Many pathogens manipulate the cAMP intracellular signaling of host cells to promote their survival and proliferation in hosts. Bacterial ExoY-like virulence factors represent a new atypical subfamily of nucleotidyl cyclase (NC) toxins. Exoenzyme Y (ExoY) was first identified as a toxin secreted via a type 3 secretion system (T3SS) by Pseudomonas aeruginosa, a major opportunistic and nosocomial human pathogen. P. aeruginosa is also responsible for progressive and severe chronic lung infections in patients with cystic fibrosis. ExoY is present in 90-98% of clinical isolates of P. aeruginosa, which suggests an important role in bacterial pathogenicity, but its role in P. aeruginosa infections remains poorly understood. Poorly characterized ExoY-related enzymatic modules or effector proteins have also been found recently in several toxins produced by various Gram-negative bacteria representing emerging human or animal pathogens. Some of these enzymes were shown to be essential for bacterial virulence. We have recently shown that these NC toxins are potently activated within the host target cells by using an original eukaryotic cofactor that is actin. Yet, our new preliminary data suggest that they may differ significantly in their substrate selectivity, their interaction with actin and activation mechanisms, impact on the actin cytoskeleton dynamics, and subcellular localizations. As a consequence, they likely perturb different biological processes in infected host cells. We aim here to characterize at the molecular and structural level and in cellular infection models the functional specificities, precise role and virulence mechanisms of this novel class of actin-activated NC toxins in bacterial infections by P. aeruginosa and various pathogenic Gram-negative organisms. To decipher their structure-function relationships we will analyze several representative members of this subfamily of actin-activated NC toxins. Our main goals are to characterize in vitro and in cells their functional specificities and impact on actin self-assembly dynamics. We will study the structural bases for their activation mechanisms and enzymatic specificities, and search for inhibitors. We will characterize ExoY phenotype and prevalence in the strains included in the P. aeruginosa reference panel collection. We will study ExoY effects on phagocytosis, inflammation, mucus production, or the integrity of cell-cell junctions in P. aeruginosa-infected cells to determine the importance of ExoY in the modulation of host airways innate immunity. We will finally analyze possible interplays between ExoY/ExoY-like virulence factors and other bacterial virulence factors that affect the actin cytoskeleton. Collectively, our proposed research will be innovative in several aspects: it will expand the fundamental knowledge on Pseudomonas aeruginosa T3SS effector virulence mechanisms in P. aeruginosa infections; it will help to identify the functional specificities of ExoY-related toxins recently found in emergent human or animal proteobacterial pathogens, and will eventually explore their potential as drug target. Finally, our project may help to understand better the pathogenicity of P. aeruginosa in diseases such as cystic fibrosis for which there is yet no effective therapy.