The zebrafish to analyze the role of extracellular vesicles and O-antigen in the virulence of Legionella pneumophila – ZebraLegion

Bacterial lipopolysaccharide (LPS) is a key virulence factor for both innate and acquired host responses to infection. Intriguingly, Legionella pneumophila (Lp) serogroup 1 (Sg1) strains, are responsible for over 80% of confirmed Legionnaires’ disease cases, a severe pneumonia that is often fatal when not treated promptly. The Sg of Lp is determined by the LPS. Here we propose to analyse the molecular mechanisms underlying the predominance of Lp Sg1 in human disease. We have recently developed a zebrafish model of Lp infection, recapitulating key features of human infection. We will exploit this powerful non-mammalian model to study immune cell behaviour and host-pathogen interactions to identify the role(s) of LPS in host-pathogen interaction and disease in vivo comparing 3 selcted strains. A Lp Sg1 and a Lp Sg 6 strain (Sg6 is the second cause of human disease) and a Sg9 strain that has the same genomic backbone as the Sg1 strain but differs only in its LPS cluster. This “isogenic pair” will allow to investigate differences in the host response that are only due to its specific LPS. In parallel we will analyse the impact of extracellular vesicles (EVs) shed by Lp. We have shown recently that Lp-EVs of Sg1 impact the host response, however it may differ depending on the strain the EVs are shed off. We will analyse virulence, trafficking and the immune response by analyzing phagocyte behavior and phagocyte Lp interaction in vivo in our zebrafish model, monitor bacterial dissemination and survival, analyze the host and bacterial response by dual RNAseq and decipher the molecular mechanisms by generating knock out zebrafish lines in key immune genes regulated differently by these strains or EVs. This knowledge will be the basis to better understand the impact of LPS and EVs on immune cells and to uncover virulence mechanisms of particularly successful bacterial clones.