FUNCTIONAL CHARACTERIZATION OF THE LEGIONELLA GENUS CORE EFFECTORS

Infection by the human pathogen Legionella pneumophila relies on the translocation of ~300 virulence proteins, termed effectors, which manipulate host cell processes. Recently we sequenced, assembled and characterized the genomes of 38 Legionella species and predicted their effector repertoires. This analysis identified 5,885 predicted effectors, and only a set of nine core effectors were shared by all the Legionella species. To characterize these core effectors, deletion mutants were constructed in the L. pneumophila homologs of these effectors and tested for intracellular growth in the environmental host Acanthamoeba castellanii. Three out of nine core effectors (33%) were found to be partially required for intracellular growth in this host and their growth defects were completely complemented by introducing the effectors on a plasmid. In addition, orthologs from five different species were examined for complementation of these deletion mutants and most of them also complemented their growth defect indicating that these core effectors perform the same function in all these species. In addition, we cloned and expressed the core effectors in the yeast Saccharomyces cerevisiae. Four out of the nine core effectors caused different degrees of growth inhibition in yeast, indicating that they affect conserved eukaryotic pathways. One of the core-effectors was found to be involved in iron acquisition and another one is probably involved in the manipulation of a MAPK pathway. In addition, examination of orthologous core effectors from five different species demonstrated that they all probably perform a similar function. To conclude, the high conservation of the core effectors and their conserved function throughout the evolution of the genus indicates that they perform critical functions during infection.