Formation and function of cell curvature in Vibrio cholerae

霍乱弧菌细胞曲率的形成和功能

• 批准号: 10443303
• 负责人: Zemer Gitai
• 金额: $ 32.46万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-07 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10443303
• 关键词: Address; Affect; Agar; Bacteria; Bacterial Proteins; Behavior; Behavioral; Binding; Biochemical; Biological Models; Biophysics; Caulobacter crescentus; Cell Shape; Cell Wall; Cell physiology; Cells; Cellular biology; Characteristics; Cryoelectron Microscopy; Cytoplasm; Cytoskeleton; Defect; Distant; Electron Microscopy; Elements; Energy-Generating Resources; Engineering; Environment; Enzymes; Escherichia coli; Face; Fluorescence Microscopy; Gel; Geometry; Growth; Guanosine Triphosphate; Higher Order Chromatin Structure; Impairment; In Vitro; Intestines; Knowledge; Learning; Mediating; Microbial Biofilms; Microbiology; Molecular; Morphogenesis; Mucous Membrane; Mucous body substance; Mus; Mutagenesis; Nature; Oxides; Pathogenesis; Pattern; Periplasmic Proteins; Polymers; Process; Proteins; Pseudomonas aeruginosa; Reporting; Rhizobium radiobacter; Rod; Role; Shapes; Structure; Surface; System; Total Internal Reflection Fluorescent; Vibrio; Vibrio cholerae; Work; base; cell behavior; cell motility; cellular imaging; copolymer; experimental study; follow-up; human pathogen; in vivo; insight; molecular scale; mutant; pathogen; periplasm; polymerization; screening; tool

Curved bacteria represent one of the most common of bacterial shapes yet the mechanisms by which bacteria become curved and the functions of curvature are largely unknown. In addition, large-scale polymers assemble in both the cytoplasm and periplasm yet only the cytoplasmic polymers have been well characterized to date. This is an important distinction, as periplasmic proteins must contend with different molecular environments like oxidizing conditions and a lack of ATP/GTP energy sources. My lab recently demonstrated that two proteins, CrvA and CrvB, assemble into periplasmic structures that are essential for establishing cell curvature in the pathogen, Vibrio cholerae. Here we propose to leverage this system to address the field’s gaps in understanding both curved shapes and periplasmic polymer formation. In previous work, we identified and characterized the first curvature determinant in V. cholerae, CrvA. CrvA forms polymers in the periplasm that pattern the insertion of new cell wall to cause these bacteria to curve. More recently we discovered a second curvature determinant, CrvB. CrvA and CrvB colocalize in the V. cholerae periplasm, and CrvA and CrvB co-expression are sufficient to induce curvature in straight Vibrio species, E. coli, P. aeruginosa, and even distantly-related C. crescentus and A. tumefaciens. Here we seek to better understand bacterial curvature determination and function by answering three outstanding questions in three aims. 1) How do CrvA/B assemble in the periplasm, whose oxidizing conditions and lack of ATP/GTP make it a very different environment from the cytoplasm where well-characterized cytoskeletons assemble? Aim 1 will answer this question by combining electron and fluorescence microscopy to determine the structure and dynamics of CrvA/CrvB assembly. 2) How do CrvAB actually generate cell curvature? CrvAB are distinct from previously-characterized shape determinants in both being periplasmic and functioning autonomously of other shape-patterning elements like MreB. Aim 2 will thus address how CrvA and CrvB induce curvature by identifying and characterizing their interactions with the cell wall and other partners. 3) How does curvature affect bacterial behaviors? Aim 3 will harness our ability to synthetically curve bacteria and use single-cell imaging to determine how curvature affects motility in gels, biofilms, and on surfaces. These experiments will dissect the biophysical basis of important behaviors that result from cell curvature. Together these studies will establish V. cholerae CrvAB as a powerful model system for studying cell shape across multiple scales, answering fundamental questions to advance several fields. First, at the molecular scale, we will learn how polymers form in the periplasm. Second, at the cellular scale, we will learn how two proteins can curve an immense range of bacteria. And third, at the behavioral/evolutionary scale, we will learn the benefits conferred by one of the most common of bacterial shapes.

弯曲的细菌代表了最常见的细菌形状之一，但其机制 细菌变得弯曲，而弯曲的功能在很大程度上是未知的。此外，大尺寸聚合物 在细胞质和周质中组装，但只有细胞质聚合物得到了很好的表征 迄今为止。这是一个重要的区别，因为周质蛋白必须与不同的分子竞争 氧化条件和缺乏 ATP/GTP 能源等环境。我的实验室最近展示了 两种蛋白质 CrvA 和 CrvB 组装成对于建立细胞至关重要的周质结构 病原体霍乱弧菌的弯曲。在这里，我们建议利用该系统来解决该领域的问题 在理解弯曲形状和周质聚合物形成方面存在差距。 在之前的工作中，我们识别并表征了霍乱弧菌的第一个曲率决定因素 CrvA。 CrvA 在周质中形成聚合物，形成新细胞壁的插入模式，从而导致这些细菌 曲线。最近我们发现了第二个曲率行列式，CrvB。 CrvA 和 CrvB 共定位于 V。 霍乱周质以及 CrvA 和 CrvB 共表达足以诱导直弧菌的弯曲 种，大肠杆菌，铜绿假单胞菌，甚至远亲新月杆菌和根癌农杆菌。 在这里，我们试图通过回答三个问题来更好地理解细菌曲率的确定和功能 三个目标中突出的问题。 1) CrvA/B如何在周质中组装，其氧化条件 ATP/GTP 的缺乏使其成为与细胞质截然不同的环境，细胞质中的环境已被充分表征 细胞骨架组装？目标 1 将通过结合电子显微镜和荧光显微镜来回答这个问题 确定 CrvA/CrvB 组装的结构和动力学。 2）CrvAB实际上是如何产生细胞的 曲率？ CrvAB 与先前表征的形状决定因素不同，因为它是周质和 独立于其他形状图案元素（例如 MreB）运行。因此，目标 2 将解决 CrvA 和 CrvB 通过识别和表征它们与细胞壁和其他伙伴的相互作用来诱导弯曲。 3）曲率如何影响细菌行为？目标 3 将利用我们综合弯曲细菌的能力 并使用单细胞成像来确定曲率如何影响凝胶、生物膜和表面的运动性。 这些实验将剖析细胞弯曲引起的重要行为的生物物理基础。 这些研究将共同​​建立霍乱弧菌 CrvAB 作为研究细胞的强大模型系统 跨多个尺度塑造，回答基本问题以推进多个领域。首先，在 分子尺度上，我们将了解聚合物如何在周质中形成。其次，在细胞尺度上，我们将学习 两种蛋白质如何控制大量细菌。第三，在行为/进化尺度上，我们 将了解最常见的细菌形状之一所带来的好处。