The role of the Type IV pili ATPase PilT as a surface sensor in regulating cell division

IV 型菌毛 ATP 酶 PilT 作为表面传感器在调节细胞分裂中的作用

• 批准号: 10308694
• 负责人: STEPHEN MELVILLE
• 金额: $ 18.67万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10308694
• 关键词: ATP phosphohydrolase; Actins; Adherence; Affinity; Animals; Bacillus subtilis; Bacteria; Binding; Biological Assay; Biological Models; Calorimetry; Cell Extracts; Cell division; Cell surface; Cells; Cellular Morphology; Chimeric Proteins; Clostridium perfringens; Co-Immunoprecipitations; Cryo-electron tomography; Disease; Family; Fiber; Flagella; Fluorescence; Gas Gangrene; Genes; Gram-Negative Bacteria; Gram-Positive Bacteria; In Vitro; Infection; Kinetics; Length; Link; Liquid substance; Location; Mediating; Methods; Modeling; Mus; Muscle Cells; Mutation; Myoblasts; Operon; Organelles; Pathogenesis; Phenotype; Physiologic pulse; Pilum; Play; Process; Proteins; Pseudomonas aeruginosa; Reporter; Role; Rotation; Sepharose; Signal Transduction; Solid; Surface; Surface Plasmon Resonance; System; Testing; Titrations; Transducers; Virulence; Virulence Factors; base; cell envelope; cell motility; cell type; crosslink; design; flexibility; in vivo; insight; member; mutant; novel; pathogen; prevent; protein protein interaction; sensor; yeast two hybrid system; z-ring

Type IV pili (TFP) play a major role in bacterial pathogenesis. Although they were originally discovered and described in Gram-negative bacteria, we discovered that nearly all Clostridia species and some other Gram- positive bacteria have TFP. We demonstrated that C. perfringens TFP are needed for bacterial adherence to mouse muscle cells (myoblasts), providing a model for their role in gas gangrene infections. For full virulence in animal hosts, bacteria need to sense the presence of a surface. Gram-negative bacteria can use TFP as surface sensing organelles, since TFP are extended from the bacterium and contact nearby cell surfaces. Surface sensing using TFP has not been studied in Gram-positive bacteria but we are using C. perfringens as a model system to determine how they do this. C. perfringens increases its cell length when grown on surfaces and we discovered that a mutation in the gene encoding the retraction ATPase, PilT, in C. perfringens lost this ability. pilT is the first gene in an operon with ftsA and ftsZ, suggesting a direct link between TFP and divisome functions. Our main hypothesis is that PilT functions as a signal transducer that communicates the presence of a surface to the components of the divisome (FtsA and FtsZ) to regulate the rate of cell division and, as consequence, cell length. PilT does this by partner switching between the PilM ring located at the base of the TFP assembly apparatus in liquid grown cells to the FtsA protein on surfaces. FtsA is important for formation of the Z-ring that is used to constrict the cell envelope during the division process and PilT binding may inactivate FtsA and inhibit cell division. We have designed two aims to test this hypothesis: Aim 1. Determine the relative affinities of the protein interactome of the retraction ATPase PilT in vitro. Purified PilT, PilM, FtsA and PilB2 will be used in co-immunoprecipitation, pull down, surface plasmon resonance, isothermal titration calorimetry and protein cross-linking assays to determine their relative binding affinity to PilT and each other. To identify other proteins that may interact with PilT, co-immunoprecipitation and pull down assays will be done in whole cell extracts. Aim 2: Define the in vivo partner switching kinetics for the PilT protein when grown in liquid media and then placed on a surface. Using a reversible split fluorescence reporter and our novel pulse-chase methods for tracking fluorescent proteins in C. perfringens, the location and protein-protein interactions of PilT will be determined when cells are grown in liquid and transferred to a solid surface. These studies will provide insights into how Gram-positive pathogens sense surfaces, which can be used to design strategies to block these sensing mechanisms and lower their virulence.

IV型Pili（TFP）在细菌发病机理中起主要作用。尽管它们最初是被发现的，并且 在革兰氏阴性细菌中描述的是，我们发现几乎所有的梭状芽胞杆菌和其他一些革兰氏种类 阳性细菌具有TFP。我们证明了细菌粘附需要的刺激性TFP需要 小鼠肌肉细胞（成肌细胞），为它们在气体坏疽感染中的作用提供了模型。为了充分的毒力 在动物宿主中，细菌需要感觉到表面的存在。革兰氏阴性细菌可以使用TFP作为 表面传感细胞器，因为TFP从细菌延伸并接触附近的细胞表面。 使用TFP的表面传感尚未在革兰氏阳性细菌中研究 一个模型系统来确定他们如何做到这一点。 C.刺激在表面上生长时会增加其细胞长度 我们发现编码回缩ATPase的基因中的突变，Pilt pilt，C。perfringens失去了 能力。 PILT是具有FTSA和FTSZ的操纵子中的第一个基因，这表明TFP与Divisome之间有直接的联系 功能。我们的主要假设是，矩形充当传达存在的信号传感器 Divisome（FTSA和FTSZ）成分的表面，以调节细胞分裂的速率，并 结果，细胞长度。 PILT通过合作伙伴在位于Pilm环之间切换来做到这一点 液体生长细胞中的TFP组装设备可在表面上ftsa蛋白。 FTSA对于形成很重要 用于在分裂过程中限制细胞包络的Z环和养殖结合可能会失活 FTSA并抑制细胞分裂。我们设计了两个目的来检验这一假设： AIM 1。确定在体外缩回ATPase PILT的蛋白质相互作用组的相对亲和力。 纯化的养殖，PILM，FTSA和PILB2将用于共免疫沉淀，下拉，表面等离子体 共振，等温滴定量热法和蛋白质交联测定，以确定其相对结合 彼此之间的亲和力。识别可能与养殖，共免疫沉淀相互作用的其他蛋白质 下拉测定将在全细胞提取物中进行。 目标2：定义体内伴侣在液体介质和 然后放在表面上。 使用可逆的分裂荧光记者和我们的新型脉搏追踪方法来跟踪荧光 在灌注梭菌中的蛋白质，将在细胞为时，将确定刺的位置和蛋白质 - 蛋白质相互作用 在液体中生长并转移到固体表面。 这些研究将提供有关革兰氏阳性病原体如何感知表面的见解，这些表面可用于 设计策略来阻止这些感应机制并降低其毒力。