Puf4 Mediates Post-transcriptional Regulation of Cell Wall Biosynthesis and Caspofungin Resistance in Cryptococcus neoformans

Abstract

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. neoformans puf4Δ 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. IMPORTANCE Cryptococcus 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.

Effects of sequence motifs in the yeast 3′ untranslated region determined from massively parallel assays of random sequences

“…6 c,d; Additional File 1 : Fig. S6f), Puf4 (UGUANANUA [ 31 , 48 , 49 ]) (Fig. 6 e,f; Additional File 1 : Fig.…”

The interactome of Cryptococcus neoformans Rmt5 reveals multiple regulatory points in fungal cell biology and pathogenesis

“…One of the RBPs that interact with Rmt5 is the pumilio/FBF RBP family member Puf4. Our previous work showed that Puf4 is critical in post-transcriptionally regulating caspofungin resistance (28). Combined with our current Rmt5 interactome data set, we speculate that Rmt5 fine-tunes virulence- and antifungal resistance-associated cellular functions via methylating Puf4 and other effectors.…”

“…Cells were grown overnight at 37°C in YPD broth and then washed with sterile distilled water. The cells were stained with calcofluor white, wheat germ agglutinin (fluorescein isothiocyanate conjugated), eosin Y, and concanavalin A (Alexa Fluor 488 conjugated) as described previously (28) to detect cell wall chitin, exposed chitooligomers, chitosan, and mannoproteins, respectively. Flow cytometry data were acquired using a BD LSRFortessa cell analyzer and analyzed using FlowJo v10.0 software.…”

Replicative Aging Remodels Cell Wall and is Associated with Increased Intracellular Trafficking in Human Pathogenic Yeasts

“…Transcript and protein levels do not always correlate, and unstable mRNA can be translated more efficiently (60). In Cn lacking post-transcriptional gene regulator PUF4 , the FKS1 mRNA and Fks1p levels did not correspond (46). Unfortunately, we were unable to quantify α-glucan with the available methods, but the α-glucan synthase AGS was upregulated in old Cn , indicating that this glucan is likely also increased.…”