Legionella pneumophila is an intracellular bacterium known to cause severe pneumonia disease. During infection, L. pneumophila establishes a replicative vacuole termed the Legionella-containing vacuole (LCV) to enable intracellular replication in alveolar macrophages. To establish the LCV, L. pneumophila uses a type IV secretion system (T4SS) called Dot/Icm to secrete around 300 effector proteins. Unfortunately, most of these effector proteins are not yet characterized. Complicating the studies of effector functions, within the large effector repertoire, there seems to be a functional redundancy, where the loss of one effector can be compensated by other effectors. In addition, interactions between effector proteins, where the action of one effector may alter or enhance the other, have also been reported. Therefore, to bypass these challenges, we aim to create and use a mutant library of L. pneumophila with large effector cluster deletions to investigate the roles of the Dot/Icm effector proteins during infection of macrophages.
We firstly grouped the nearby effectors, avoiding essential non-effector genes and genes that encode for the Dot/Icm T4SS, to form effector clusters. Currently, we have succeeded in deleting 9 effector clusters, which altogether contain 68 effector genes. Additionally, one mutant strain lacking 5 effector clusters, which encode for 47 effector genes, has also been created. These mutant strains are currently being subjected to replication assays in macrophages. Further, a competition assay will also be done in vivo to determine which mutant(s) show the least ability to survive inside the host cells. In addition, as the Dot/Icm T4SS is indispensable for the biogenesis of LCV, the ability of these mutants to form an LCV will also be assessed through confocal microscopy. Together, these will allow us to identify the Dot/Icm effectors essential for the bacterial intracellular replication and to understand their roles during L. pneumophila infection.