Puf4 Mediates Post-transcriptional Regulation of Caspofungin Resistance inCryptococcus neoformans

Abstract

24Echinocandins have been on the market for 20 years, yet they are the newest class of 25 antifungal drugs. The human fungal pathogen Cryptococcus neoformans is intrinsically resistant 26 to the echinocandin antifungal drug caspofungin, which targets the b-1,3-glucan synthase 27 encoded by the FKS1. Analysis of a C. neoformans puf4∆ mutant, lacking the pumilio/FBF RNA 28 binding protein family member Puf4, revealed exacerbated caspofungin resistance. In contrast, 29 overexpression of PUF4 resulted in caspofungin sensitivity. The FKS1 mRNA contains three 30 Puf4-binding elements (PBEs) in its 5' untranslated region. Puf4 binds with specificity to this 31 region of the FKS1. The FKS1 mRNA was destabilized in the puf4∆ mutant, and the abundance 32 of the FKS1 mRNA was reduced compared to wild type, suggesting that Puf4 is a positive 33 regulator FKS1 mRNA stability. In addition to FKS1, the abundance of additional cell wall 34 biosynthesis genes, including chitin synthases (CHS3, CHS4, CHS6) and deacetylases (CDA1, 35 CDA2, CDA3) as well as a b-1,6-glucan synthase gene (SKN1) was regulated by Puf4 during a 36 caspofungin time course. The use of fluorescent dyes to quantify cell wall components revealed 37 that the puf4∆ mutant had increased chitin content, suggesting a cell wall composition that is 38 less reliant on b-1,3-glucan. Overall, our findings suggest a mechanism by which caspofungin 39 resistance, and more broadly, cell wall biogenesis, is regulated post-transcriptionally by Puf4. 40 41 Importance 42 Cryptococcus neoformans is an environmental fungus that causes pulmonary and central 43 nervous system infections. It is also responsible for 15% of AIDS-related deaths. A major 44 contributor to the high morbidity and mortality statistics is the lack of safe and effective 45 antifungal therapies, especially in resource-poor settings. Yet, antifungal drug development has 46 stalled in the pharmaceutical industry. Therefore, it is of importance to understand the mechanism by which C. neoformans is resistant to caspofungin in order to design adjunctive 48 therapies to potentiate its activity toward this important pathogen. 50 Introduction 51Invasive deep mycoses primarily impact immunocompromised populations causing high rates of 52 morbidity and mortality (1, 2). The pathogenic fungus Cryptococcus neoformans is the causative 53 agent of fatal meningitis most often in patients with defects in cell mediated immunity, including 54 transplant recipients and those living with HIV/AIDS (3-6). C. neoformans is responsible of the 55 15% of AIDS-related deaths (6). Treatment of cryptococcosis is difficult and therapeutic options 56 are limited. Even the best combination treatment using Amphotericin B with 5-fluorocytosine (5-57 FC) is not well tolerated, and 5-FC is often unavailable in resource-poor areas (7). Some of the 58 largest clinal challenges to invasive fungal infections are poor efficacy of the drugs, emerging 59 resistance issues, narrow selection and availability of antifungals especially in the areas where...

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

“…In contrast, Puf3 (UGUANAUA [22,29,33]) (Fig. 6c,d), Puf4 (UGUANANUA [22,34,35]) (Fig. 6e,f) and Puf5 (UGUANNNNUA [22,34]) (Fig.…”

Biocidal Resistance in Clinically Relevant Microbial Species: A Major Public Health Risk

“…MDR in clinical isolates as observed in C. albicans , C. glabrata and C. auris can be intrinsic or acquired. Candida krusei and C. auris are intrinsically resistant to fluconazole, whereas Cryptococcus species are intrinsically resistant to caspofungin [ 54 ]. Acquired resistance is a result of prolonged exposure to antifungal therapeutics where subtoxic concentrations of biocides may also induce resistance.…”