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

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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.