Identification of Legionella pneumophila Effectors Regulated by the LetAS- RsmYZ- CsrA Regulatory Cascade, Many of Which Modulate Vesicular Trafficking

Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular human pathogen that utilizes the Icm/ Dot type IVB secretion system to translocate a large repertoire of effectors into host cells. To find coregulated effectors, we performed a bioinformatic genomic screen with the aim of identifying effector-encoding genes containing putative CsrA regulatory elements. The regulation of these genes by the LetAS-RsmYZ-CsrA regulatory cascade was experimentally validated by examining their levels of expression in deletion mutants of relevant regulators and by site-directed mutagenesis of the putative CsrA sites. These analyses resulted in the identification of 26 effector-encoding genes regulated by the LetAS- RsmYZ-CsrA regulatory cascade, all of which were expressed at higher levels during the stationary phase. To determine if any of these effectors is involved in modulating the secretory pathway, they were overexpressed in wild-type yeast as well as in a yeast sec22 deletion mutant, which encodes an R-SNARE that participates in the endoplasmic reticulum (ER)-Golgi trafficking. This examination identified many novel LetAS- RsmYZ-CsrA regulated effectors which are involved in this process. To further characterize the role of these 26 effectors in vesicular trafficking, they were examined in yeast arf and arl deletion mutants, which encode small GTPases that regulate ER-Golgi trafficking. This analysis revealed that the effectors examined manipulate different processes of the secretory pathway. Collectively, our results demonstrate that several of the L. pneumophila effectors which are coregulated in the bacterial cell are involved in the modulation of the same eukaryotic pathway.