Characterisation of explosive cell lysis in Pseudomonas aeruginosa

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Pseudomonas aeruginosa is a Gram-negative pathogen commonly associated with nosocomial infections and implanted medical devices. It causes both acute and chronic infections, which are often associated with the formation of biofilms. Extracellular DNA (eDNA) is a major component of these biofilms and is involved in adhesion, intercellular connectivity and facilitating cell migration. eDNA is also essential for the formation of biofilms, however the mechanism of its production at the early stages of development is unknown. At the initiation of this Thesis, it was observed that during the early stages of submerged and interstitial biofilm development, a small proportion of cells spontaneously transform into round cells before rapidly lysing and releasing eDNA and cellular content into the extracellular milieu. This phenomenon is termed ‘explosive cell lysis’. This process accounts for all eDNA produced in these biofilms as well as a number of other ‘public goods’ including cytosolic proteins and membrane vesicles (MVs) that may benefit the biofilm community. As eDNA, MVs and extracellular proteins contribute to biofilm development and virulence, explosive cell lysis may be a critical process for P. aeruginosa. Therefore, it is important to identify the mechanism mediating this process. The aim of this Thesis was to identify and characterise the genes and cellular processes that lead to explosive cell lysis and eDNA release, and to better understand the role of eDNA in early stages of biofilm development. This Thesis identifies an endolysin of a cryptic bacteriophage as the enzyme responsible for this phenomenon and reports that explosive cell lysis is also under the control of the SOS response regulator RecA. Investigations into the induction of explosive cell lysis determined that intrinsic stressors like the formation of inclusion bodies and exogenous stressors that cause DNA damage induce explosive cell lysis and eDNA release. This Thesis also identifies that explosive cell lysis is mediated by the action of three independent holins that work in coordination to translocate the endolysin responsible for explosive cell lysis. Exploration into the role of explosive cell lysis in submerged biofilm formation determined that explosive cell lysis is essential for the formation of microcolonies, microcolony formation is spatially and temporally correlated to explosive cell lysis, and sequential explosive events are required for building microcolonies. Overall the results presented in this Thesis add to our understanding of the complex nature of eDNA production during biofilm development and have identified a novel role for cryptic bacteriophage.
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