Interplay between T3SS effectors, ExoY activation, and cGMP signaling in Pseudomonas aeruginosa infection

Abstract The Type III Secretion System (T3SS) of Pseudomonas aeruginosa injects effector proteins into host cells to subvert cellular processes and promote infection. While the roles of several T3SS effectors in virulence are well established, the function of the nucleotidyl cyclase ExoY has remained elusive and debated. Here, we show that ExoY-produced cyclic GMP (cGMP) regulates the cytotoxic activity of the co-injected effector ExoT, thereby modulating host cell damage. Using engineered P. aeruginosa strains expressing ExoY variants with distinct substrate preferences, we demonstrate that cGMP production by ExoY limits ExoT-mediated dephosphorylation of the host adaptor protein CrkII, a downstream consequence of ExoT’s ADP-ribosyltransferase activity. This attenuation reduces ExoT-induced cell retraction and decreases bacterial virulence. In contrast, we find that ExoT and another T3SS effector, ExoS, can inhibit ExoY activity and cGMP production in certain cell types, thus limiting ExoY’s regulatory influence. These findings highlight the intricate interplay between T3SS effectors within host cells and reveal an unrecognized layer of ExoY-based complexity in P. aeruginosa ’s pathogenic strategy.