Legionella pneumophila (L.pneumophila) is a gram-negative bacterium that replicates in fresh water amoeba and mammalian macrophage. In the environment, L.pneumophila is phagocytosized by Acanthamoeba, such as Acanthamoeba castellanii. However, L.pneumophila has developed the ability to hijack cellular machinery to support their intracellular replication inside the host cell. With the help of over 330 effector proteins translocated via Dot/Icm type Ⅳ secretion system, L.pneumophila establishes the Legionella containing vacuole (LCV) to support the intracellular replication niche. While a subset of effector proteins have been shown to be involved in intracellular processes such as ER recruitment and post-translational modifications in macrophages, the majority remain uncharacterized, especially in Acanthamoeba. A major limitation of these studies has been the functional redundancy of effector proteins.
Acanthamoeba life cycle presents two different stages: the motile trophozoite and the resistant cyst. The active stage of Acanthamoeba is trophozoite with typical tapering pseudopodia, which plays a vital role in the metabolic activities including locomotion, eating and dividing. In harsh, nutrient limited conditions, trophozoites change to the double walled cysts, resistant to long-term starvation, as well as the chemical and physical disinfectant treatments. When conditions improve, the amoeba recovers from the cyst and recommences growth. However, the life cycle of Acanthamoeba influenced by L.pneumophila infection is not well studied.
In this study, we aim to investigate the host-pathogen interaction between A. castellanii and L.pneumophila. Therefore, we constructed a library of L. pneumophila mutants carrying large genomic region deletions. Recently, 10 mutants have been created, resulting in the deletion of 68 effector proteins collectively. Defect intracellular replication in amoeba has been observed among the mutants. The mutants are also used to explore the changes in A. castellanii caused by L.pneumophila infection. RNA sequencing has been done to explore the function of effector protein in cluster 4.