Pseudomonas aeruginosa is a human pathogen that causes life-threatening nosocomial infections. This organism has a range of virulence mechanisms, however its secretion systems play a key role in the transport of effectors, toxins and other virulence factors which are important for colonisation of niches including the human host. One newly identified secretion system is the Type VI secretion system (T6SS) which has been identified in a number of Gram-negative bacteria. This system is a specialised transmembrane complex which secretes toxins and other effectors into eukaryotic and prokaryotic cells. The H1-T6SS of P. aeruginosa is one of the best characterised systems and has been shown to target non-immune prokaryotic cells. To date, three toxins/immunity pairs (Tse1/Tsi1, Tse2/Tsi2 and Tse3/Tsi3) have been identified in P. aeruginosa. Importantly, in a P. aeruginosa triple toxin mutant, T6SS-dependent killing still occurs, suggesting more toxins are involved. The current research will identify novel T6SS toxins and elucidate their transport. The use of transposon directed insertion sequencing (TraDIS) will uncover the full P. aeruginosa toxin repertoire.The mechanism of T6SS target cell puncture and effector secretion is currently unclear. One possibility is that effectors are linked to the cell-puncturing device, VgrG. This hypothesis will be investigated by testing the ability of VgrG-toxin chimeras to mediate T6SS-dependent cell killing. To identify any non-toxin proteins that are secreted by VgrG, a Pseudomonas two-hybrid library will be screened, using full length VgrG or the C terminus of VgrG as bait. Finally, the puncture and delivery of effectors by the H1-T6SS will be examined using in situ live fluorescent microscopy.Overall, the proposed research will provide breakthrough insight into the mechanism of action of this important virulence factor in P. aeruginosa which could be exploited for the development of targeted therapeutic options.