Abstract Sensory adaptation is a short-term memory process by which a signaling system returns to its pre-stimulus level despite ongoing exposure to the input signal. Although well-characterized in bacterial chemotaxis, little is known about how adaptation operates in the other bacterial signaling systems. A better understanding of how adaptation operates in these systems will provide new fundamental knowledge and could identify new therapeutic approaches. We focus on mechanosensing, which is critical for surface colonization and infection in Pseudomonas aeruginosa (PA), a leading cause of multi-drug resistant nosocomial infections and a significant health threat. PA uses the Pil-Chp mechanosensing system to transduce a mechanical signal that drives twitching motility and cAMP production to modulate a virulence program upon surface contact. How adaptation functions in this system, or mechanosensing in general, is unexplored. We propose to dissect the mechanism and role of adaptation in the Pil-Chp system because (i) The core enzymatic machinery of the mechanosensory adaptation system, the methylating enzyme PilK and demethylating enzyme ChpB, are conserved but its regulation appears to be distinct from the E. coli chemotaxis system; (ii) This system affords the opportunity to understand how adaptation is deployed in response to surface contact; and (iii) The Pil-Chp system contributes to virulence in a murine model of acute pneumonia, demonstrating its relevance to human PA infections. We discovered that PilK acts as a methylase and ChpB acts as a demethylase for the PilJ chemoreceptor to control the two outputs. Unlike in chemotaxis, the methylase and demethylase exhibit inverse spatial localization. Our studies support a model in which the PilK methylase localizes to the lagging pole, where the PilJ chemoreceptor would be methylated and poised to be activated. In contrast, the ChpB demethylase is recruited to the leading pole by interactions with the response regulator PilG. PilG is required for coordinating TFP extension and retraction at the leading pole. This localization would lead to temporally and spatially restricted PilJ demethylation at the leading pole. PilJ activity would be dampened, potentially facilitating PilG relocalization to the lagging pole and reversals. Thus, sensory adaptation in the Pil-Chp system is fundamentally different from adaptation in E. coli chemotaxis in that it uses temporal AND spatial cues. We will test this hypothesis as follows: Aim 1. Link PilJ methylation states to PilJ activity and outputs. Aim 2. Determine how the response regulator PilG regulates the ChpB demethylase. Aim 3. Test whether the PilK methylase is regulated by MapZ, a c-di-GMP binding protein, to link twitching and flagellar motility.

Project relevance Pseudomonas aeruginosa is an environmental bacterium that is a leading cause of hospital acquired infections. Antibiotic resistance is rampant in P. aeruginosa, leading to limited therapeutic options. This grant seeks to understand how this bacterium responds to rapidly changing environments, which may lead to new therapeutic approaches to treat human infections.