Legionella pneumophila is an accidental human pathogen that causes the severe pneumonia known as Legionnaire’s Disease. L. pneumophila is found in the aquatic environment, often as a parasite of a diverse range of freshwater amoebae. During infection, L. pneumophila establishes a replicative vacuole termed the Legionella-containing vacuole (LCV) that sustains intracellular replication in both macrophages and amoebae. Establishment of the LCV requires the Dot/Icm type IV secretion system (T4SS), that injects more than 300 effector proteins into the infected host cell. Despite their central role in LCV biogenesis, to date most effector proteins remain uncharacterized. Therefore, to aid in the study of effector-associated phenotypes, in this study, we generated fourteen genomic deletions in L. pneumophila, which resulted in the deletion of 81 effector genes and 147 non-effector genes collectively.
These mutants were then used to identify the genomic regions important for bacterial replication in vitro and in vivo. Despite the loss of up to 42 effector-encoding genes within a single mutant, most mutants can replicate efficiently in THP1 macrophages. In contrast, in the protozoan host, Acanthamoeba castellanii, at least six mutants showed a severe replication defect. Interestingly, in a mouse lung infection model, four mutants displayed an unexpected increase in bacterial burden, while one mutant showed a reduction in bacterial load. We are currently studying the molecular effects of these large deletions on the host response during Legionella infection, in particular the impact of effector region deletions on the host transcriptome.