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 eIF2α (α subunit of eukaryotic initiation factor 2) by a sole kinase. Preventing eIF2α-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 levels of select oxidative stress response factors. Our results illustrate translational suppression as a critical determinant of select mRNA decay, gene expression, and subsequent survival in response to oxidative stress.IMPORTANCE Fungal 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 in 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 not only furthers our understanding of 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.
Abstract 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 β -1,3-glucan synthase encoded by the FKS1 . Analysis of a C. neoformans puf4 Δ 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 Δ 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 β -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 Δ 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 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.
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.
Abstract A common feature shared by systemic fungal pathogens of environmental origin, such as Cryptococcus neoformans , is their ability to adapt to mammalian core body temperature. In C. neoformans , this adaptation is accompanied by Ccr4-mediated decay of ribosomal protein mRNAs. Here we use the related, but thermo-intolerant species Cryptococcus amylolentus to demonstrate that this response contributes to host-temperature adaptation and pathogenicity of cryptococci. In a C. neoformans ccr4 Δ mutant, stabilized ribosomal protein mRNAs are retained in the translating pool, and stress-induced transcriptomic changes are reduced in comparison with the wild type strain, likely due to ineffective translation of transcription factors. In addition, the mutant displays increased exposure of cell wall glucans, and recognition by Dectin-1 results in increased phagocytosis by lung macrophages, linking mRNA decay to adaptation and immune evasion.
Fungi are ubiquitous in the environment and humans constantly encounter them in the soil, air, water, and food. The vast majority of these interactions are inconsequential. However, in the context of immunodeficiency precipitated by HIV infection, hematologic malignancy, or transplantation, a small subset of fungi can cause devastating, systemic infection. The most deadly of the opportunistic environmental fungi, Cryptococcus neoformans , is estimated to cause hundreds of thousands of deaths per year, mostly in the context of HIV co‐infection. The cellular processes that mediate adaptation to the host environment are of great interest as potential novel therapeutic targets. One such cellular process important for host adaptation is mRNA decay, which mediates the specific degradation of subsets of functionally related mRNAs in response to stressors relevant to pathogenesis, including human core body temperature, carbon limitation, and reactive oxygen stress. Thus, for C. neoformans , host adaptation requires mRNA decay to mediate rapid transcriptome remodeling in the face of stressors encountered in the host. Several nodes of stress‐responsive signaling that govern the stress‐responsive transcriptome also control the decay rate of mRNAs cleared from the ribosome during stress, suggesting an additional layer of coupling between mRNA synthesis and decay that allows C. neoformans to be a successful pathogen of humans. WIREs RNA 2017, 8:e1424. doi: 10.1002/wrna.1424 This article is categorized under: RNA Turnover and Surveillance > Regulation of RNA Stability
Stress adaptation is fundamental to the success of Cryptococcus neoformans as a human pathogen and requires a reprogramming of the translating pool of mRNA. This reprogramming begins with the regulated degradation of mRNAs encoding the translational machinery. The mechanism by which these mRNAs are specified has not been determined. This study has identified a cis element within a G-quadruplex structure that binds two C. neoformans homologues of cellular nucleic acid binding protein (CNBP). These proteins regulate the polysome association of the target mRNA but perform functions related to sterol homeostasis which appear independent of ribosomal protein mRNAs. The presence of two CNBP homologues in C. neoformans suggests a diversification of function of these proteins, one of which appears to regulate sterol biosynthesis and fluconazole sensitivity.
Abstract 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 elF2α by a sole kinase. Preventing elF2α mediated translational suppression resulted in growth sensitivity to hydrogen peroxide (H 2 O 2 ). Our work suggests that translational repression in response to H 2 O 2 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 elF2α, partly restored transcript decay and the expression of the critical oxidative stress response transcript Thioredoxin Reductase 1 (T RR1 ). Our results illustrate translational suppression as a key determinant of select mRNA decay, gene expression, and subsequent survival in response to oxidative stress. Importance Fungal 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 H 2 O 2 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.
As free living organisms, fungi are challenged with a variety of environmental insults that threaten their cellular processes. In some cases, these challenges mimic conditions present within mammals resulting in the accidental selection of virulence factors over evolutionary time. Be it within a host or the soil, fungi must contend with environmental challenges through the production of stress effector proteins while maintaining factors required for viability in any condition. Initiation and upkeep of this balancing act is mainly under the control of kinases that affect the propensity and selectivity of protein translation. This review will focus on kinases in pathogenic fungi that facilitate a virulence phenotype through translational control.
