Antagonistic second-messenger signalling coordinates Pseudomonas aeruginosa surface colonization and virulence

Abstract Efficient colonisation of host tissue surfaces is a prerequisite for bacterial infection. In the human pathogen Pseudomonas aeruginosa , the second messengers cAMP and c-di-GMP regulate surface behaviours and virulence, but how these pathways are coordinated during tissue infection has remained unclear. Here we demonstrate that cAMP and c-di-GMP establish mutually exclusive signalling states at the single-cell level, generating distinct subpopulations of motile and surface-attached cells through pathway-specific control of type IV pili (T4P). Initial surface contact triggers an increase in c-di-GMP, activating FimW and driving stable attachment via bipolar T4P formation. This state is progressively replaced by cAMP, which inactivates FimW-dependent attachment and induces FimX-mediated T4P polarisation, surface motility and virulence activation. The resulting signalling transition enforces coexisting subpopulations, with attached cells gradually displaced by spreading cells. Using a human airway tissue model, we show that both T4P-mediated adhesion and twitching motility are required for invasion and tissue damage. Based on this, we propose that reciprocal cAMP–c-di-GMP signalling coordinates complementary T4P functions to optimise P. aeruginosa surface colonisation and infection.