Legionella pneumophila is an accidental human pathogen that causes the severe pneumonia known as Legionnaire’s Disease. Naturally, 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 nine genomic deletions in L. pneumophila, which resulted in the deletion of 68 effector genes and 138 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, all mutants can replicate efficiently in THP1 macrophages. Meanwhile, in the protozoan host, at least six mutants showed a severe replication defect. Interestingly, in the mouse model, four mutants displayed an unexpected increase in bacterial burden, while one mutant showed a reduction in bacterial replication. We have successfully isolated one putative Dot/Icm effector that when deleted displayed an increase in survival in A-strain mice. Interestingly, strain missing this putative effector also triggered lower IFNγ production in the murine lungs, perhaps suggesting an effector-triggered immunity (ETI) phenotype. Together, these highlight the differential requirements for infecting different hosts, and that L. pneumophila may be better adapted to infecting the protozoan hosts rather than mammalian hosts.