Prp8 Intein-Targeted Fluorescent Probe for Specific Detection of <i> <i>Cryptococcus neoformans</i> </i> and Inhibitor Screening

<i><i>Cryptococcus neoformans</i></i> (<i><i>C. neoformans</i></i>) 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 <i><i>C. neoformans</i></i> remains challenging. The Prp8 intein, a unique self-splicing protein in <i><i>C. neoformans</i></i>, 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, <b>H2</b> enables specific detection of <i><i>C. neoformans</i></i> with minimal interference from <i>Candida</i> species. A dual-staining approach combining capsule staining with intracellular Prp8 intein labeling significantly improves the accuracy of <i><i>C. neoformans</i></i> detection. Probe <b>H2</b> was also successfully applied for fluorescence imaging of fungal infections in a <i>Galleria mellonella</i> model, demonstrating potential for assessing fungal infections in <i>vivo</i>. 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.