The human fungal pathogen Cryptococcus neoformans is intrinsically resistant to the echinocandin antifungal drug caspofungin, which targets the β-1,3-glucan synthase encoded by FKS1 Echinocandins have been on the market for 20 years, yet they are the newest class of antifungal drugs. Analysis of a C. neoformanspuf4Δ mutant, lacking the pumilio/FBF RNA binding protein family member Puf4, revealed exacerbated caspofungin resistance. In contrast, overexpression of PUF4 resulted in caspofungin sensitivity. The FKS1 mRNA contains three Puf4-binding elements (PBEs) in its 5' untranslated region. Puf4 binds with specificity to this region of FKS1 The FKS1 mRNA was destabilized in the puf4Δ mutant, and the abundance of the FKS1 mRNA was reduced compared to wild type, suggesting that Puf4 is a positive regulator of FKS1 mRNA stability. In addition to FKS1, the abundance of additional cell wall biosynthesis genes, including chitin synthases (CHS3, CHS4, and CHS6) and deacetylases (CDA1, CDA2, and CDA3) as well as a β-1,6-glucan synthase gene (SKN1), was regulated by Puf4. The use of fluorescent dyes to quantify cell wall components revealed that the puf4Δ mutant had increased chitin content, suggesting a cell wall composition that is less reliant on β-1,3-glucan. Overall, our findings suggest a mechanism by which caspofungin resistance, and more broadly, cell wall biogenesis, is regulated post-transcriptionally by Puf4.IMPORTANCECryptococcus neoformans is an environmental fungus that causes pulmonary and central nervous system infections. It is also responsible for 15% of AIDS-related deaths. A significant contributor to the high morbidity and mortality statistics is the lack of safe and effective antifungal therapies, especially in resource-poor settings. Yet, antifungal drug development has stalled in the pharmaceutical industry. Therefore, it is essential to understand the mechanism by which C. neoformans is resistant to caspofungin to design adjunctive therapies to potentiate the drug's activity toward this important pathogen.

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