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.

弯曲细菌代表了最常见的细菌形状之一 细菌变得弯曲，曲率的功能在很大程度上未知。此外，大规模聚合物 在细胞质和周围组装中，但只有细胞质聚合物才得到很好的特征 迄今为止。这是一个重要的区别，因为外周蛋白必须包含不同的分子 诸如氧化条件和缺乏ATP/GTP能源之类的环境。我的实验室最近证明了 两种蛋白质CRVA和CRVB组装成周围结构，这些结构对于建立细胞至关重要 病原体中的曲率，弧菌霍乱。在这里，我们建议利用该系统来解决该领域的 理解弯曲形状和外围聚合物形成方面的差距。 在先前的工作中，我们确定并表征了CRVA霍乱弧菌中确定的第一条曲率。 CRVA在周围形成聚合物，使新细胞壁的插入使这些细菌插入 曲线。最近，我们发现了确定的第二个曲率CRVB。 CRVA和CRVB在V中进行共定位。 霍乱脑周期，CRVA和CRVB共表达足以诱导直弧菌中的曲率 物种，大肠杆菌，铜绿假单胞菌，甚至遥远相关的C. C. c.c。tumefaciens。 在这里，我们试图通过回答三个 三个目标中的出色问题。 1）CRVA/B如何在Pariplasm中组装，其氧化条件 缺乏ATP/GTP，使其与良好的细胞质完全不同 细胞骨骼组装？ AIM 1将通过组合电子和荧光显微镜来回答这个问题 确定CRVA/CRVB组件的结构和动力学。 2）CRVAB如何实际生成单元格 曲率？ CRVAB与前表征的确定剂不同，均为周质和 诸如MREB之类的其他形状图案元素自动起作用。 AIM 2因此将解决CRVA和 CRVB通过识别和表征其与细胞壁和其他伴侣的相互作用来诱导曲率。 3）曲率如何影响细菌行为？ AIM 3将利用我们合成细菌的能力 并使用单细胞成像来确定曲率如何影响凝胶，生物膜和表面上的运动。 这些实验将剖析细胞曲率引起的重要行为的生物物理基础。 这些研究将共同​​建立V. Cholerae CRVAB作为研究细胞的强大模型系统 跨多个尺度的形状，回答基本问题以提高几个领域。首先，在 分子尺度，我们将学习聚合物如何在周期中形成。第二，在细胞尺度上，我们将学习 两种蛋白如何弯曲巨大的细菌。第三，在行为/进化量表上，我们 将学习最常见的细菌形状之一赋予的好处。