Effect of insertion of intein to Cryptococcus amylolentus, a nonpathogenic fungus closely related to causative agents of cryptococcosis.

Inteins are mobile elements within a host protein, with flanking exteins. Autocleavage of intein results in the fusion of exteins, leading to activation of protein. The presence of intein is species dependent. Pathogenic fungi Cryptococcus neoformans (Cne) and C. gattii (Cga) contain inteins in their inactive Prp8 protein precursor, whereas closely related nonpathogenic C. amylolentus (Cam) lacks inteins. Handling pathogenic fungi requires additional safety requirements. Studies on nonpathogenic but closely related fungal strains can expedite research on the role of inteins and potential changes in virulence or pathology. In this report, we have genetically modified and characterized Cam to possess intein (Cam-int). First, we inserted a selection marker into the Prp8 intein of Cne using an MIG vector and tested intein splicing efficiency in E. coli. The intein-selection marker fragment was then integrated into the prp8 gene of Cam, demonstrating in vivo splicing within Cam without affecting certain virulence factors. Intein splicing inhibitors, cisplatin and 6G-318S, showed increased sensitivity to Cam-int compared to the wild-type strain without the intein. This Cam-int fungal strain can serve as a valuable tool for further studying the role of inteins and holds potential for screening intein splicing inhibitors.