Leipheimer, J. | Search bioRxiv

bioRxiv2020

Puf4 Mediates Post-transcriptional Regulation of Caspofungin Resistance in Cryptococcus neoformans

Echinocandins have been on the market for 20 years, yet they are the newest class of antifungal drugs. The human fungal pathogen Cryptococcus neoformans is intrinsically resistant to the echinocandin antifungal drug caspofungin, which targets the {beta}-1,3-glucan synthase encoded by the FKS1. Analysis of a C. neoformans puf4{Delta} 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 the FKS1. The FKS1 mRNA was destabilized in the puf4{Delta} mutant, and the abundance of the FKS1 mRNA was reduced compared to wild type, suggesting that Puf4 is a positive regulator FKS1 mRNA stability. In addition to FKS1, the abundance of additional cell wall biosynthesis genes, including chitin synthases (CHS3, CHS4, CHS6) and deacetylases (CDA1, CDA2, CDA3) as well as a {beta}-1,6-glucan synthase gene (SKN1) was regulated by Puf4 during a caspofungin time course. The use of fluorescent dyes to quantify cell wall components revealed that the puf4{Delta} mutant had increased chitin content, suggesting a cell wall composition that is less reliant on {beta}-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 major 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 of importance to understand the mechanism by which C. neoformans is resistant to caspofungin in order to design adjunctive therapies to potentiate its activity toward this important pathogen.

microbiology↗

bioRxiv2019

Translational Regulation Promotes Oxidative Stress Resistance in the Human Fungal Pathogen Cryptococcus neoformans

Cryptococcus neoformans is one of the few environmental fungi that can survive within a mammalian host and cause disease. Although many of the factors responsible for establishing virulence have been recognized, how they are expressed in response to certain host derived cellular stresses is rarely addressed. Here we characterize the temporal translational response of C. neoformans to oxidative stress. We find that translation is largely inhibited through the phosphorylation of the critical initiation factor elF2 by a sole kinase. Preventing elF2 mediated translational suppression resulted in growth sensitivity to hydrogen peroxide (H2O2). Our work suggests that translational repression in response to H2O2 partly facilitates oxidative stress adaptation by accelerating the decay of abundant non-stress related transcripts while facilitating the proper expression of critical oxidative stress response factors. Carbon starvation, which seems to induce translational suppression that is independent elF2, partly restored transcript decay and the expression of the critical oxidative stress response transcript Thioredoxin Reductase 1 (TRR1). Our results illustrate translational suppression as a key determinant of select mRNA decay, gene expression, and subsequent survival in response to oxidative stress.\n\nImportanceFungal survival in a mammalian host requires the coordinated expression and downregulation of a large cohort of genes in response to cellular stresses. Initial infection with C. neoformans occurs at the lungs, where it interacts with host macrophages. Surviving macrophage derived cellular stresses, such as the production of reactive oxygen and nitrogen species, is believed to promote dissemination into the central nervous system. Therefore, investigating how an oxidative stress resistant phenotype is brought about in C. neoformans furthers our understanding of not only fungal pathogenesis but also unveils mechanisms of stress induced gene reprogramming. We discovered that H2O2 derived oxidative stress resulted in severe translational suppression and that this suppression was necessary for the accelerated decay and expression of tested transcripts. Surprisingly, compounding oxidative stress with carbon starvation resulted in a decrease in peroxide mediated killing, revealing unexpected synergy between stress responses.

microbiology↗