Cryptococcus neoformans Prp8 Intein: An In Vivo Target-Based Drug Screening System in Saccharomyces cerevisiae to Identify Protein Splicing Inhibitors and Explore Its Dynamics.

Inteins are genetic mobile elements that are inserted within protein-coding genes, which are usually housekeeping genes. They are transcribed and translated along with the host gene, then catalyze their own splicing out of the host protein, which assumes its functional conformation thereafter. As Prp8 inteins are found in several important fungal pathogens and are absent in mammals, they are considered potential therapeutic targets since inhibiting their splicing would selectively block the maturation of fungal proteins. We developed a target-based drug screening system to evaluate the splicing of Prp8 intein from the yeast pathogen Cryptococcus neoformans (CnePrp8i) using Saccharomyces cerevisiae Ura3 as a non-native host protein. In our heterologous system, intein splicing preserved the full functionality of Ura3. To validate the system for drug screening, we examined cisplatin, which has been described as an intein splicing inhibitor. By using our system, new potential protein splicing inhibitors may be identified and used, in the future, as a new class of drugs for mycosis treatment. Our system also greatly facilitates the visualization of CnePrp8i splicing dynamics in vivo.

PRP8 intein in Ajellomycetaceae family pathogens: sequence analysis, splicing evaluation and homing endonuclease activity.

Inteins are intervening sequences that are transcribed and translated with flanking host protein sequences and then self-excised by protein splicing. Bi-functional inteins also contain a homing endonuclease responsible for their genetic mobility. The PRP8 intein, the most widespread among fungi, occurs in important pathogens such as Histoplasma capsulatum and Paracoccidioides brasiliensis, from the Ajellomycetaceae family. Herein, we describe the bi-functional PRP8 intein in two other Ajellomycetacean pathogens, Blastomyces dermatitidis and Emmonsia parva. Sequence analysis and experimental evidence suggest that the homing endonuclease from PbrPRP8 is inactive. The splicing activity of the PRP8 intein from the B. dermatitidis, E. parva and P. brasiliensis species complex was demonstrated in a non-native protein context in Escherichia coli. Since the PRP8 intein is located in a functionally essential nuclear protein, it can be considered a promising therapeutic target for anti-fungal drugs, because inhibition of intein splicing should inhibit proliferation of intein-containing pathogens.