Investigating the role of a novel Type IV secretion system in Legionella pneumophila

A novel Type IV secretion system (T4SS) was identified in Legionella pneumophila isolates from the Edinburgh Legionnaires’ disease outbreak in 2012. A phylogenetic reconstruction of the isolates shows four distinct clades, two of which share a region thought to encode the novel T4SS. In a Galleria mellonella infection model, strains with this T4SS caused more rapid killing than those without it. Furthermore, patients infected with isolates containing the novel T4SS required more clinical care intervention. This project aims to dissect the role of the novel T4SS in intracellular survival. Bioinformatic analysis showed that the T4SS is closely related to the Legionella genomic island-associated T4SS (LGI-T4SS) of L. longbeachae. Other bioinformatic tools were used to identify neighbouring genes predicted to encode T4SS-secreted effector proteins. The expression of these genes was then detected during broth culture and intracellular growth using RT-PCR. A range of techniques were employed in order to compare the intracellular survival, replication and virulence of representative isolates from each of the four clades. Macrophage-like cells were infected in a gentamicin protection assay to compare intracellular replication. DNA extracted from the infected cells at four time points was quantified by qPCR to measure replication of the bacteria over time. In addition, the level of host cell death and autophagy was compared in macrophage-like cells infected with representative isolates from each clade. The results indicate that the region encoding the novel T4SS originated in L. longbeachae and encodes at least one putative T4SS effector protein. Although no difference was observed in host cell death and autophagy during infection with representative isolates, the presence of the novel region correlates with a reduced rate of intracellular replication.