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.

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.

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.

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.

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.

Uncoupling of mRNA synthesis and degradation impairs adaptation to host temperature in Cryptococcus neoformans.

The pathogenic fungus Cryptococcus neoformans must overcome multiple stressors to cause disease in its human host. In this study, we report that C. neoformans rapidly and transiently repressed ribosomal protein (RP) transcripts during a transition from 30°C to host temperature. This repression was accompanied by accelerated mRNA degradation mediated by the major deadenylase, Ccr4, and influenced by the dissociable RNA polymerase II subunit, Rpb4. Destabilization and deadenylation of RP transcripts were impaired in an rpb4Δ mutant, suggesting that Rpb4 may be involved in host temperature-induced Ccr4-mediated decay. Accelerated decay of ER stress transcripts 1 h following a shift to host temperature was also impaired in the rpb4Δ mutant. In response to host temperature, Rpb4 moved from the nucleus to the cytoplasm, supporting a role for Rpb4 in coupling transcription and degradation. The PKH signalling pathway was implicated as a regulator of accelerated degradation of the RP transcripts, but not of the ER stress transcripts, revealing a further level of specificity. When transcription and degradation were uncoupled by deletion of Rpb4, growth at host temperature was impaired and virulence was attenuated. These data suggest that mRNA synthesis and decay are coupled in C. neoformans via Rpb4, and this tight coordination promotes host-temperature adaptation and pathogenicity.