Pseudomonas aeruginosa is an opportunistic pathogen, causing both acute and chronic infections. This bacterium displays remarkable adaptability and potential for virulence, partly due to its arsenal of protein secretion systems. The type VI secretion system (T6SS) is a contractile injection apparatus, firing a spear-like structure into target cells to deliver its cargo of effector proteins. P. aeruginosa encodes three such systems, denoted H1-, H2- and H3-T6SS. This dissertation discloses work focused on progressing our understanding of the H2-T6SS in this pathogen.

We reveal that the H2-T6SS is controlled by the Gac/Rsm pathway, a major regulatory network in this pathogen responsible for the lifestyle switch between motile and sessile bacteria. Quorum sensing, the sophisticated signalling network governing social behaviour, is responsible for the expression of this secretion system in a growth-phase dependent manner, while temperature also has an input in a strain-dependent fashion.

We advance our understanding of the composition of the H2-T6SS nanomachine, identifying multiple components of the spear-like delivery device, comprising an Hcp tube capped with a spike structure composed of three VgrGs and one PAAR protein. Importantly, we begin to decipher the payload of this secretion system, describing several phospholipase family effectors which confer a significant advantage to P. aeruginosa during bacterial competition. Building upon this, we propose a hierarchy of effector delivery determined by the VgrG/PAAR composition of the spike.

Finally, we characterise a specific H2-T6SS effector: the C-terminal extension of the VgrG2b spike protein. Although we initially investigate its reported role within eukaryotic cells, we determine that this metallopeptidase-like effector is part of a wider antibacterial T6SS toxin family. We describe its cognate periplasmic immunity determinant and progress the elucidation of the target of the effector. Overall, we advance our understanding of the H2-T6SS of P. aeruginosa in terms of its regulation, organisation and cargo.