Linking apparatus dynamics to interbacterial intoxication by type VI secretion

描述(由申请人提供)： 细菌进化出了相互交流和竞争的复杂途径。这种机制最终决定了微生物群落的组成，这在许多环境和医学相关的背景下都很重要。VI型分泌系统(T6SS)是一种新兴的研究细菌相互作用的模型系统。我们最近证明，T6SS有助于将蛋白质效应物输送到其他革兰氏阴性细菌。该系统在数百个已测序的蛋白质细菌基因组中发现，其活性可以对目标细胞群产生重大影响。尽管T6SS经常被描述为一种类似噬菌体的途径，它“穿透”受体细胞并注入毒素，但一个正常工作的T6S装置及其对受体细胞的影响尚未被直接观察到。这项拟议的合作项目寻求使用定量时间推移荧光显微镜来直接可视化和定量表征基于T6S的细菌中毒的作用和效力，并以单细胞分辨率进行。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。我们的方法学将使我们能够确定靶向后果的光谱，并测量接触依赖型杀伤率，这是细胞适合性的关键决定因素，无法在传统的液体竞争平板分析中直接测量。该提案的第二个目的是通过定义靶细胞中毒与所定义的T6S组分的局部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别，我们将破译T6S装置的靶向机制(随机和定向)，并识别效应器传递的事件诊断。重要的是，这项提议支持的研究小组汇集了成功完成这项工作所需的跨学科和互补性专门知识。该提案包括强有力的初步数据，证明了利用单细胞分辨率对细菌相互作用进行量化的创新战略的可行性和概念验证。我们预计这项工作将对细菌分泌产生重要的见解，并开发出一个分析框架，该框架将普遍适用于细菌细胞相互作用的定量分析。

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.