Ready, Aim, Fire: understanding Sfa2-mediated control of the Type VI secretion system for interbacterial competition and invasion

准备、瞄准、开火：了解 Sfa2 介导的 VI 型分泌系统对细菌间竞争和入侵的控制

• 批准号: BB/Y00048X/1
• 负责人: Luke Allsopp
• 金额: $ 73.23万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FY00048X%2F1
• 关键词: Ready; Aim; Fire; understanding; Sfa2

Summary: One fundamental offensive strategy used by almost all bacteria is to secrete toxins to help them survive or kill competing organisms. Some of these bacterial secretion systems act like poisoned spears delivering toxic proteins directly into competing bacteria to kill them. This enables one bacterium to takeover a location. Bacteria can also use these poisoned spears to deliver toxins into animal and plant cells. This helps the bacteria to invade eukaryotic cells or manipulate them for bacterial gain.The type VI secretion system (T6SS) is a spring-loaded spear or harpoon for delivery of these toxic effector proteins and was discovered in 2006. It is found in 25% of Gram-negative bacteria. It is important for bacterial interactions in diverse environments from our intestines through to soil microbes. We currently do not know the signals that bacteria respond to for activation of these bacterial weapons.This project is focused on these bacterial secretion weapons in the organism Pseudomonas aeruginosa. P. aeruginosa is a widely found pathogen of humans, animals and plants. P. aeruginosa has three T6SSs and multiple smaller T6SS gene clusters spread throughout its genome. This proposal is centred on the most potent of these, the H2-T6SS that kills bacteria, helps P. aeruginosa enter eukaryotic cells and is deployed against host organisms. However, we have no understanding of the mechanisms that regulate when the H2-T6SS is switched 'on' or 'off'. This knowledge is important because we need to understand the signals for deployment of antimicrobial weapons to combat them.My laboratory has recently shown a critical role for two key regulatory proteins RpoN and Sfa2 on gene expression and function of the H2-T6SS. RpoN is a global regulator that controls 25% of the genes in P. aeruginosa. However, it cannot act alone for gene activation. It requires a sigma factor activator protein to help it. We have identified that Sfa2 is the one that helps RpoN to specifically activate the H2-T6SS genes leading to activation of this delivery system. Remarkably, it also allows RpoN to activate orphan T6SS genes throughout the genome that each encode a toxic effector. This allows this bacterial weapon to have more poisoned spear tips and a larger arsenal of toxic bacterial effector proteins that can be loaded and fired. As the effector proteins are the true mediators of T6SS functions its important to understand what they do. Additionally, we have identified new potential T6SS effectors that are controlled by Sfa2 that have not been characterised.We know that Sfa2 is essential for this system to get switched 'on' but we do not know what each of the parts or domains of Sfa2 do, what all the genes that Sfa2 controls do, and we do not know the signal that Sfa2 responds to activate this bacterial weapon.This project will provide fundamental knowledge on how Sfa2 functions, what it controls and what it responds to. Specifically, during this three-year New Investigator Award we will 1) define the role of key Sfa2 domains in functional assays; 2) characterise two newly identified Sfa2 regulated toxic effector encoding gene for their role in H2-T6SS interbacterial competition; 3) determine the signal for Sfa2 sigma factor activation through biochemical assays and validate its impact upon H2-T6SS phenotypes. These aims will deliver knowledge on the rules of life that enable bacteria to deploy their antimicrobial weapons linked with interbacterial competition, eukaryotic cell invasion and severity in host infection.Our work will reveal the signal bacteria use to deploy these weapons. This will lead the way to strategies to target the T6SS with drugs to disable them or ways to use the T6SS in synthetic biology applications in the future against pathogens as novel therapies to combat bacteria.

摘要：几乎所有细菌都使用的一种基本进攻策略是分泌毒素来帮助它们生存或杀死竞争生物。其中一些细菌分泌系统就像毒矛一样，将有毒蛋白质直接传递到竞争细菌中以杀死它们。这使得一种细菌能够占据一个位置。细菌还可以利用这些有毒的矛将毒素输送到动植物细胞中。这有助于细菌侵入真核细胞或操纵真核细胞以获得细菌。VI 型分泌系统 (T6SS) 是一种弹簧加载的矛或鱼叉，用于传递这些有毒效应蛋白，于 2006 年被发现。它存在于 25% 的细菌中。革兰氏阴性细菌。它对于从肠道到土壤微生物的不同环境中的细菌相互作用非常重要。我们目前还不知道细菌响应哪些信号来激活这些细菌武器。该项目的重点是铜绿假单胞菌中的这些细菌分泌武器。铜绿假单胞菌是人类、动物和植物中广泛发现的病原体。铜绿假单胞菌在其基因组中分布有三个 T6SS 和多个较小的 T6SS 基因簇。该提案的重点是其中最有效的 H2-T6SS，它可以杀死细菌，帮助铜绿假单胞菌进入真核细胞，并针对宿主生物体进行部署。然而，我们不了解调节 H2-T6SS 何时“打开”或“关闭”的机制。这些知识很重要，因为我们需要了解部署抗菌武器来对抗它们的信号。我的实验室最近证明了两种关键调节蛋白 RpoN 和 Sfa2 对 H2-T6SS 的基因表达和功能的关键作用。 RpoN 是一个全局调节因子，控制着铜绿假单胞菌 25% 的基因。然而，它不能单独作用于基因激活。它需要西格玛因子激活蛋白来帮助它。我们已经确定 Sfa2 可以帮助 RpoN 特异性激活 H2-T6SS 基因，从而激活该传递系统。值得注意的是，它还允许 RpoN 激活整个基因组中的孤儿 T6SS 基因，每个基因都编码一个毒性效应子。这使得这种细菌武器拥有更多的有毒矛尖和更大的可装载和发射的有毒细菌效应蛋白库。由于效应蛋白是 T6SS 功能的真正调节者，因此了解它们的作用非常重要。此外，我们还发现了由 Sfa2 控制但尚未表征的新的潜在 T6SS 效应器。我们知道 Sfa2 对于该系统“开启”至关重要，但我们不知道 Sfa2 的每个部分或域的作用，Sfa2 控制的所有基因都做什么，我们不知道 Sfa2 响应激活这种细菌武器的信号。该项目将提供有关 Sfa2 如何发挥作用、它控制什么以及它响应什么的基础知识。具体来说，在为期三年的新研究者奖期间，我们将 1) 定义关键 Sfa2 结构域在功能测定中的作用； 2) 表征两个新鉴定的Sfa2调节毒性效应子编码基因在H2-T6SS细菌间竞争中的作用； 3) 通过生化测定确定 Sfa2 sigma 因子激活信号并验证其对 H2-T6SS 表型的影响。这些目标将提供有关生命规则的知识，使细菌能够部署与细菌间竞争、真核细胞入侵和宿主感染严重程度相关的抗菌武器。我们的工作将揭示细菌用于部署这些武器的信号。这将引导我们制定用药物来靶向 T6SS 使其失效的策略，或者在未来将 T6SS 用于对抗病原体的合成生物学应用中，作为对抗细菌的新疗法。