Gcn2 rescues reprogramming in the absence of Hog1/p38 signaling in C. neoformans during thermal stress.

The fungus Cryptococcus neoformans is an opportunistic pathogen of people that reprograms its translatome to facilitate adaptation and virulence within the host. We studied the role of Hog1/p38 in reprogramming translation during thermal stress adaptation, and found that this pathway acts on translation via crosstalk with the Gcn2 pathway, a well-studied regulator of general translation control. Using a combination of molecular assays and phenotypic analysis, we show that increased output from the Gcn2 pathway in a Hog1 deletion mutant is associated with rescue of thermal stress adaptation at both molecular and phenotypic scales. We characterize known outputs of the Hog1 pathway during thermal stress as either Gcn2-dependent or Gcn2-independent, and demonstrate that Hog1 activation regulates the Gcn2 pathway even in the absence of thermal stress. Finally, we implicate this phenomenon in another Hog1-regulated process, morphogenesis, and recapitulate Hog1-Gcn2 crosstalk in the distantly related fungal pathogen, Candida albicans. Our results point to an important link between the stress response machinery and translation control, and clarify the etiology of phenotypes associated with Hog1 deletion. More broadly, this study highlights complex interplay between core conserved signal transduction pathways and the utility of molecular assays to better understand how these pathways are connected.

Contributions of Ccr4 and Gcn2 to the Translational Response of C. neoformans to Host-Relevant Stressors and Integrated Stress Response Induction.

In response to the host environment, the human pathogen Cryptococcus neoformans must rapidly reprogram its translatome from one which promotes growth to one which is responsive to host stress. In this study, we investigate the two events which comprise translatome reprogramming: the removal of abundant, pro-growth mRNAs from the translating pool, and the regulated entry of stress-responsive mRNAs into the translating pool. Removal of pro-growth mRNAs from the translating pool is controlled primarily by two regulatory mechanisms, repression of translation initiation via Gcn2, and decay mediated by Ccr4. We determined that translatome reprogramming in response to oxidative stress requires both Gcn2 and Ccr4, whereas the response to temperature requires only Ccr4. Additionally, we assessed ribosome collision in response to host-relevant stress and found that collided ribosomes accumulated during temperature stress but not during oxidative stress. The phosphorylation of eIF2α that occurred as a result of translational stress led us to investigate the induction of the integrated stress response (ISR). We found that eIF2α phosphorylation varied in response to the type and magnitude of stress, yet all tested conditions induced translation of the ISR transcription factor Gcn4. However, Gcn4 translation did not necessarily result in canonical Gcn4-dependent transcription. Finally, we define the ISR regulon in response to oxidative stress. In conclusion, this study begins to reveal the translational regulation in response to host-relevant stressors in an environmental fungus which is capable of adapting to the environment inside the human host. IMPORTANCE Cryptococcus neoformans is a human pathogen capable of causing devastating infections. It must rapidly adapt to changing environments as it leaves its niche in the soil and enters the human lung. Previous work has demonstrated a need to reprogram gene expression at the level of translation to promote stress adaptation. In this work, we investigate the contributions and interplay of the major mechanisms that regulate entry of new mRNAs into the pool (translation initiation) and the clearance of unneeded mRNAs from the pool (mRNA decay). One result of this reprogramming is the induction of the integrated stress response (ISR) regulon. Surprisingly, all stresses tested led to the production of the ISR transcription factor Gcn4, but not necessarily to transcription of ISR target genes. Furthermore, stresses result in differential levels of ribosome collisions, but these are not necessarily predictive of initiation repression as has been suggested in the model yeast.

Long Non-Coding RNAs in Cryptococcus neoformans: Insights Into Fungal Pathogenesis.

Long non-coding RNAs (lncRNAs) are highly expressed and can modulate multiple cellular processes including transcription, splicing, translation, and many diverse signaling events. LncRNAs can act as sponges for miRNAs, RNA and DNA binding proteins, functioning as competitive endogenous RNAs. The contribution of lncRNAs to microbial pathogenesis is largely neglected in eukaryotic pathogens despite the abundance of RNA sequencing datasets encompassing conditions of stress, gene deletions and conditions that mimic the host environment. The human fungal pathogen Cryptococcus neoformans encodes 6975 (84%) protein-coding and 1359 (16%) non-protein-coding RNAs, of which 1182 (14.2%) are lncRNAs defined by a threshold of greater than 200 nucleotides in length. Here, we discuss the current state of knowledge in C. neoformans lncRNA biology. Utilizing existing RNA seq datasets, we examine trends in lncRNA expression and discuss potential implications for pathogenesis.

A Conserved Gcn2-Gcn4 Axis Links Methionine Utilization and the Oxidative Stress Response in Cryptococcus neoformans.

The fungal pathogen Cryptococcus neoformans relies on post-transcriptional mechanisms of gene regulation to adapt to stressors it encounters in the human host, such as oxidative stress and nutrient limitation. The kinase Gcn2 regulates translation in response to stress by phosphorylating the initiation factor eIF2, and it is a crucial factor in withstanding oxidative stress in C. neoformans, and amino acid limitation in many fungal species. However, little is known about the role of Gcn2 in nitrogen limitation in C. neoformans. In this study, we demonstrate that Gcn2 is required for C. neoformans to utilize methionine as a source of nitrogen, and that the presence of methionine as a sole nitrogen source induces eIF2 phosphorylation. The stress imposed by methionine leads to an oxidative stress response at both the levels of transcription and translation, as seen through polysome profiling as well as increased abundance of select oxidative stress response transcripts. The transcription factor Gcn4 is also required for methionine utilization and oxidative stress resistance, and RT-qPCR data suggests that it regulates expression of certain transcripts in response to oxidative stress. The results of this study suggest a connection between nitrogen metabolism and oxidative stress in C. neoformans that is mediated by Gcn4, possibly indicating the presence of a compound stress response in this clinically important fungal pathogen.

Cryptococcus neoformans Coinfection Dampens the TNF-α Response in HIV-1-Infected Human THP-1 Macrophages.

Cryptococcus neoformans is a devastating opportunistic fungal pathogen. It mostly impacts people in an immunocompromised state, such as people living with HIV/AIDS and following organ transplantation. Macrophages, in addition to being a major cellular reservoir of HIV-1, represent a unique niche in which both C. neoformans and HIV-1 can coinhabit in the course of natural infection. Here, we report the observation that HIV-1 infection of THP-1 macrophages increases the rate at which they phagocytose C. neoformans cells. We investigated the tumor necrosis factor alpha (TNF-α) signaling and nuclear factor kappa B (NF-κB) activation in human monocyte-derived macrophages infected with HIV-1 alone, as well as those coinfected with HIV-1 and C. neoformans Our findings showed that while HIV-1 infection alone upregulates TNF-α production and activates NF-κB signaling, C. neoformans coinfection drastically and rapidly dampens this proinflammatory response. These data suggest an antagonism between two important human pathogens during coinfection of macrophages.IMPORTANCE Fungal infections are one of the leading causes of death for people who live with HIV/AIDS. Even though these pathogens are independently well studied, it is still enigmatic how coinfection with HIV-1 and C. neoformans alters gene expression and cellular processes, especially in clinically relevant cell types. Understanding the interplay between these two pathogens is especially critical because C. neoformans mortality largely depends on the host's immunocompromised state during viral infection. Studying this coinfection is challenging since HIV-1 only infects human cells, and the modified murine HIV-1 virus does not reproduce the clinical landmarks of HIV-1 infection or AIDS in mice. Our observations shed light on how these two pathogens trigger opposing trends in TNF-α and NF-κB signaling in human monocyte-derived macrophages.

Tools for Assessing Translation in Cryptococcus neoformans.

Cryptococcus neoformans is a ubiquitous environmental fungus capable of establishing an infection in a human host. Rapid changes in environments and exposure to the host immune system results in a significant amount of cellular stress, which is effectively combated at the level of translatome reprogramming. Repression of translation following stress allows for the specific reallocation of limited resources. Understanding the mechanisms involved in regulating translation in C. neoformans during host infection is critical in the development of new antifungal drugs. In this review, we discuss the main tools available for assessing changes in translation state and translational output during cellular stress.

Glucan Unmasking Identifies Regulators of Temperature-Induced Translatome Reprogramming in C. neoformans.

The cell walls of fungi are critical for cellular structure and rigidity but also serve as a major communicator to alert the cell to the changing environment. In response to stresses encountered in human hosts, pathogenic fungi remodel their cell walls. Masking the ß-1,3-glucan component of the cell wall is critical to escape detection by innate immune cells. We previously demonstrated that ß-1,3-glucan is unmasked in response to host temperature stress when translatome reprogramming is defective in Cryptococcus neoformans Here, we used ß-1,3-glucan unmasking as an output to identify signaling modules involved both in masking and in translatome reprogramming in response to host temperature stress. We reveal that the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway is involved in translatome reprogramming and that mutants in this pathway display moderate unmasking when grown at 37°C. Additionally, we show that mutants of the cell wall integrity (CWI)/Mpk1 MAPK pathway extensively unmask ß-1,3-glucan. While the CWI pathway does not impact translatome reprogramming, our data suggest that it may play a role in the posttranslational regulation of transcription factors that govern masking.IMPORTANCECryptococcus neoformans is a fungal pathogen that causes devastating morbidity and mortality in immunocompromised individuals. It possesses several virulence factors that aid in its evasion from the host immune system, including a large polysaccharide capsule that cloaks the antigenic cell wall. Studies investigating how the cell wall is remodeled to keep this pathogen disguised in response to stress have been limited. We previously found that host temperature stress results in translatome reprogramming that is necessary for keeping the highly antigenic ß-(1, 3)-glucan component masked. Our data reveal signaling modules that trigger these responses and suggest the points of regulation at which these pathways act in achieving masking. Understanding these mechanisms may allow for therapeutic manipulation that may promote the immune recognition and clearance of this fungal pathogen.

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

The human fungal pathogen Cryptococcus neoformans is intrinsically resistant to the echinocandin antifungal drug caspofungin, which targets the ß-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 ß-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 ß-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.

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 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.

Thermotolerance in the pathogen Cryptococcus neoformans is linked to antigen masking via mRNA decay-dependent reprogramming.

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 ccr4Δ 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.

Protein Kinases at the Intersection of Translation and Virulence.

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.

CNBP Homologues Gis2 and Znf9 Interact with a Putative G-Quadruplex-Forming 3' Untranslated Region, Altering Polysome Association and Stress Tolerance in Cryptococcus neoformans.

In Cryptococcus neoformans, mRNAs encoding ribosomal proteins (RP) are rapidly and specifically repressed during cellular stress, and the bulk of this repression is mediated by deadenylation-dependent mRNA decay. A motif-finding approach was applied to the 3' untranslated regions (UTRs) of RP transcripts regulated by mRNA decay, and a single, significant motif, GGAUG, was identified. Znf9, a small zinc knuckle RNA binding protein identified by mass spectrometry, was found to interact specifically with the RPL2 3'-UTR probe. A second, homologous protein, Gis2, was identified in the genome of C. neoformans and also bound the 3'-UTR probe, and deletion of both genes resulted in loss of binding in cell extracts. The RPL2 3' UTR contains four G-triplets (GGG) that have the potential to form a G-quadruplex, and temperature gradient gel electrophoresis revealed a potassium-dependent structure consistent with a G-quadruplex that was abrogated by mutation of G-triplets. However, deletion of G-triplets did not abrogate the binding of either Znf9 or Gis2, suggesting that these proteins either bind irrespective of structure or act to prevent structure formation. Deletion of both GIS2 and ZNF9 resulted in a modest increase in basal stability of the RPL2 mRNA which resulted in an association with higher-molecular-weight polysomes under unstressed conditions. The gis2Δ mutant and gis2Δ znf9Δ double mutant exhibited sensitivity to cobalt chloride, fluconazole, and oxidative stress, and although transcriptional induction of ERG25 was similar to that of the wild type, analysis of sterol content revealed repressed levels of sterols in the gis2Δ and gis2Δ znf9Δ double mutant, suggesting a role in translational regulation of sterol biosynthesis.IMPORTANCE 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.

mRNA decay: an adaptation tool for the environmental fungal pathogen Cryptococcus neoformans.

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 For further resources related to this article, please visit the WIREs website.

De Novo Pyrimidine Biosynthesis Connects Cell Integrity to Amphotericin B Susceptibility in Cryptococcus neoformans.

The use of amphotericin B (AmB) in conjunction with 5-fluorocytosine (5-FC) is known to be the optimal therapy for treating cryptococcosis, but the mechanism by which 5-FC synergizes with AmB is unknown. In this study, we generated a Cryptococcus neoformans ura1Δ mutant lacking dihydroorotate dehydrogenase (DHODH), which demonstrated temperature-sensitive growth due to a defect in cell integrity and sensitivity to cell wall-damaging agents. In addition, sensitivity to AmB was greatly increased. Inclusion of uracil or uridine in the medium did not suppress the cell wall or AmB phenotype, whereas complementation with the wild-type URA1 gene complemented the mutant phenotype. As a measure of membrane accessibility, we assayed the rate of association of the lipid-binding dye 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and saw more rapid association in the ura1Δ mutant. We likewise saw an increased rate of DiOC6 association in other AmB-sensitive mutants, including a ura- spontaneous URA5 mutant made by 5-fluoroorotic acid (5-FOA) selection and a bck1Δ mutant defective in cell integrity signaling. Similar results were also obtained by using a specific plasma membrane-binding CellMask live stain, with cell integrity mutants that exhibited increased and faster association of the dye with the membrane. Chitin synthase mutants (chs5Δ and chs6Δ) that lack any reported cell wall defects, in turn, demonstrate neither any increased susceptibility to AmB nor a greater accessibility to either of the dyes. Finally, perturbation of the cell wall of the wild type by treatment with the ß-1,6-glucan synthase inhibitor caspofungin was synergistic with AmB in vitro. IMPORTANCE Synergy between AmB and nucleotide biosynthetic pathways has been documented, but the mechanism of this interaction has not been delineated. Results from this study suggest a correlation between uridine nucleotide biosynthesis and cell integrity likely mediated through the pool of nucleotide-sugar conjugates, which are precursor molecules for both capsule and cell wall of C. neoformans. Thus, we propose a mechanism by which structural defects in the cell wall resulting from perturbation of pyrimidine biosynthesis allow faster and increased penetration of AmB molecules into the cell membrane. Overall, our work demonstrates that impairment of pyrimidine biosynthesis in C. neoformans could be a potential target for antifungal therapy, either alone or in combination with AmB.

Opposing PKA and Hog1 signals control the post-transcriptional response to glucose availability in Cryptococcus neoformans.

The pathogenic fungus Cryptococcus neoformans must adapt to glucose-limited conditions in the lung and glucose replete conditions upon dissemination to the brain. We report that glucose controls ribosome biogenesis and translation by modulating mRNA decay through a balance of PKA and Hog1 signalling. Glucose signalling through PKA stabilized ribosomal protein (RP) mRNAs whereas glucose starvation destabilized RP transcripts through Hog1. Glucose starvation-induced oxidative stress response genes, and treatment of glucose-fed cells with reactive oxygen species (ROS) generating compounds repressed RP transcripts, both of which were dependent on Hog1. Stabilization of RP transcripts led to retention of polysomes in a hog1Δ mutant, whereas stabilization of RP transcripts by cyclic AMP did not affect translation repression, suggesting that Hog1 alone signals translation repression. In sum, this work describes a novel antagonism between PKA and Hog1 controlling ribosome biogenesis via mRNA stability in response to glucose availability in this important human pathogen.

Morphotype-specific effector functions of Cryptococcus neoformans PUM1.

The basidiomycete fungal pathogen Cryptococcus neoformans requires the PUF protein, Pum1, for hyphal morphogenesis during sexual development. In this study we found that Pum1 was auto-repressive under growth as yeast, but that auto-repression was relieved during filamentous growth through utilization of an alternative transcription start site driven by the master filamentation regulator Znf2. In addition, Pum1 was required to stabilize the ZNF2 mRNA through an indirect mechanism suggesting that Znf2 and Pum1 each positively regulate the expression of the other to achieve the filamentous morphotype required for sexual development in Cryptococcus.

Puf4 regulates both splicing and decay of HXL1 mRNA encoding the unfolded protein response transcription factor in Cryptococcus neoformans.

The endoplasmic reticulum (ER) responds to errors in protein folding or processing by induction of the unfolded protein response (UPR). During conditions of ER stress, unconventional splicing of an mRNA encoding the UPR-responsive transcription factor occurs at the ER surface, resulting in activation of the UPR. UPR activation is necessary for adaptation to ER stress and for the pathogenic fungus Cryptococcus neoformans is an absolute requirement for temperature adaptation and virulence. In this study, we have determined that C. neoformans has co-opted a conserved PUF RNA binding protein to regulate the posttranscriptional processing of the HXL1 mRNA encoding the UPR transcription factor. PUF elements were identified in both the 5' and 3' untranslated regions of the HXL1 transcript, and both elements bound Puf4. Deletion of PUF4 resulted in delayed unconventional splicing of HXL1 mRNA and delayed induction of Hxl1 target genes. In addition, the HXL1 transcript was stabilized in the absence of Puf4. The puf4Δ mutant exhibited temperature sensitivity but was as virulent as the wild type, despite a reduction in fungal burden in the brains of infected mice. Our results reveal a novel regulatory role in which a PUF protein influences the unconventional splicing of the mRNA encoding the UPR-responsive transcription factor. These data suggest a unique role for a PUF protein in controlling UPR kinetics via the posttranscriptional regulation of the mRNA encoding the UPR transcription factor Hxl1.

RNA biology and the adaptation of Cryptococcus neoformans to host temperature and stress.

Cryptococcus neoformans is an environmental fungus that can cause severe disease in humans. C. neoformans encounters a multitude of stresses within the human host to which it must adapt in order to survive and proliferate. Upon stressful changes in the external milieu, C. neoformans must reprogram its gene expression to properly respond to and combat stress in order to maintain homeostasis. Several studies have investigated the changes that occur in response to these stresses to begin to unravel the mechanisms of adaptation in this organism. Here, we review studies that have explored stress-induced changes in gene expression with a focus on host temperature adaptation. We compare global messenger RNA (mRNA) expression data compiled from several studies and identify patterns that suggest that orchestrated, transient responses occur. We also utilize the available expression data to explore the possibility of a common stress response that may contribute to cellular protection against a variety of stresses in C. neoformans. In addition, we review studies that have revealed the significance of post-transcriptional mechanisms of mRNA regulation in response to stress, and discuss how these processes may contribute to adaptation and virulence.

Inhibition of nucleotide biosynthesis potentiates the antifungal activity of amphotericin B.

The polyene antifungal agent Amphotericin B exhibits potent and broad spectrum fungicidal activity. However, high nephrotoxicity can hinder its administration in resource poor settings. Quantification of early fungicidal activity in studies of HIV patients with cryptococcosis demonstrate that 5-Fluorocytosine therapy in combination with Amphotericin B results in faster clearance than with Amphotericin B alone. In vitro synergy between the two drugs has also been reported but the mechanism by which 5-Fluorocytosine synergizes with Amphotericin B has not been delineated. In this study we set out to investigate the effect of genetic mutation or pharmacologic repression of de novo pyrimidine and purine biosynthesis pathways on the Amphotericin B susceptibility of Cryptococcus neoformans. We demonstrate that a ura- derivative of wild type Cryptococcus neoformans strain H99 is hypersensitive to Amphotericin B. This sensitivity is remediated by re-introduction of a wild type URA5 gene, but not by addition of exogenous uracil to supplement the auxotrophy. Repression of guanine biosynthesis by treatment with the inosine monophosphate dehydrogenase inhibitor, mycophenolic acid, was synergistic with Amphotericin B as determined by checkerboard analysis. As in Cryptococcus neoformans, a ura(-) derivative of Candida albicans was also hypersensitive to Amphotericin B, and treatment of Candida albicans with mycophenolic acid was likewise synergistic with Amphotericin B. In contrast, neither mycophenolic acid nor 5-FC had an effect on the Amphotericin B susceptibility of Aspergillus fumigatus. These studies suggest that pharmacological targeting of nucleotide biosynthesis pathways has potential to lower the effective dose of Amphotericin B for both C. neoformans and C. albicans. Given the requirement of nucleotide and nucleotide sugars for growth and pathogenesis of Cryptococcus neoformans, disrupting nucleotide metabolic pathways might thus be an effective mechanism for the development of novel antifungal drugs.