EXOY, AN ADENYLATE-CYCLASE SECRETED BY THE PSEUDOMONAS-AERUGINOSA TYPE-III SYSTEM

The exoenzyme S regulon is a set of coordinately regulated virulence g enes of Pseudomonas aeruginosa, Proteins encoded by the regulon includ e a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins, The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an ac ute cytotoxin (ExoU). In this study, we identified ExoY as a fourth ef fector protein of the regulon, ExoY is homologous to the extracellular adenylate cyclases of Bordetella pertussis (CyaA) and Bacillus anthra cis (EF), The homology among the three adenylate cyclases is limited t o two short regions, one of which possesses an ATP-binding motif, In a ssays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyz ed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated qv calmodulin. A 500-fold stimulation of activ ity was detected following the addition of a cytosolic extract from Ch inese hamster ovary (CHO) cells. These results indicate that a eukaryo tic factor, distinct from calmodulin, enhances rExoY catalysis. Site-d irected mutagenesis of residues within the putative active site of Exo Y abolished adenylate cyclase activity. Infection of CHO cells with Ex oY-producing strains of P, aeruginosa resulted in the intracellular ac cumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is di rectly translocated into the eukaryotic cytosol.