Detection of Bacterial Neutral Ceramidase in Diabetic Foot Ulcers with an Optimized Substrate and Chemoenzymatic Probes.

Abstract

Ceramidases (CDases) are important in controlling skin barrier integrity by regulating ceramide composition and affording downstream signal molecules. While the functions of epidermal CDases are known, roles of neutral CDases secreted by skin-residing microbes are undefined. Here, we developed a one-step fluorogenic substrate, S-B, for specific detection of bacterial CDase activity and inhibitor screening. We identified a non-hydrolyzable substrate mimic C6 as the best hit. Based on C6, we designed JX-1, a photoaffinity probe and observed an efficient detection of bacterial CDases. Using JX-1 we identified endogenous low-abundant PaCDase in P. aeruginosa monoculture and mixed skin bacteria culture. Harnessing both S-B and JX-1, we observed in clinical diabetic foot ulcers patient samples that CDase activity positively correlates to P. aeruginosa relative abundance and is negatively associated with wound area reduction. Overall, our study demonstrates that bacterial CDases are important regulators of skin ceramides and potentially play a role in wound healing.