Legionella are ubiquitous environmental organisms that cause Legionnaires’ disease in susceptible humans. During infection, Legionella establishes an intracellular niche, known as Legionella containing vacuole (LCV), in lung phagocytes. Biogenesis of the LCV is dependent on the Dot/Icm secretion system which translocates ~300 bacterial effectors into the host cells during Legionella infection. These effectors modulate the host metabolism and cell autonomous defence, in order to ensure intracellular survival and replication of the bacteria.
During Legionella infection the host mounts a strong inflammatory response, producing cytokines such as interferon-gamma (IFNg), to control the infection. We showed that IFNg is crucial for the clearance of L. pneumophila and L. longbeachae in C57BL/6 mice. This is because IFNg-/- mice are unable to control and clear a Legionella infection effectively. Similar disease progressions have been observed in IFNg-/- mice infected with Mycobacterium tuberculosis, Salmonella typhimurium, Leishmania major and Listeria monocytogenes. Therefore this study aims to further characterise the antimicrobial mechanisms of IFNg during Legionella infections.
Preliminary results show that upon IFNg stimulation, we observe a significant reduction in levels of Dot/Icm translocated effector proteins in mouse macrophages. Since translocation of effectors into host cell is essential for the intracellular survival of Legionella, we aim to further characterise this phenomenon and identify IFNg stimulated factors that modulate the function of the Dot/Icm secretion system. In order to identify these factors, we will utilize a high–throughput CRISPR screen. For this purpose, macrophages in which IFNg induced genes have been knocked out individually will be infected with Legionella and the amount of effector proteins translocated into the host cells will be quantified. The identification of IFNg induced factors which inhibit the Dot/Icm effector translocation process will pave the way for development of new treatment strategies.