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：几乎所有细菌使用的一种基本进攻策略是分泌毒素，以帮助它们生存或杀死竞争性生物。这些细菌分泌系统中的一些作用着中毒的长矛，将有毒蛋白直接用于竞争细菌以杀死它们。这使一个细菌可以接管位置。细菌还可以使用这些中毒的长矛将毒素输送到动物和植物细胞中。这有助于细菌侵袭真核细胞或操纵它们以获得细菌增益。VI型分泌系统（T6SS）是一种弹簧弹性的长矛或鱼叉，用于递送这些有毒效应蛋白，并于2006年发现。它在革兰氏阴性细菌的25％中发现。这对于从肠道到土壤微生物的各种环境中的细菌相互作用很重要。目前，我们不知道细菌对这些细菌武器的激活作出反应的信号。该项目重点介绍了有机体假单胞菌铜绿假单胞菌的这些细菌分泌武器。铜绿假单胞菌是人类，动物和植物的一种广泛发现的病原体。铜绿假单胞菌具有三个T6SS，多个较小的T6S基因簇分布在其整个基因组中。该提案以最有效的速度为中心，即杀死细菌的H2-T6S，有助于铜绿假单胞菌进入真核细胞，并针对宿主生物进行部署。但是，我们对当“ On”或“ Off”切换H2-T6S时调节的机制没有理解。这些知识很重要，因为我们需要了解部署抗菌武器以对抗它们的信号。我的实验室最近显示了两种关键调节蛋白RPON和SFA2在H2-T6SS的基因表达和功能方面表现出至关重要的作用。 RPON是一个全球调节剂，可控制铜绿假单胞菌中25％的基因。但是，它不能单独起作用基因激活。它需要Sigma因子激活蛋白来帮助它。我们已经确定SFA2是帮助RPON专门激活H2-T6SS基因的一种，导致该输送系统的激活。值得注意的是，它还允许RPON在整个基因组中激活每个编码有毒效应子的孤儿T6SS基因。这使得这种细菌武器可以具有更多中毒的长矛尖和更大的有毒细菌效应蛋白的库，可以加载和发射。由于效应子蛋白是T6SS的真正介体的作用，因此了解其所做的事情很重要。此外，我们已经确定了由SFA2控制的新的潜在T6SS效应子，这些效应子尚未具有特征。我们知道SFA2对于该系统而言，要切换为“ ON”至关重要，但我们不知道SFA2的每个部分或域都可以做什么，而SFA2控件的所有基因都不知道SFA2控制能力，并且SFA2的知识将供您提供依据。 SFA2功能，它控制的内容以及它的响应。具体而言，在这三年的新调查员奖中，我们将1）定义关键SFA2域在功能测定中的作用； 2）表征两个新鉴定的SFA2调节的毒性效应子编码基因在H2-T6SS间竞争中的作用； 3）通过生化测定确定SFA2 Sigma因子激活的信号，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中严重程度相关的抗菌武器。这将导致采取策略，以使用药物将其靶向T6S，或者将来将T6SS用于合成生物学应用中的方法，以针对病原体作为对抗细菌的新型疗法。