养物中渗透诱导的溶质交换的全面现象学描述。 大肠杆菌、细胞包膜力学以及通道门控的空间和热力学特性。 (1) 我们将 确定渗透过程中通过特定通道离开细胞的主要渗透剂的身份和可用性 使用现代代谢组学进行休克。我们将研究主要渗透性和不可渗透性渗透剂的影响 关于 MscS 和 MscL 门控并可视化可能影响 MD 状态分布的渗透和相互作用 模拟。 （二）解决MSCS开放机制遗留的若干问题 失活后，我们将确定具有稳定静息和开放状态的突变体的晶体结构。这 然后，状态之间的过渡路径将在模拟中重建。 (3) 我们将聘用 停流技术记录活体培养物中光散射的动力学并评估细胞的渗透性 细胞膜对水和渗透剂的作用；我们将把汇率与渗透细胞活力联系起来。使用 流体学和视频显微镜我们将确定细胞壁的弹性和膜的数量 可拉伸肽聚糖内的储库。并行电生理分析将提供通道 门控和灭活的密度和参数。两人非一致行动的详细情况 渗透率响应过程中的冗余通道和一组“重要”参数将提供 定量模型的基础，该模型可以预测特定的渗透程度和速度是否 降档将是可以容忍的或致命的。