Characterising the role of TssA proteins in the assembly and dynamics of the type VI secretion system

Abstract

The gram-negative, opportunistic pathogen Pseudomonas aeruginosa encodes many weapons for virulence and interbacterial warfare, including the type VI secretion system (T6SS), a contractile nanomachine delivering effector proteins and antibacterial toxins either into the extracellular environment or directly into neighbouring cells. The T6SS component TssA is variable in size, domain organisation, symmetry and structure. Some TssA proteins are proposed to coordinate the assembly of the T6SS contractile sheath, but the exact role of TssA proteins remains unclear, and understanding is complicated by this relatively low level of conservation of TssA proteins. This project aimed to characterise the three TssA proteins of P. aeruginosa to assess their contribution to, and positioning within, their respective T6SSs and determine whether these were influenced by TssA variation. To achieve this, functional assays were optimised to monitor the activity of the three P. aeruginosa T6SSs and assess the requirement for TssA proteins. Interactions of both full length TssA proteins and individual domains with other T6SS proteins were also characterised and in silico approaches allowed the position of TssA proteins within the T6SS to be modelled. It was identified that each P. aeruginosa TssA protein was specifically required for the activity of its respective T6SS. Interactions of these TssA proteins with T6SS components were identified including specific interactions with the cognate contractile sheath, where this specificity could be altered by the exchange of domains. This work conclusively demonstrated specificity determinants for TssA proteins, which allowed the generation of functional chimeric TssA proteins, even with components from different TssA sub-groups. From this work a new docking has been proposed for TssA proteins with the contractile sheath and a model has been developed for how sheath assembly is coordinated by TssA proteins from this position.