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

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 a photoaffinity probe, JX-1, which efficiently detects bacterial CDases. Using JX-1, we identified endogenous low-abundance PaCDase in a P. aeruginosa monoculture and in a mixed skin bacteria culture. Harnessing both S-B and JX-1, we found that CDase activity positively correlates with the relative abundance of P. aeruginosa and is negatively associated with wound area reduction in clinical diabetic foot ulcer patient samples. Overall, our study demonstrates that bacterial CDases are important regulators of skin ceramides and potentially play a role in wound healing.

Automated summary

Bacterial ceramidases play important roles in controlling skin barrier integrity and wound healing. In this study, a specific substrate and a photoaffinity probe were developed to detect bacterial ceramidase activity. The activity of ceramidase positively correlated with the abundance of P. aeruginosa and was negatively associated with wound area reduction in clinical diabetic foot ulcer patients. These findings highlight the importance of bacterial ceramidases in regulating skin ceramides and their potential role in wound healing.