BackgroundPseudomonas aeruginosais an important opportunisticpathogen causing serious pulmonary, urogenital and sys-temic infections. P. aeruginosa injects four effector pro-teins into host cells via the type III secretion system. ExoYis one of these proteins.ExoY was originally classified as adenylyl cyclase withhomology to the typical calmodulin-stimulated adenylylcyclase exotoxins CyaA fromBordetella pertussis andedema factor from Bacillus anthracis, but the pathophy-siologial function of ExoY has remained elusive [1,2].Recently, our group showed that CyaA and edema factorpossess a rather broad base specifity (ATP >> CTP >UTP) [3,4], raising the question of wether ExoY may alsobind and metabolize nucleoside 5’- triphosphates otherthan ATP.MethodsWe determined cyclic nucleotide concentrations in cellstransfected with ExoY plasmid or infected with P. aerugi-nosa with a highly sensitive HPLC-MS/MS method. More-over, we determined the catalytic activity of purified ExoY.ResultsIn mammalian cells transfected with ExoY plasmid andinfected with ExoY-encoding P. aeruginosa, massive pro-duction of cGMP and cUMP was observed, with littleproduction of cAMP. Purified ExoY was a highly effectivenucleotidyl cyclase with the substrate preference GTP >>UTP ~ ATP > CTP. Fluorescence resonance energytransfer studies with methylanthraniloyl-substitutednucleotides corroborated the preference of ExoY forGTP. In contrast to ExoY, CyaA induced accumulation