Abstract
Bacterial nucleotidyl cyclase toxins are potent virulence factors that upon entry into eukaryotic cells are stimulated by endogenous cofactors to catalyze the production of large amounts of 3'5'-cyclic nucleoside monophosphates. The activity of the effector ExoY from Pseudomonas aeruginosa is stimulated by the filamentous form of actin (F-actin). Utilizing yeast phenotype analysis, site-directed mutagenesis, functional biochemical assays, and confocal microscopy, we demonstrate that the last nine amino acids of the C terminus of ExoY are crucial for the interaction with F-actin and, consequently, for ExoY's enzymatic activity in vitro and toxicity in a yeast model. We observed that isolated C-terminal sequences of P. aeruginosa ExoY that had been fused to a carrier protein bind to F-actin and that synthetic peptides corresponding to the extreme ExoY C terminus inhibit ExoY enzymatic activity in vitro and compete with the full-length enzyme for F-actin binding. Interestingly, we noted that various P. aeruginosa isolates of the PA14 family, including highly virulent strains, harbor ExoY variants with a mutation altering the C terminus of this effector. We found that these naturally occurring ExoY variants display drastically reduced enzymatic activity and toxicity. Our findings shed light on the molecular basis of the ExoY-F-actin interaction, revealing that the extreme C terminus of ExoY is critical for binding to F-actin in target cells and that some P. aeruginosa isolates carry C-terminally mutated, low-activity ExoY variants.
Highlights
Bacterial nucleotidyl cyclase toxins are potent virulence factors that upon entry into eukaryotic cells are stimulated by endogenous cofactors to catalyze the production of large amounts of 35-cyclic nucleoside monophosphates
A yeast strain expressing an ExoY variant lacking the 30 N-terminal amino acids displayed a moderate growth on galactose-containing media indicating loss of toxicity of this variant
Whereas the N-terminal 20 amino acids are clearly dispensable for ExoY toxicity in yeast ( these residues may correspond to a type 3 secretion system (T3SS) secretion signal [27]), deletion of 10 additional amino acids at the N terminus likely impairs the catalytic core of ExoY
Summary
Bacterial nucleotidyl cyclase toxins are potent virulence factors that upon entry into eukaryotic cells are stimulated by endogenous cofactors to catalyze the production of large amounts of 35-cyclic nucleoside monophosphates. We noted that various P. aeruginosa isolates of the PA14 family, including highly virulent strains, harbor ExoY variants with a mutation altering the C terminus of this effector. ExoY was identified by Yahr et al [19] as a 378-amino acidlong protein that shares sequence similarity with well-characterized calmodulin-activated adenylyl cyclases from Bordetella pertussis (CyaA) and Bacillus anthracis (edema factor). These toxins share highly conserved regions responsible for catalysis but are different in regions predicted to bind the activator [19, 20]. In this study we characterize an ExoY region located at the C terminus of the enzyme, which is required for the interaction with F-actin and enzymatic activity in vitro
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