Dissecting the disarming of macrophages by the respiratory pathogen Legionella pneumophila

Abstract

Legionella pneumophila is a Gram-negative bacterium that infects protists and humans causing a disease known as Legionellosis. Severe Legionellosis, which can progress to a potentially fatal pneumonia called Legionnaires’ disease (LD), is increasing in incidence likely due to aging populations and global warming. The major virulence factors that L. pneumophila and related species use to affect host cell processes are effector proteins secreted by the Defect in organelle trafficking/ intracellular multiplication (Dot/Icm) type 4 secretion system (T4SS). The individual functions of most effectors remain unknown. Within this study, I aimed to characterise via both bioinformatic and experimental methods a family of effectors founded by the effector LtpB. Using comparative genomics and homology search it was established that the LtpB family is widely distributed in Legionella species. In L. pneumophila isolates the homologue LegA2 is the most conserved family member, whereas LtpB and some other homologues such as Lpp2048 are found sporadically together with LegA2. While legA2 seems to be part of the core genome, ltpB is located in a putative genomic island suggesting that it might move via horizontal gene transfer. Analysis of the predicted structure and biophysical properties of the proteases suggests that they localise to a mostly hydrophilic environment such as the host cell cytosol. Immunofluorescence microscopy experiments demonstrated the localisation of the putative proteases in the cytosol of host cells during infection. Ectopic-expression experiments recapitulated the cytoplasmic localisation and showed that the effectors do not cause cell rounding or cytotoxicity. L. pneumophila ΔltpB knockout mutants showed a statistically significant reduction in virulence compared to a wildtype strain in the Galleria mellonella infection model. From this, we can surmise that LtpB and LegA2 are Dot/Icm T4SS effectors that localise to the host cell cytosol and in the case of LtpB contribute to the virulence of L. pneumophila.

Thesis is embargoed until 31 December 2029.

Date of Award	Dec 2024
Original language	English
Awarding Institution	Queen's University Belfast
Supervisor	Gunnar Neels Schroeder (Supervisor), Jose Bengoechea (Supervisor) & Paul Moynagh (Supervisor)