Prp8 Intein-Targeted Fluorescent Probe for Specific Detection of Cryptococcus neoformans and Inhibitor Screening.

Cryptococcus neoformans (C. neoformans) infections pose a critical threat to global health, with high mortality rates observed among immunocompromised individuals. Rapid, cost-effective diagnostics are essential for preventive treatment and reducing mortality in resource-scarce settings, yet the development of selective fluorescent probes for C. neoformans remains challenging. The Prp8 intein, a unique self-splicing protein in C. neoformans, represents an ideal target for developing fluorescent probes and antifungal drugs. In this study, a series of fluorescent probes integrating Prp8 intein inhibitors with an environment-sensitive fluorophore was developed for selective covalent binding to the Prp8 intein. Among these probes, H2 enables specific detection of C. neoformans with minimal interference from Candida species. A dual-staining approach combining capsule staining with intracellular Prp8 intein labeling significantly improves the accuracy of C. neoformans detection. Probe H2 was also successfully applied for fluorescence imaging of fungal infections in a Galleria mellonella model, demonstrating potential for assessing fungal infections in vivo. Furthermore, the fluorescence intensity of the probe exhibited a dose-dependent relationship with Prp8 inhibitor activity, enabling rapid screening of intein-splicing inhibitors within 2 h, thereby improving efficiency and reducing assay time.