Linking apparatus dynamics to interbacterial intoxication by type VI secretion

描述（由申请人提供）： 细菌进化出了复杂的途径来相互交流和竞争。这些机制最终决定了在丰富的环境和医学相关背景下重要的微生物群落的组成。VI型分泌系统（T6SS）是研究细菌相互作用的一个新兴模型系统。我们最近证明了T6SS有助于将蛋白质效应物递送至其他革兰氏阴性细菌。该系统存在于数百个已测序的变形菌基因组中，其活性可对靶细胞群体产生显著影响。虽然T6SS通常被描述为“刺穿”受体细胞并注入毒素的噬菌体样途径，但功能性T6S装置及其对受体细胞的影响尚未被直接观察到。该拟议的合作项目旨在使用定量延时荧光显微镜直接可视化和定量表征基于T6S的细菌中毒的作用和效力，具有单细胞分辨率。在第一个目标中，我们将利用单细胞分析来鉴定和定量描述T6S对受体细菌的影响。我们的方法将使我们能够确定靶向结果的谱并测量接触依赖性杀伤率，这是细胞适应性的关键决定因素，不能在传统的液体竞争测定板中直接测量。该提案的第二个目的是通过定义靶细胞中毒与定义的T6S组分的定位和动态行为之间的因果关系来建立T6S装置的细胞靶向机制。通过研究与T6S亚结构相关的蛋白质类，我们将破译T6S装置的靶向机制（随机与定向），并确定诊断效应传递的事件。至关重要的是，该提案支持的研究小组汇集了成功完成工作所需的跨学科和互补专业知识。该提案包括强有力的初步数据，证明了用单细胞分辨率定量细菌相互作用的创新策略的可行性和概念验证。我们期望这项工作既能产生对细菌分泌的重要见解，又能开发出一种普遍适用于细菌细胞相互作用定量分析的分析框架。

DESCRIPTION (provided by applicant): Bacteria have evolved complex pathways for communicating and competing with each other. Such mechanisms ultimately dictate the composition of microbial communities important in a wealth of environmentally and medically relevant contexts. The type VI secretion system (T6SS) is an emerging model system for the study of bacterial interactions. We recently demonstrated that the T6SS facilitates delivery of protein effectors to other Gram-negative bacteria. The system is found in hundreds of sequenced proteobacterial genomes and its activity can have dramatic consequence on target cell populations. Though the T6SS is often described as a bacteriophage-like pathway that "punctures" recipient cells and injects toxins, a functioning T6S apparatus and the effects it exerts on recipient cells have yet to be directly observed. This proposed collaborative project seeks to use quantitative time-lapse fluorescence microscopy to directly visualize and quantitatively characterize the action and potency of T6S-based bacterial intoxication with single- cell resolution. In the first aim, we will utilize single cell analyses t identify and quantitatively describe the effects of T6S on recipient bacteria. Our methodology will allow us to determine the spectrum of targeting consequences and measure the contact-dependent killing rate, which is a key determinant of cell fitness, cannot be directly measured in traditional plate of liquid competition assays. The second aim of the proposal is to establish the cellular targeting mechanism of the T6S apparatus by defining a causal relationship between target cell intoxication and the localization and dynamic behavior of defined T6S components. By studying classes of proteins associated with T6S substructures, we will decipher the targeting mechanism of the T6S apparatus (random versus directed) and identify events diagnostic of effector delivery. Critically, the research groups supported by this proposal bring together the interdisciplinary and complementary expertise needed to successfully complete the work. The proposal includes strong preliminary data demonstrating feasibility and proof-of-concept of an innovative strategy for the quantitation of bacterial interactions with single-cell resolution. We expect this work to both generate important insights into bacterial secretion as well as develop an analytical framework that will be generally applicable to the quantitative analysis of bacterial cellular interactions.