Legionella pneumophila pathogenesis : establishment of a new insect infection model and characterisation of the effector protein LtpD

Abstract

Legionella pneumophila is the causative agent of Legionnaires’ disease, severe pneumonia acquired from inhalation of contaminated water droplets. In the lung, L. pneumophila infects alveolar macrophages and creates a compartment named the Legionella containing vacuole (LCV), which avoids degradation and recruits components of the secretory pathway. LCV creation depends on the action of the Dot/Icm system that translocates over 275 effectors into the host cell. To study the function of these effectors, models that approximate human disease are required. Here, I characterise the larvae of Galleria mellonella as an infection model for L. pneumophila. Infection resulted in larval mortality and bacterial replication in a strain- and Dot/Icm-dependent manner. Flagella expression was dispensable for bacterial virulence, however secreted phospholipases and the Dot/Icm effector SdhA were shown to be important in virulence. Deletion of SdhA resulted in disruption of the LCV membrane and destruction of haemocytes. The importance of SdhA expression was confirmed in a mammalian model, validating the utility of G. mellonella. In the second part of this study, the novel protein LtpD was characterised. LtpD was translocated via the Dot/Icm secretion system and localised to the LCV. A series of truncation mutants defined a C-terminal 153 amino acid domain as required for LCV localisation. This region was shown to bind directly to the lipid phosphoinositide 3-phosphate. Further analysis revealed that LtpD also interacted with the enzyme inositol monophosphatase 1, however did not change the enzyme’s activity in vitro. Deletion of LtpD resulted in a subtle growth defect in mammalian macrophages at late time points during infection. This growth defect was also seen the G. mellonella and mouse lungs, confirming that LtpD is a virulence factor of L. pneumophila. In summary, here I present an infection model to investigate L. pneumophila virulence and further characterisation of the Dot/Icm effector LtpD.