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

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对手。其中一些细菌分泌系统就像有毒的长矛，将有毒蛋白质直接输送到竞争细菌中，杀死它们。这使得一种细菌能够占据一个位置。细菌也可以利用这些有毒的矛将毒素输送到动物和植物细胞中。这有助于细菌侵入真核细胞或操纵它们以获得细菌。VI型分泌系统（T6SS）是一种弹簧装载的矛或鱼叉，用于输送这些有毒的效应蛋白，于2006年被发现。它存在于25%的革兰氏阴性细菌中。从我们的肠道到土壤微生物，这对不同环境中的细菌相互作用很重要。我们目前还不知道细菌对激活这些细菌武器的信号作出反应。本项目主要研究这些细菌在铜绿假单胞菌中的分泌武器。铜绿假单胞菌是广泛存在于人类、动物和植物中的病原体。P. aeruginosa具有三个T6SS和多个较小的T6SS基因簇分布在其基因组中。这一建议集中在其中最有效的H2-T6SS上，它能杀死细菌，帮助铜绿假单胞菌进入真核细胞，并被部署来对抗宿主生物。然而，我们不了解调节H2-T6SS何时“打开”或“关闭”的机制。这方面的知识很重要，因为我们需要了解部署抗菌武器以对抗它们的信号。我的实验室最近发现了两个关键调控蛋白RpoN和Sfa2对H2-T6SS基因表达和功能的关键作用。RpoN是控制铜绿假单胞菌中25%基因的全球性调节因子。然而，它不能单独作用于基因激活。它需要一个因子激活蛋白来帮助它。我们已经确定Sfa2是一个帮助RpoN特异性激活H2-T6SS基因的基因，从而激活该传递系统。值得注意的是，它还允许RpoN激活整个基因组中编码毒性效应的孤儿T6SS基因。这使得这种细菌武器有更多的有毒矛尖和更大的有毒细菌效应蛋白库，可以装载和发射。由于效应蛋白是T6SS功能的真正介质，因此了解它们的作用非常重要。此外，我们还发现了由Sfa2控制的新的潜在T6SS效应物，这些效应物尚未被表征。我们知道Sfa2对于这个系统的开启至关重要，但我们不知道Sfa2的每个部分或区域的作用，Sfa2控制的所有基因的作用，我们也不知道Sfa2响应激活这种细菌武器的信号。这个项目将提供关于Sfa2如何工作、控制什么以及响应什么的基本知识。具体来说，在这个为期三年的新研究者奖期间，我们将1)定义关键Sfa2结构域在功能分析中的作用；2)分析两个新发现的Sfa2调控的毒性效应编码基因在H2-T6SS菌间竞争中的作用；3)通过生化检测确定Sfa2 sigma因子激活信号，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来部署这些武器的信号。这将引导我们制定针对T6SS的药物来禁用它们的策略，或者在未来的合成生物学应用中使用T6SS作为对抗细菌的新疗法来对抗病原体。

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.