Pil-Chp orchestrates a multi-level regulatory system to control type IV pilus dynamics in Pseudomonas aeruginosa

The cyclical extension and retraction dynamics of type IV pili (T4P) mediate critical virulence traits in many bacterial species, yet how these dynamics are regulated remain poorly understood. Here, by analyzing individual pilus dynamics across mutants covering the entire T4P system of Pseudomonas aeruginosa , we identified active pili in several mutants previously classified as non-piliated. Correlating these dynamics with intracellular cAMP levels, transcriptional profiles, and T4P protein abundances reveals how the chemosensory Pil-Chp system controls T4P fiber length, count, extension rate, and polarity in surface-naïve cells by modulating cAMP levels. Specifically, fiber length is tuned by modulating the effective association and dissociation rates of the extension ATPase PilB; fiber count is regulated by controlling machine abundance; and extension rate is limited by the abundance of the major pilin (PilA). Furthermore, we demonstrate that these regulatory mechanisms of T4P length and count differentially impact T4P-dependent functions: phage infection efficiency increases continuously with fiber count or length, whereas twitching motility exhibits step-like activation thresholding. This indicates that the functional output of T4P dynamics is strictly context dependent. Because the Pil-Chp system is conserved across diverse human and plant pathogens, this multi-level regulatory logic may represent a widespread mechanism controlling T4P-mediated virulence.