Mechanical compression activates cAMP signaling in Pseudomonas aeruginosa

In Pseudomonas aeruginosa, cyclic AMP (cAMP) is a central regulator of virulence, including the type III secretion system (T3SS), yet the signals that activate cAMP remain incompletely understood. Using the real-time cAMP biosensor Gflamp1, we show that mechanical compression elevates cAMP, with an estimated activation force of approximately 3-30 nN. This mechanoresponse requires the Pil-Chp system, the FimV-FimL module, and the adenylate cyclase CyaB. Productive surface piliation and pilus extension or retraction are dispensable, whereas the major pilin PilA remains required. We further show that the histidine kinase ChpA interacts with the polar scaffold FimV and that FimL modulates FimV-dependent ChpA polar localization. Fluorescence lifetime imaging microscopy-Förster resonance energy transfer (FLIM-FRET) analysis supports compression-dependent changes in the apparent proximity of ChpA to PilG and FimL. Finally, compressed-state growth increases T3SS-associated transcription, suggesting that compression-induced cAMP signaling may link confined growth to virulence-associated gene expression.