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During chromosome segregation, the spindle assembly checkpoint (SAC) detects errors in kinetochore-microtubule attachments. Timely activation and maintenance of the SAC until defects are corrected is essential for genome stability. Here, we show that shugoshin (Sgo1), a conserved tension-sensing protein, ensures the maintenance of SAC signals in response to unattached kinetochores during mitosis in a basidiomycete budding yeast Cryptococcus neoformans. Sgo1 maintains optimum levels of Aurora B kinase Ipl1 and protein phosphatase 1 (PP1) at kinetochores. The absence of Sgo1 results in the loss of Aurora BIpl1 with a concomitant increase in PP1 levels at kinetochores. This leads to a premature reduction in the kinetochore-bound Bub1 levels and early termination of the SAC signals. Intriguingly, the kinase function of Bub1 is dispensable for shugoshin's subcellular localization. Sgo1 is predominantly localized to spindle pole bodies (SPBs) and along the mitotic spindle with a minor pool at kinetochores. In the absence of proper kinetochore-microtubule attachments, Sgo1 reinforces the Aurora B kinaseIpl1-PP1 phosphatase balance, which is critical for prolonged maintenance of the SAC response. Author summary: Precise segregation of the genome to daughter cells is the key to cell growth, survival, and proliferation. A proper attachment of spindle microtubules to the kinetochore of each eukaryotic chromosome is required for the faithful segregation of the genome. Error correction machinery such as the spindle assembly checkpoint (SAC) prevents chromosome mis-segregation by monitoring and correcting improper attachments by delaying the cell cycle progression. In this study, we probed the role of an evolutionarily conserved protein, shugoshin, in the basidiomycete budding yeast Cryptococcus neoformans in monitoring the kinetochore-MT attachments during mitosis. We show atypical localization of shugoshin to spindle pole bodies (SPBs) and along spindle MTs in this organism. Shugoshin, in C. neoformans, plays a role in maintaining the SAC response to prolong the mitotic arrest in MT depolymerizing conditions. Most strikingly, unlike other systems, the kinase activity of Bub1 is dispensable for the shugoshin's localization and function during mitosis in C. neoformans. Overall, this work reveals the mitotic role of shugoshin in monitoring kinetochore-MT attachments in one of the most critically important human fungal pathogens. [ABSTRACT FROM AUTHOR]

Cryptococcosis is a lethal mycosis instigated by the pathogenic species Cryptococcus neoformans and Cryptococcus gattii, primarily affects the lungs, manifesting as pneumonia, and the brain, where it presents as meningitis. Mortality rate could reach 100% if infections remain untreated in cryptococcal meningitis. Treatment options for cryptococcosis are limited and and there are no licensed vaccines clinically available to treat or prevent cryptococcosis. Our study utilizes an integrated bioinformatics approaches to develop a polyvalent multiepitope subunit vaccine focusing on the key virulent proteins Heat shock transcription factor and Chaperone DnaK of both C. neoformans and C. gatti. Then in silico analysis was done to predict highly antigenic epitopes by assessing antigenicity, transmembrane topology screening, allergenecity, toxicity, and molecular docking approaches. Following this analysis, we designed two vaccine constructs integrating a compatible adjuvant and suitable linkers. These constructs exhibited notable characteristics including high antigenicity, non-toxicity, solubility, stability, and compatibility with Toll-like receptors (TLRs). The interaction between both vaccine constructs and TLR2, TLR3, and TLR9 was assessed through molecular docking analysis. Molecular dynamics simulations and MM-PBSA calculations suggest the substantial stabilizing property and binding affinity of Vaccine Construct V1 against TLR9. Both the vaccines revealed to have a higher number of interchain hydrogen bond with TLR9. These findings serve as a crucial stepping stone towards a comprehensive solution for combating cryptococcus infections induced by both C. neoformans and C. gattii. Further validation through in vivo studies is crucial to confirm the effectiveness and potential of the vaccine to curb the spread of cryptococcosis. Subsequent validation through in vivo studies is paramount to confirm the effectiveness and potential of the vaccine in reducing the spread of cryptococcosis. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is a pathogenic basidiomycetous yeast that primarily infects immunocompromised individuals. Fatal outcome of cryptococcosis depends on the ability of C. neoformans to sense and adapt to 37°C. A complex of conserved filament forming GTPases, called septins, composed of Cdc3, Cdc10, Cdc11, and Cdc12, assembles at the mother-bud neck in C. neoformans. Septins Cdc3 and Cdc12 are essential for proliferation of C. neoformans at 37°C and for virulence in the Galleria mellonella model of infection, presumably due to their requirement for septin complex formation, and the involvement in cytokinesis. However, how exactly Cdc3, and Cdc12 contribute to C. neoformans growth at 37°C remains unknown. Based on studies investigating roles of septins in Saccharomyces cerevisiae, septin complex at the mother-bud neck of C. neoformans is predicted to interact with proteins involved in cell cycle control, morphogenesis, and cytokinesis, but the septin-associated proteome in C. neoformans has not been investigated. Here, we utilized tandem mass spectrometry to define C. neoformans proteins that associate with either Cdc3 or Cdc10 at ∼25°C or after the shift to 37°C. Our findings unveil a diverse array of septin-associated proteins, highlighting potential roles of septins in cell division, and stress response. Two proteins, identified as associated with both Cdc3 and Cdc10, the actin-binding protein profilin, which was detected at both temperatures, and ATP-binding multi-drug transporter Afr1, which was detected exclusively at 37°C, were further confirmed by co-immunoprecipitation. We also confirmed that association of Cdc3 with Afr1 was enhanced at 37°C. Upon shift to 37°C, septins Cdc3 and Cdc10 exhibited altered localization and Cdc3 partially co-localized with Afr1. In addition, we also investigated changes to levels of individual C. neoformans proteins upon shift from ∼25 to 37°C in exponentially grown culture and when cells entered stationary phase at ∼25°C. Our study reveals changes to C. neoformans proteome associated with heat and nutrient deprivation stresses and provides a landscape of septin-associated C. neoformans proteome, which will facilitate elucidating the biology of septins and mechanisms of stress response in this fungal pathogen. [ABSTRACT FROM AUTHOR]

Immune responses during acute infection often contain canonical elements which are shared across the responses to an array of agents within a given pathogen class (i.e., respiratory viral infection). Identification of these shared, canonical elements across similar infections offers the potential for impacting development of novel diagnostics and therapeutics. In this way, analysis of host gene expression patterns ('signatures') in white blood cells has been shown to be useful for determining the etiology of some acute viral and bacterial infections. In order to study conserved immune elements shared across the host response to related pathogens, we performed in vitro human PBMC challenges with common fungal pathogens (Candida albicans, Cryptococcus neoformans and gattii); four strains of influenza virus (Influenza A/Puerto Rico/08/34 [H1N1, PR8], A/Brisbane/59/2007 [H1N1], A/Solomon Islands/3/2006 [H1N1], and A/Wisconsin/67/2005 [H3N2]); and gram-negative (Escherichia coli) and gram-positive (Streptococcus pneumoniae) bacteria. Exposed human cells were then analyzed for differential gene expression utilizing Affymetrix microarrays. Analysis of pathogen exposure of PBMCs revealed strong, conserved gene expression patterns representing these canonical immune response elements to each broad pathogen class. A 41-gene multinomial signature was developed which correctly classified fungal, viral, or bacterial exposure with 94–98% accuracy. Furthermore, a 21-gene signature consisting of a subset of the discriminatory PBMC-derived genes was capable of accurately differentiating human patients with invasive candidiasis, acute viral infection, or bacterial infection (AUC 0.94, 0.83, and 0.96 respectively). These data reinforce the conserved nature of the genomic responses in human peripheral blood cells upon exposure to infectious agents and highlight the potential for in vitro models to augment our ability to develop novel diagnostic classifiers for acute infectious diseases, particularly devastating fungal infections. [ABSTRACT FROM AUTHOR]

The eukaryotic serine/threonine protein phosphatase PP2A is a heterotrimeric enzyme composed of a scaffold A subunit, a regulatory B subunit, and a catalytic C subunit. Of the four known B subunits, the B"' subunit (known as striatin) interacts with the multi-protein striatin-interacting phosphatase and kinase (STRIPAK) complex. Orthologs of STRIPAK components were identified in Cryptococcus neoformans, namely PP2AA/Tpd3, PP2AC/Pph22, PP2AB/Far8, STRIP/Far11, SLMAP/Far9, and Mob3. Structural modeling, protein domain analysis, and detected protein-protein interactions suggest C. neoformans STRIPAK is assembled similarly to the human and fungal orthologs. Here, STRIPAK components Pph22, Far8, and Mob3 were functionally characterized. Whole-genome sequencing revealed that mutations in STRIPAK complex subunits lead to increased segmental and chromosomal aneuploidy, suggesting STRIPAK functions in maintaining genome stability. We demonstrate that PPH22 is a haploinsufficient gene: heterozygous PPH22/pph22Δ mutant diploid strains exhibit defects in hyphal growth and sporulation and have a significant fitness disadvantage when grown in competition against a wild-type diploid. Deletion mutants pph22Δ, far8Δ, and mob3Δ exhibit defects in mating and sexual differentiation, including impaired hyphae, basidia, and basidiospore production. Loss of either PPH22 or FAR8 in a haploid background leads to growth defects at 30°C, severely reduced growth at elevated temperature, abnormal cell morphology, and impaired virulence. Additionally, pph22Δ strains frequently accumulate suppressor mutations that result in overexpression of another putative PP2A catalytic subunit, PPG1. The pph22Δ and far8Δ mutants are also unable to grow in the presence of the calcineurin inhibitors cyclosporine A or FK506, and thus these mutations are synthetically lethal with loss of calcineurin activity. Conversely, mob3Δ mutants display increased thermotolerance, capsule production, and melanization, and are hypervirulent in a murine infection model. Taken together, these findings reveal that the C. neoformans STRIPAK complex plays an important role in genome stability, vegetative growth, sexual development, and virulence in this prominent human fungal pathogen. Author summary: This study focused on a highly conserved protein signaling complex known as STRIPAK, which is important for various developmental processes in fungi and humans. By investigating the functions of this complex in Cryptococcus neoformans, it was discovered to play crucial roles in maintaining genome stability, sexual development, and pathogenesis. In particular, mutations in the genes encoding two subunits of the STRIPAK complex were found to lead to significant defects, including abnormal growth and cell morphology, compromised stress response, and impaired virulence. Interestingly, mutation of a third STRIPAK complex subunit resulted in hypervirulence, characterized by increased thermotolerance, enhanced production of melanin pigment and polysaccharide capsule, and reduced survival in infected animals. Our findings reveal that the STRIPAK complex is an important regulator of growth and virulence of C. neoformans, highlighting its potential as a target for therapies aimed at combating fungal infections. This work furthers understanding of how the STRIPAK complex functions in Cryptococcus, and also in other organisms including humans, where related protein phosphatase complexes govern key cellular processes. [ABSTRACT FROM AUTHOR]

Nickel (Ni) is an abundant element on Earth and it can be toxic to all forms of life. Unlike our knowledge of other metals, little is known about the biochemical response to Ni overload. Previous studies in mammals have shown that Ni induces various physiological changes including redox stress, hypoxic responses, as well as cancer progression pathways. However, the primary cellular targets of nickel toxicity are unknown. Here, we used the environmental fungus Cryptococcus neoformans as a model organism to elucidate the cellular response to exogenous Ni. We discovered that Ni causes alterations in ergosterol (the fungal equivalent of mammalian cholesterol) and lipid biosynthesis, and that the Sterol Regulatory Element-Binding transcription factor Sre1 is required for Ni tolerance. Interestingly, overexpression of the C-4 methyl sterol oxidase gene ERG25, but not other genes in the ergosterol biosynthesis pathway tested, increases Ni tolerance in both the wild type and the sre1Δ mutant. Overexpression of ERG25 with mutations in the predicted binding pocket to a metal cation cofactor sensitizes Cryptococcus to nickel and abolishes its ability to rescue the Ni-induced growth defect of sre1Δ. As overexpression of a known nickel-binding protein Ure7 or Erg3 with a metal binding pocket similar to Erg7 does not impact on nickel tolerance, Erg25 does not appear to simply act as a nickel sink. Furthermore, nickel induces more profound and specific transcriptome changes in ergosterol biosynthetic genes compared to hypoxia. We conclude that Ni targets the sterol biosynthesis pathway primarily through Erg25 in fungi. Similar to the observation in C. neoformans, Ni exposure reduces sterols in human A549 lung epithelial cells, indicating that nickel toxicity on sterol biosynthesis is conserved. Author summary: Nickel is commonly known as an allergen and toxin for humans, but the way in which nickel causes adverse effects is unknown. We sought to use C. neoformans as a model to investigate the primary targets of nickel and how cells tolerate this commonly occurring metal. We found that in both mammalian cells and fungal cells, exposure to nickel causes sterol deficiency. We discovered that Erg25, an essential enzyme key to the production of ergosterol (fungal equivalent of cholesterol), was critical for cryptococcal cells to tolerate nickel. Cells unable to increase production of this enzyme in response to nickel exposure, such as the sre1Δ mutant with the Sterol Regulatory Element-Binding regulator disrupted, were incapable of growing in the presence of nickel. Therefore, it appears that both cells react to nickel through upregulating a conserved biochemical pathway and particularly the Erg25 enzyme. This work could guide future investigations into novel approaches to manage nickel toxicity. [ABSTRACT FROM AUTHOR]

This article discusses the population heterogeneity in Cryptococcus neoformans and its impact on pathogenesis. Cryptococcus neoformans is a prevalent global threat, especially for people living with HIV. The article explores the different ways in which the human-Cryptococcus interaction can progress, including latent/dormant cryptococcosis, pulmonary cryptococcosis, disseminated cryptococcosis, and cryptococcal relapse. The article also examines population heterogeneity in C. neoformans, which provides benefits to the pathogen in coping with changing conditions and contributes to its survival. The specific manifestations of population heterogeneity discussed in the article include heteroresistance, viable but nonculturable state, antifungal persistence, and biofilm formation. The article highlights the influence of population heterogeneity on the survival and pathogenesis of C. neoformans. [Extracted from the article]

Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle checkpoint which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1Δ or mad2Δ strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrest Cryptococcus cells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effective in vitro substrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. We conclude that CnMps1 protein kinase, Mad1 and Mad2 proteins have all conserved their important, spindle checkpoint signalling roles helping ensure high fidelity chromosome segregation. Author summary: Cryptococcus neoformans is an environmental fungus that kills a large fraction of AIDS patients through meningitis. The World Health Organisation recently published a report on disease-causing fungi and ranked C. neoformans at the top, in the critical priority group of fungal pathogens. This position reflects the public health importance of this organism, the lack of effective drugs for fighting its infection, and the need to prioritise its research and development. Cryptococcus undergoes fascinating changes during its life and infection cycle. Here we study a control process regulating normal cell divisions, monitoring the movement of DNA between mother and daughter cells. This surveillance system is known as the spindle checkpoint and we demonstrate that its components (Mps1, Mad1 and Mad2), and their mechanism of action, are well conserved. Knowing this, we can now target subtle changes in this pathway for future drug development. [ABSTRACT FROM AUTHOR]

The position of the nucleus before it divides during mitosis is variable in different budding yeasts. Studies in the pathogenic intron-rich fungus Cryptococcus neoformans reveal that the nucleus moves entirely into the daughter bud before its division. Here, we report functions of a zinc finger motif containing spliceosome protein C. neoformans Slu7 (CnSlu7) in cell cycle progression. The budding yeast and fission yeast homologs of Slu7 have predominant roles for intron 3' splice site definition during pre-mRNA splicing. Using a conditional knockdown strategy, we show CnSlu7 is an essential factor for viability and is required for efficient cell cycle progression with major role during mitosis. Aberrant nuclear migration, including improper positioning of the nucleus as well as the spindle, were frequently observed in cells depleted of CnSlu7. However, cell cycle delays observed due to Slu7 depletion did not activate the Mad2-dependent spindle assembly checkpoint (SAC). Mining of the global transcriptome changes in the Slu7 knockdown strain identified downregulation of transcripts encoding several cell cycle regulators and cytoskeletal factors for nuclear migration, and the splicing of specific introns of these genes was CnSlu7 dependent. To test the importance of splicing activity of CnSlu7 on nuclear migration, we complemented Slu7 knockdown cells with an intron less PAC1 minigene and demonstrated that the nuclear migration defects were significantly rescued. These findings show that CnSlu7 regulates the functions of diverse cell cycle regulators and cytoskeletal components, ensuring timely cell cycle transitions and nuclear division during mitosis. Author summary: Nuclear position in eukaryotic cells is spatio-temporally regulated during the cell cycle. Unlike in Saccharomyces cerevisiae, where nuclear migration to mother-daughter bud neck precedes mitotic segregation, in Cryptococcus neoformans, the nucleus moves entirely into the daughter bud before division. Transcription dynamics during the C. neoformans cell cycle show periodic expression changes and since all nascent pre-mRNAs must be processed to mRNA, splicing is indispensable for gene expression. Yet, how evolutionarily conserved splicing factors modulate the complex intron-rich C. neoformans transcriptome and their impacts on atypical mitotic nuclear position is unexplored. Here, we show CnSlu7 a zinc finger motif spliceosome factor is essential for viability and for cell cycle progression with a major mitotic role. Live imaging of the nucleus, the spindle and other factors revealed significant abnormalities in nuclear migration from mother to daughter cells, as well as nuclear and spindle position defects upon CnSlu7 depletion. Depletion of CnSlu7 altered the transcript status of various cell cycle players, including those critical for nuclear migration, as their introns were CnSlu7 dependent for splicing. Together, these findings highlight the roles of C. neoformans Slu7 in fine-tuning gene expression levels of transcripts that ensures timely cell cycle progression and spatial control of nuclear position. [ABSTRACT FROM AUTHOR]

Elucidating gene function is a major goal in biology, especially among non-model organisms. However, doing so is complicated by the fact that molecular conservation does not always mirror functional conservation, and that complex relationships among genes are responsible for encoding pathways and higher-order biological processes. Co-expression, a promising approach for predicting gene function, relies on the general principal that genes with similar expression patterns across multiple conditions will likely be involved in the same biological process. For Cryptococcus neoformans, a prevalent human fungal pathogen greatly diverged from model yeasts, approximately 60% of the predicted genes in the genome lack functional annotations. Here, we leveraged a large amount of publicly available transcriptomic data to generate a C. neoformans Co-Expression Network (CryptoCEN), successfully recapitulating known protein networks, predicting gene function, and enabling insights into the principles influencing co-expression. With 100% predictive accuracy, we used CryptoCEN to identify 13 new DNA damage response genes, underscoring the utility of guilt-by-association for determining gene function. Overall, co-expression is a powerful tool for uncovering gene function, and decreases the experimental tests needed to identify functions for currently under-annotated genes. Author summary: A central problem in genetics is the connection between genotype and phenotype. Computational approaches to predict gene function can be especially useful for non-model organisms where extensive functional testing has not yet been performed. Co-expression to predict gene function is based on the principle that genes that share similar expression patterns across multiple environmental conditions or perturbations are likely to be involved in the same biological process. Here, we collected transcriptomic data from the Cryptococcus neoformans field and built a robust co-expression network for predicting gene function, especially biological process information. Not only are we able to use this network for retrospective analysis of known gene clusters, but we are also able to make prospective predictions about gene function, including the well-studied processes of capsule and ergosterol biosynthesis. We also discovered a new role for 13 genes in the response to DNA damaging agents, showing that co-expression can reveal new players in conserved biological processes. [ABSTRACT FROM AUTHOR]

Macrophages play a key role in disseminated cryptococcosis, a deadly fungal disease caused by Cryptococcus neoformans. This opportunistic infection can arise following the reactivation of a poorly characterized latent infection attributed to dormant C. neoformans. Here, we investigated the mechanisms underlying reactivation of dormant C. neoformans using an in vitro co-culture model of viable but non-culturable (VBNC; equivalent of dormant) yeast cells with bone marrow-derived murine macrophages (BMDMs). Comparative transcriptome analysis of BMDMs incubated with log, stationary phase or VBNC cells of C. neoformans showed that VBNC cells elicited a reduced transcriptional modification of the macrophage but retaining the ability to regulate genes important for immune response, such as NLRP3 inflammasome-related genes. We further confirmed the maintenance of the low immunostimulatory capacity of VBNC cells using multiplex cytokine profiling, and analysis of cell wall composition and dectin-1 ligands exposure. In addition, we evaluated the effects of classic (M1) or alternative (M2) macrophage polarization on VBNC cells. We observed that intracellular residence sustained dormancy, regardless of the polarization state of macrophages and despite indirect detection of pantothenic acid (or its derivatives), a known reactivator for VBNC cells, in the C. neoformans-containing phagolysosome. Notably, M0 and M2, but not M1 macrophages, induced extracellular reactivation of VBNC cells by the secretion of extracellular vesicles and non-lytic exocytosis. Our results indicate that VBNC cells retain the low immunostimulatory profile required for persistence of C. neoformans in the host. We also describe a pro-pathogen role of macrophage-derived extracellular vesicles in C. neoformans infection and reinforce the impact of non-lytic exocytosis and the macrophage profile on the pathophysiology of cryptococcosis. Author summary: Dormancy enables human opportunistic pathogens to persist in the host in a limited replicative state, establishing a latent infection. Upon immunosuppression, dormant cells can reactivate and resume growth, leading to an active infection. Cryptococcus neoformans is the causative agent of cryptococcosis, a deadly yeast infection characterized by a latent phase. Building upon previous evidence that macrophages serve as a cellular reservoir for inducing and hosting dormant C. neoformans cells, we demonstrated that these phagocytes not only support dormancy, but can also paradoxically promote reactivation. Specifically, non-activated and anti-inflammatory macrophages promote the reactivation of a subset of dormant cells by the release of extracellular vesicles, as well as by yeast expulsion through a phenomenon known as non-lytic exocytosis. Importantly, dormant C. neoformans infection maintains macrophages in a non-inflammatory state required for reactivation. Our findings establish a determining role of macrophages, which is dependent on macrophage phenotype, in the maintenance or reactivation of dormant C. neoformans infection. In addition, we have uncovered a pro-pathogen role of non-lytic exocytosis and extracellular vesicle release in facilitating the reactivation of this model dormant yeast. Our study contributes to the understanding of the pathophysiology of cryptococcosis and potentially other latent infections. [ABSTRACT FROM AUTHOR]

The "Amoeboid Predator-Fungal Animal Virulence Hypothesis" posits that interactions with environmental phagocytes shape the evolution of virulence traits in fungal pathogens. In this hypothesis, selection to avoid predation by amoeba inadvertently selects for traits that contribute to fungal escape from phagocytic immune cells. Here, we investigate this hypothesis in the human fungal pathogens Cryptococcus neoformans and Cryptococcus deneoformans. Applying quantitative trait locus (QTL) mapping and comparative genomics, we discovered a cross-species QTL region that is responsible for variation in resistance to amoeba predation. In C. neoformans, this same QTL was found to have pleiotropic effects on melanization, an established virulence factor. Through fine mapping and population genomic comparisons, we identified the gene encoding the transcription factor Bzp4 that underlies this pleiotropic QTL and we show that decreased expression of this gene reduces melanization and increases susceptibility to amoeba predation. Despite the joint effects of BZP4 on amoeba resistance and melanin production, we find no relationship between BZP4 genotype and escape from macrophages or virulence in murine models of disease. Our findings provide new perspectives on how microbial ecology shapes the genetic architecture of fungal virulence, and suggests the need for more nuanced models for the evolution of pathogenesis that account for the complexities of both microbe-microbe and microbe-host interactions. Author summary: A prominent hypothesis for the evolution of many environmental pathogens proposes that opportunistic pathogenesis is an "accidental" by-product of selection to survive encounters with microbial predators. Chief among the predators that have been suggested as relevant to the evolution of virulence are phagocytic amoebae. Amoebae share many characteristics with macrophages and other primary immune cells that microbial pathogens encounter during infection of animal hosts. This has led to the suggestion that amoebae may act as "training grounds" for both bacterial and fungal pathogens. In this study we test key tenets of the accidental pathogen hypothesis by examining two related questions: "Do alleles important for survival in the face of amoeba predation correspond to known virulence genes? And does genetic variation that increases resistance to amoeba predation increase virulence potential?" We carried out quantitative trait locus (QTL) mapping in two species of the human fungal pathogen Cryptococcus and identified an orthologous QTL, shared by the two species, where allelic variation is a key predictor of resistance to amoeba predation. In C. neoformans we show that this QTL corresponds to a deletion upstream of a transcription factor gene, BZP4. Variation at BZP4 also predicts melanin synthesis, another trait implicated in Cryptococcus virulence. Although BZP4 genotype is a strong predictor of resistance to amoeba predation, we find no correlation between genetic variation at this locus and the ability to proliferate in macrophages or to kill animal hosts. Our findings suggest that the evolutionary landscape of fungal virulence is complex, and highlights the importance of accounting for natural genetic variation when evaluating evolutionary hypotheses. [ABSTRACT FROM AUTHOR]

V-ATPase, which comprises 13–14 subunits, is essential for pH homeostasis in all eukaryotes, but its proper function requires a regulator to assemble its subunits. While RAVE (regulator of H+-ATPase of vacuolar and endosomal membranes) and Raboconnectin-3 complexes assemble V-ATPase subunits in Saccharomyces cerevisiae and humans, respectively, the function of the RAVE complex in fungal pathogens remains largely unknown. In this study, we identified two RAVE complex components, Rav1 and Wdr1, in the fungal meningitis pathogen Cryptococcus neoformans, and analyzed their roles. Rav1 and Wdr1 are orthologous to yeast RAVE and human Rabconnectin-3 counterparts, respectively, forming the hybrid RAVE (hRAVE) complex. Deletion of RAV1 caused severe defects in growth, cell cycle control, morphogenesis, sexual development, stress responses, and virulence factor production, while the deletion of WDR1 resulted in similar but modest changes, suggesting that Rav1 and Wdr1 play central and accessary roles, respectively. Proteomics analysis confirmed that Wdr1 was one of the Rav1-interacting proteins. Although the hRAVE complex generally has V-ATPase-dependent functions, it also has some V-ATPase-independent roles, suggesting a unique role beyond conventional intracellular pH regulation in C. neoformans. The hRAVE complex played a critical role in the pathogenicity of C. neoformans, and RAV1 deletion attenuated virulence and impaired blood-brain barrier crossing ability. This study provides comprehensive insights into the pathobiological roles of the fungal RAVE complex and suggests a novel therapeutic strategy for controlling cryptococcosis. Author summary: Although the assembly regulator of the V-ATPase is critical for its normal function, its pathobiological roles in fungal pathogens are largely unknown, with only yeast and human studies available for structural and functional analyses. In this study, we investigated the function of the RAVE complex in C. neoformans. We found that the cryptococcal RAVE complex had a hybrid format of yeast RAVE and human Raboconnectin-3 complexes and played diverse roles in growth, cell cycle control, morphogenesis, sexual reproduction, virulence factor production, stress responses, and virulence. Furthermore, we demonstrated that the hRAVE complex acts as an assembly regulator of the V-ATPase to maintain pH homeostasis in intra- and extracellular compartments and has unique roles in several stress responses in C. neoformans. These findings provide novel insights into the pathobiological functions of the RAVE complex and suggest its potential as a promising antifungal drug target. [ABSTRACT FROM AUTHOR]

Fungi often adapt to environmental stress by altering their size, shape, or rate of cell division. These morphological changes require reorganization of the cell wall, a structural feature external to the cell membrane composed of highly interconnected polysaccharides and glycoproteins. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that are typically secreted into the extracellular space to catalyze initial oxidative steps in the degradation of complex biopolymers such as chitin and cellulose. However, their roles in modifying endogenous microbial carbohydrates are poorly characterized. The CEL1 gene in the human fungal pathogen Cryptococcus neoformans (Cn) is predicted by sequence homology to encode an LPMO of the AA9 enzyme family. The CEL1 gene is induced by host physiological pH and temperature, and it is primarily localized to the fungal cell wall. Targeted mutation of the CEL1 gene revealed that it is required for the expression of stress response phenotypes, including thermotolerance, cell wall integrity, and efficient cell cycle progression. Accordingly, a cel1Δ deletion mutant was avirulent in two models of C. neoformans infection. Therefore, in contrast to LPMO activity in other microorganisms that primarily targets exogenous polysaccharides, these data suggest that CnCel1 promotes intrinsic fungal cell wall remodeling events required for efficient adaptation to the host environment. Author summary: Fungi need to adapt quickly to environmental stresses to thrive. The fungal cell wall, which supplies support and integrity to the cell, is an essential compartment to react and interact with the surrounding environment. Rapid changes within the carbohydrate composition and architecture occur in response to environmental stresses. Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper-enzymes, secreted by microbes to assist in the first steps of remodeling and degradation of complex and recalcitrant carbohydrates. In this study we explore the role of the putative AA9 family LPMO CnCel1 for growth and virulence of the fungal human pathogen Cryptococcus neoformans. The CEL1 gene is highly up-regulated in presence of host stresses and a cel1Δ mutant strain is avirulent in a murine model of infection. Downstream analysis of virulence-associated phenotypes identified the CEL1 gene to be required for thermotolerance as well as cell wall integrity, and efficient cell cycle progression in presence of host-mimicking stresses. Based upon those findings, we propose that CnCel1 likely promotes intrinsic fungal cell wall remodeling events essential for adaptation to the host environment. [ABSTRACT FROM AUTHOR]

The encapsulated fungus Cryptococcus neoformans is the most common cause of fungal meningitis, with the highest rate of disease in patients with AIDS or immunosuppression. This microbe enters the human body via inhalation of infectious particles. C. neoformans capsular polysaccharide, in which the major component is glucuronoxylomannan (GXM), extensively accumulates in tissues and compromises host immune responses. C. neoformans travels from the lungs to the bloodstream and crosses to the brain via transcytosis, paracytosis, or inside of phagocytes using a "Trojan horse" mechanism. The fungus causes life-threatening meningoencephalitis with high mortality rates. Hence, we investigated the impact of intranasal exogenous GXM administration on C. neoformans infection in C57BL/6 mice. GXM enhances cryptococcal pulmonary infection and facilitates fungal systemic dissemination and brain invasion. Pre-challenge of GXM results in detection of the polysaccharide in lungs, serum, and surprisingly brain, the latter likely reached through the nasal cavity. GXM significantly alters endothelial cell tight junction protein expression in vivo, suggesting significant implications for the C. neoformans mechanisms of brain invasion. Using a microtiter transwell system, we showed that GXM disrupts the trans-endothelial electrical resistance, weakening human brain endothelial cell monolayers co-cultured with pericytes, supportive cells of blood vessels/capillaries found in the blood-brain barrier (BBB) to promote C. neoformans BBB penetration. Our findings should be considered in the development of therapeutics to combat the devastating complications of cryptococcosis that results in an estimated ~200,000 deaths worldwide each year. Author summary: Cryptococcus neoformans infection of the central nervous system (CNS) typically begins by inhalation of fungal spores and results in devastating mortality rates worldwide. Over 200,000 deaths have been reported annually, with cryptococcal meningoencephalitis being the most severe form of the disease. This study investigates the ability of the fungus to invade, colonize, and cause damage to the host through properties of the fungal polysaccharide capsule, which allows the microbe a variety of both protective and offensive abilities. This capsule, made primarily of the polysaccharide glucuronoxylomannan (GXM), has been implicated in the progression and severity of cryptococcal infection in the CNS. We determined that GXM increases the fungal burden in the lungs of mice and enhances fungal migration to the brain. Interaction of GXM with the blood-brain barrier, which is a protective structure that regulates movement of particles into the CNS, demonstrated that GXM can disrupt the integrity of this barrier, compromising the delicate balances of fluids, immune cells, and other factors vital to the maintenance of the CNS. The findings of this study reveal the substantial role of GXM in establishing C. neoformans infection in the brain and necessitate future studies to further understand these interactions. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans (CN) cells survive within the acidic phagolysosome of macrophages (MΦ) for extended times, then escape without impacting the viability of the host cell via a phenomenon that has been coined 'vomocytosis'. Through this mechanism, CN disseminate throughout the body, sometimes resulting in a potentially fatal condition—Cryptococcal Meningitis (CM). Justifiably, vomocytosis studies have focused primarily on MΦ, as alveolar MΦ within the lung act as first responders that ultimately expel this fungal pathogen. Herein, we hypothesize that dendritic cells (DCs), an innate immune cell with attributes that include phagocytosis and antigen presentation, can also act as 'vomocytes'. Presciently, this report shows that vomocytosis of CN indeed occurs from murine, bone marrow-derived DCs. Primarily through time-lapse microscopy imaging, we show that rates of vomocytosis events from DCs are comparable to those seen from MΦ and further, are independent of the presence of the CN capsule and infection ratios. Moreover, the phagosome-altering drug bafilomycin A inhibits this phenomenon from DCs. Although DC immunophenotype does not affect the total number of vomocytic events, we observed differences in the numbers of CN per phagosome and expulsion times. Interestingly, these observations were similar in murine, bone marrow-derived MΦ. This work not only demonstrates the vomocytic ability of DCs, but also investigates the complexity of vomocytosis regulation in this cell type and MΦ under multiple modulatory conditions. Understanding the vomocytic behavior of different phagocytes and their phenotypic subtypes is needed to help elucidate the full picture of the dynamic interplay between CN and the immune system. Critically, deeper insight into vomocytosis could reveal novel approaches to treat CM, as well as other immune-related conditions. [ABSTRACT FROM AUTHOR]

Background: Cryptococcosis is a devastating opportunistic infection in immunocompromised individuals, primarily in people living with HIV/AIDS. This study evaluated a protocol for the early diagnosis of meningitis due to C. neoformans, utilizing established molecular techniques from serum and CSF samples. Methods: The 18S and 5.8S (rDNA-ITS) sequence-specific nested PCR assays were compared with direct India ink staining and the latex agglutination test for detection of C. neoformans in serum and cerebrospinal fluid (CSF) from 49 Brazilian suspected meningitis patients. Results were validated with samples obtained from 10 patients negative for cryptococcosis and HIV, and by analysis of standard C. neoformans strains. Principal findings: The 5.8S DNA-ITS PCR was more sensitive (89–100%) and specific (100%) than the 18S rDNA PCR and conventional tests (India ink staining and latex agglutination) for identification of C. neoformans. While the 18S PCR exhibited a sensitivity (72%) similar to that of the latex agglutination assay in serum samples, it was superior to the latex agglutination assay when testing CSF, with a sensitivity of 84%. However, the latex agglutination was superior to the 18SrDNA PCR in specificity in CSF (92%). The 5.8S DNA-ITS PCR yielded the highest levels of accuracy (96–100%) of any test for detection (serological and mycological) of C. neoformans in both serum and CSF. Conclusion: Use of the nested 5.8S PCR was superior to other techniques for the diagnosis of cryptococcosis. The possibility of using serum, a non-invasively collected material, in a targeted 5.8S PCR analysis to identify Cryptococcus spp. is recommended, especially in immunosuppressed patients. Our results indicate that nested 5.8S PCR can increase the diagnostic capability of cryptococcosis, and we suggest its use to monitor patients in the future. Author summary: Cryptococcal meningitis is an infectious disease of global importance with high morbidity and mortality, especially among individuals with HIV/AIDS. While there have been improvements in the last two decades in the diagnosis of Cryptococcus neoformans, the methods presently employed are problematic for public hospitals in Brazil and other locations due to their extreme cost. In this study, we present a low-cost option for detection and identification of C. neoformans in noninvasive serum sample in immunosuppressed individuals, including those with HIV/AIDS. A nested PCR (5.8SrDNA-ITS) associated with the latex agglutination test has high precision in detection of suspected Cryptococcus spp. [ABSTRACT FROM AUTHOR]

Basidiospores Basidia are specialized cells that are generated from the apical cell in the hyphae. Morphological transition to titan cells One of the most unique and characteristic abilities of cryptococcal cells is the capacity for dramatic changes in size of the encapsulated cells (Fig 1). Cryptococcal cell morphology affects host cell interactions and pathogenicity. In I Cryptococcus i , pseudohyphae are chains of incompletely separated yeast cells that resemble true hyphae but are separated by constrictions between cells rather than septa [[9]]. [Extracted from the article]

Cryptococcosis is a potentially lethal fungal infection of humans caused by organisms within the Cryptococcus neoformans/gattii species complex. Whilst C. neoformans is a relatively common pathogen of immunocompromised individuals, C. gattii is capable of acting as a primary pathogen of immunocompetent individuals. Within the host, both species undergo morphogenesis to form titan cells: exceptionally large cells that are critical for disease establishment. To date, the induction, defining attributes, and underlying mechanism of titanisation have been mainly characterized in C. neoformans. Here, we report the serendipitous discovery of a simple and robust protocol for in vitro induction of titan cells in C. gattii. Using this in vitro approach, we reveal a remarkably high capacity for titanisation within C. gattii, especially in strains associated with the Pacific Northwest Outbreak, and characterise strain-specific differences within the clade. In particular, this approach demonstrates for the first time that cell size changes, DNA amplification, and budding are not always synchronous during titanisation. Interestingly, however, exhibition of these cell cycle phenotypes was correlated with genes associated with cell cycle progression including CDC11, CLN1, BUB2, and MCM6. Finally, our findings reveal exogenous p-Aminobenzoic acid to be a key inducer of titanisation in this organism. Consequently, this approach offers significant opportunities for future exploration of the underlying mechanism of titanisation in this genus. Author summary: Cryptococcus gattii is a fungal pathogen that causes lethal infections in humans and other animals. Upon entry to the lung, some (but not all) cryptococcal cells are induced to become so-called 'titan cells'; huge cells with thickened cell walls, altered capsule and highly duplicated DNA. As a key virulence determinant, Titan cells can manipulate the host immune system (avoiding phagocytosis and triggering a non-protective immune response) and enhance dissemination to the brain. Thus far, the process of forming titan cells has been largely studied in the related species C. neoformans. Here we describe a new in vitro method to induce titan cells in C. gattii. Using this approach, we discovered the novel, asynchronous progression of cell cycle phenotypes during titanisation and showed how titan formation is strongly influenced by population density as well as the external environment; in particular, the presence of the molecule p-aminobenzoic acid. [ABSTRACT FROM AUTHOR]

The fungus Cryptococcus neoformans is a major human pathogen with a remarkable intracellular survival strategy that includes exiting macrophages through non-lytic exocytosis (Vomocytosis) and transferring between macrophages (Dragotcytosis) by a mechanism that involves sequential events of non-lytic exocytosis and phagocytosis. Vomocytosis and Dragotcytosis are fungal driven processes, but their triggers are not understood. We hypothesized that the dynamics of Dragotcytosis could inherit the stochasticity of phagolysosome acidification and that Dragotcytosis was triggered by fungal cell stress. Consistent with this view, fungal cells involved in Dragotcytosis reside in phagolysosomes characterized by low pH and/or high oxidative stress. Using fluorescent microscopy, qPCR, live cell video microscopy, and fungal growth assays we found that the that mitigating pH or oxidative stress reduced Dragotcytosis frequency, whereas ROS susceptible mutants of C. neoformans underwent Dragotcytosis more frequently. Dragotcytosis initiation was linked to phagolysosomal pH, oxidative stresses, and macrophage polarization state. Dragotcytosis manifested stochastic dynamics thus paralleling the dynamics of phagosomal acidification, which correlated with the inhospitality of phagolysosomes in differently polarized macrophages. Hence, randomness in phagosomal acidification randomly created a population of inhospitable phagosomes where fungal cell stress triggered stochastic C. neoformans non-lytic exocytosis dynamics to escape a non-permissive intracellular macrophage environment. Author summary: Host macrophages do not have prior information about the pathogens they ingest. Hence, they have no information about the threats from or vulnerabilities of microbes they phagocytose. Consequently, macrophages must hedge bets in their defense strategies when combatting pathogens with unknown vulnerabilities. This results in a wide variety of phagosomal environments which maximizes the overall antimicrobial ability of the entire population of phagosomes but individual phagosomes may or may not be hostile to a specific pathogen. Using live cell microscopy to track in vitro infections, viability staining, qPCR, and enzyme activity assays we investigated the relationship between phagosomal cell stress and Dragotcytosis, or fungal macrophage-to-macrophage transfer. We have found that Cryptococcus neoformans game this system by transferring to new phagosomes when it finds itself in too hostile of an environment. Specifically, this process is triggered when the yeast experiences overwhelming cellular stress. These findings illustrate a system in which C. neoformans is detecting cellular damage as a trigger for the transfer process and the stochastic nature of microbe-macrophage interactions at the cellular level. [ABSTRACT FROM AUTHOR]

As is the case globally, Cryptococcus gattii is a less frequent cause of cryptococcosis than Cryptococcus neoformans in South Africa. We performed multilocus sequence typing (MLST) and fluconazole susceptibility testing of 146 isolates randomly selected from 750 South African patients with C. gattii disease identified through enhanced laboratory surveillance, 2005 to 2013. The dominant molecular type was VGIV (101/146, 70%), followed by VGI (40/146, 27%), VGII (3/146, 2%) and VGIII (2/146, 1%). Among the 146 C. gattii isolates, 99 different sequence types (STs) were identified, with ST294 (14/146, 10%) and ST155 (10/146, 7%) being most commonly observed. The fluconazole MIC50 and MIC90 values of 105 (of 146) randomly selected C. gattii isolates were 4 μg/ml and 16 μg/ml, respectively. VGIV isolates had a lower MIC50 value compared to non-VGIV isolates, but these values were within one double-dilution of each other. HIV-seropositive patients had a ten-fold increased adjusted odds of a VGIV infection compared to HIV-seronegative patients, though with small numbers (99/136; 73% vs. 2/10; 20%), the confidence interval (CI) was wide (95% CI: 1.93–55.31, p = 0.006). Whole genome phylogeny of 98 isolates of South Africa's most prevalent molecular type, VGIV, identified that this molecular type is highly diverse, with two interesting clusters of ten and six closely related isolates being identified, respectively. One of these clusters consisted only of patients from the Mpumalanga Province in South Africa, suggesting a similar environmental source. This study contributed new insights into the global population structure of this important human pathogen. Author summary: Cryptococcus is the most common cause of meningitis among adults in South Africa. Most human disease is caused by the members of two species complexes within the genus, Cryptococcus neoformans and Cryptococcus gattii. The environmental range of these species complexes, both found in soil, overlaps in southern Africa though C. gattii is a less common human pathogen. C. gattii is divided into six molecular types: VGI, VGII, VGIII, VGIV, VGV and VGVI. In earlier molecular epidemiology studies including relatively few isolates, most southern African isolates were confirmed as molecular type VGIV. We aimed to determine the molecular diversity of C. gattii in South Africa by genotyping patient isolates obtained through laboratory surveillance, 2005–2013. We confirmed that VGIV was the dominant molecular type and that HIV-seropositive patients were more likely to be infected with VGIV compared to those HIV-seronegative. Analysis of the genomes of South African VGIV isolates revealed that they spanned the whole VGIV clade and confirmed that most isolates did not cluster specifically. However, we observed two interesting clusters of closely related isolates, consisting of patients from three neighbouring provinces in South Africa, suggesting a similar environmental source. Further studies of clinical and environmental African C. gattii isolates are needed to gain a better understanding of this pathogen. [ABSTRACT FROM AUTHOR]

Copper homeostasis mechanisms are essential for microbial adaption to changing copper levels within the host during infection. In the opportunistic fungal pathogen Cryptococcus neoformans (Cn), the Cn Cbi1/Bim1 protein is a newly identified copper binding and release protein that is highly induced during copper limitation. Recent studies demonstrated that Cbi1 functions in copper uptake through the Ctr1 copper transporter during copper limitation. However, the mechanism of Cbi1 action is unknown. The fungal cell wall is a dynamic structure primarily composed of carbohydrate polymers, such as chitin and chitosan, polymers known to strongly bind copper ions. We demonstrated that Cbi1 depletion affects cell wall integrity and architecture, connecting copper homeostasis with adaptive changes within the fungal cell wall. The cbi1Δ mutant strain possesses an aberrant cell wall gene transcriptional signature as well as defects in chitin / chitosan deposition and exposure. Furthermore, using Cn strains defective in chitosan biosynthesis, we demonstrated that cell wall chitosan modulates the ability of the fungal cell to withstand copper stress. Given the previously described role for Cbi1 in copper uptake, we propose that this copper-binding protein could be involved in shuttling copper from the cell wall to the copper transporter Ctr1 for regulated microbial copper uptake. Author summary: Microorganisms must be equipped to readily acquire essential micro-nutrients like copper from nutritionally poor environments while simultaneously shielding themselves from conditions of metal excess. We explored mechanisms of microbial copper homeostasis in the human opportunistic fungal pathogen Cryptococcus neoformans (Cn) by defining physiological roles of the newly described copper-binding and release protein Cn Cbi1/Bim1. Highly induced during copper limitation, Cbi1 has been shown to interact with the high-affinity copper transporter Ctr1. We defined Cbi1-regulated changes in the fungal cell wall, including controlling levels and surface exposure of the structural carbohydrates chitin and chitosan during copper and host-derived stress. These polysaccharides are embedded deeply in the cell wall and are known to avidly bind copper. Our data suggest a model in which the fungal cell wall, especially the chito-oligomer layer, serves as a copper-binding structure. Also, alterations in the cell wall polysaccharide content affect the ability to withstand excess copper. Given its ability to bind and release copper, the Cbi1 protein likely shuttles copper from the cell wall to copper transporters for regulated copper acquisition. [ABSTRACT FROM AUTHOR]

Pathogenic Potential (PP) is a mathematical description of an individual microbe, virus, or parasite's ability to cause disease in a host, given the variables of inoculum, signs of disease, mortality, and in some instances, median survival time of the host. We investigated the relationship between pathogenic potential (PP) and infective inoculum (I) using two pathogenic fungi in the wax moth Galleria mellonella with mortality as the relevant outcome. Our analysis for C. neoformans infection revealed negative exponential relationship between PP and I. Plotting the log(I) versus the Fraction of animals with signs or symptoms (Fs) over median host survival time (T) revealed a linear relationship, with a slope that varied between the different fungi studied and a y-intercept corresponding to the inoculum that produced no signs of disease. The I vs Fs/T slope provided a measure of the pathogenicity of each microbial species, which we call the pathogenicity constant or kPath. The kPath provides a new parameter to quantitatively compare the relative virulence and pathogenicity of microbial species for a given host. In addition, we investigated the PP and Fs/T from values found in preexisting literature. Overall, the relationship between Fs/T and PP versus inoculum varied among microbial species and extrapolation to zero signs of disease allowed the calculation of the lowest pathogenic inoculum (LPI) of a microbe. Microbes tended to fall into two groups: those with positive linear relationships between PP and Fs/T vs I, and those that had a negative exponential PP vs I relationship with a positive logarithmic Fs/T vs I relationship. The microbes with linear relationships tended to be bacteria, whereas the exponential-based relationships tended to be fungi or higher order eukaryotes. Differences in the type and sign of the PP vs I and Fs/T vs I relationships for pathogenic microbes suggest fundamental differences in host-microbe interactions leading to disease. Author summary: The ability of a microbe, virus, or parasite to cause disease is dependent on multiple factors, virulence factors. host immune defenses, the infective inoculum, and the type of immune response. For many microbes their capacity for causing disease is highly dependent on the inoculum. The mathematical formula for Pathogenic Potential (PP) is a way to compare the ability of an organism to have a pathogenic effect, as measured by Fraction with signs or symptoms (Fs), mortality (M), and inoculum (I), and can include the median survival time of the host (T). Increasing inoculum of the fungus Cryptococcus neoformans for a moth host resulted in exponentially smaller pathogenic potential, and the Fs/T versus inoculum plot showed a logarithmic relationship. Together, these relationships show diminishing returns with increasing cryptococcal inoculum, in which each individual fungus plays a smaller role in pathogenicity. Literature data shows that other microbes, mostly bacteria, had linear Fs/T versus inoculum relationships, which indicate that each bacterium contributed an equal amount to pathogenicity. These differences in relationships can point to differences in host-microbe interactions and suggest new ways in which the organism causes disease. [ABSTRACT FROM AUTHOR]

Meningitis caused by infectious pathogens is associated with vessel damage and infarct formation, however the physiological cause is often unknown. Cryptococcus neoformans is a human fungal pathogen and causative agent of cryptococcal meningitis, where vascular events are observed in up to 30% of patients, predominantly in severe infection. Therefore, we aimed to investigate how infection may lead to vessel damage and associated pathogen dissemination using a zebrafish model that permitted noninvasive in vivo imaging. We find that cryptococcal cells become trapped within the vasculature (dependent on their size) and proliferate there resulting in vasodilation. Localised cryptococcal growth, originating from a small number of cryptococcal cells in the vasculature was associated with sites of dissemination and simultaneously with loss of blood vessel integrity. Using a cell-cell junction tension reporter we identified dissemination from intact blood vessels and where vessel rupture occurred. Finally, we manipulated blood vessel tension via cell junctions and found increased tension resulted in increased dissemination. Our data suggest that global vascular vasodilation occurs following infection, resulting in increased vessel tension which subsequently increases dissemination events, representing a positive feedback loop. Thus, we identify a mechanism for blood vessel damage during cryptococcal infection that may represent a cause of vascular damage and cortical infarction during cryptococcal meningitis. Author summary: Meningitis is a life threatening form of infection in the brain that is difficult to treat. How infection spreads from the blood to cause meningitis is not well understood. Here we have shown how infection with the fungus Cryptococcus neoformans can be spread from the blood by blocking and bursting blood vessels. Using zebrafish larvae, we were able to follow the same infections over a period of days to understand how this infection behaves in blood vessels. We found that fungal cells become stuck within blood vessels depending on their size. These cells grow within blood vessels, resulting in the blood vessels becoming wider. We measured increased tension in blood vessels suggesting that, with the bloackage and widening of vessels, there was increased local blood pressure. We found that vessel blockage was associated with their rupture and spreading of fungus into the surround tissue. Finally, by increasing the tension in vessels we could increase the number of blood bursting events supporting our conclusion that blood vessel blockage leads to the spread of the infection outside of blood vessels. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is a fungus that is able to survive abnormally high levels of ionizing radiation (IR). The radiolysis of water by IR generates reactive oxygen species (ROS) such as H2O2 and OH-. C. neoformans withstands the damage caused by IR and ROS through antioxidant production and enzyme-catalyzed breakdown of ROS. Given these particular cellular protein needs, questions arise whether transfer ribonucleic acids molecules (tRNAs) undergo unique chemical modifications to maintain their structure, stability, and/or function under such environmental conditions. Here, we investigated the effects of IR and H2O2 exposure on tRNAs in C. neoformans. We experimentally identified the modified nucleosides present in C. neoformans tRNAs and quantified changes in those modifications upon exposure to oxidative conditions. To better understand these modified nucleoside results, we also evaluated tRNA pool composition in response to the oxidative conditions. We found that regardless of environmental conditions, tRNA modifications and transcripts were minimally affected. A rationale for the stability of the tRNA pool and its concomitant profile of modified nucleosides is proposed based on the lack of codon bias throughout the C. neoformans genome and in particular for oxidative response transcripts. Our findings suggest that C. neoformans can rapidly adapt to oxidative environments as mRNA translation/protein synthesis are minimally impacted by codon bias. [ABSTRACT FROM AUTHOR]

The yeast-to-hypha transition is tightly associated with pathogenicity in many human pathogenic fungi, such as the model fungal pathogen Cryptococcus neoformans, which is responsible for approximately 180,000 deaths annually. In this pathogen, the yeast-to-hypha transition can be initiated by distinct stimuli: mating stimulation or glucosamine (GlcN), the monomer of cell wall chitosan. However, it remains poorly understood how the signal specificity for Cryptococcus morphological transition by disparate stimuli is ensured. Here, by integrating temporal expression signature analysis and phenome-based clustering evaluation, we demonstrate that GlcN specifically triggers a unique cellular response, which acts as a critical determinant underlying the activation of GlcN-induced filamentation (GIF). This cellular response is defined by an unusually hyperactive cell wall synthesis that is highly ATP-consuming. A novel cell surface protein Gis1 was identified as the indicator molecule for the GlcN-induced cell wall response. The Mpk1-directed cell wall pathway critically bridges global cell wall gene induction and intracellular ATP supply, ensuring the Gis1-dependent cell wall response and the stimulus specificity of GIF. We further reveal that the ability of Mpk1 to coordinate the cell wall response and GIF activation is conserved in different Cryptococcus pathogens. Phosphoproteomics-based profiling together with genetic and phenotypic analysis revealed that the Mpk1 kinase mediates the regulatory specificity of GIF through a coordinated downstream regulatory network centered on Skn7 and Crz1. Overall, our findings discover an unprecedented and conserved cell wall biosynthesis-dependent fungal differentiation commitment mechanism, which enables the signal specificity of pathogenicity-related dimorphism induced by GlcN in Cryptococcus pathogens. Author summary: Many human fungal pathogens can undergo dimorphic transition between yeast and hyphal forms in response to different external stimuli, and this morphological transition is generally and critically linked with their infections. In Cryptococcus neoformans, a model pathogenic fungus, the yeast-to-hypha transition can be elicited by mating stimulation or glucosamine (GlcN), the monomer of cell wall chitosan. Here, we show that GlcN specifically evokes a unique hyperactive cell wall synthetic response, which determines GlcN-induced filamentation (GIF) as a key commitment event. The Mpk1-directed cell wall signaling pathway as a core and conserved cascade connects the cell wall synthetic response and GIF activation in different Cryptococcus pathogens. Overall, the findings reveal a previously unrecognized function of GlcN in stimulating cell wall signaling and biosynthetic machinery, which enables a unique dimorphism commitment mechanism underlying the signal specificity of the mating-independent yeast-to-hypha transition in Cryptococcus pathogens. [ABSTRACT FROM AUTHOR]

Hybridization has resulted in the origin and variation in extant species, and hybrids continue to arise despite pre- and post-zygotic barriers that limit their formation and evolutionary success. One important system that maintains species boundaries in prokaryotes and eukaryotes is the mismatch repair pathway, which blocks recombination between divergent DNA sequences. Previous studies illuminated the role of the mismatch repair component Msh2 in blocking genetic recombination between divergent DNA during meiosis. Loss of Msh2 results in increased interspecific genetic recombination in bacterial and yeast models, and increased viability of progeny derived from yeast hybrid crosses. Hybrid isolates of two pathogenic fungal Cryptococcus species, Cryptococcus neoformans and Cryptococcus deneoformans, are isolated regularly from both clinical and environmental sources. In the present study, we sought to determine if loss of Msh2 would relax the species boundary between C. neoformans and C. deneoformans. We found that crosses between these two species in which both parents lack Msh2 produced hybrid progeny with increased viability and high levels of aneuploidy. Whole-genome sequencing revealed few instances of recombination among hybrid progeny and did not identify increased levels of recombination in progeny derived from parents lacking Msh2. Several hybrid progeny produced structures associated with sexual reproduction when incubated alone on nutrient-rich medium in light, a novel phenotype in Cryptococcus. These findings represent a unique, unexpected case where rendering the mismatch repair system defective did not result in increased meiotic recombination across a species boundary. This suggests that alternative pathways or other mismatch repair components limit meiotic recombination between homeologous DNA and enforce species boundaries in the basidiomycete Cryptococcus species. Author summary: Several mechanisms enforce species boundaries by either preventing the formation of hybrid zygotes, known as pre-zygotic barriers, or preventing the viability and fecundity of hybrids, known as post-zygotic barriers. Despite these barriers, interspecific hybrids form at an appreciable frequency, such as hybrid isolates of the human fungal pathogenic species, Cryptococcus neoformans and Cryptococcus deneoformans, which are regularly isolated from both clinical and environmental sources. C. neoformans x C. deneoformans hybrids are typically highly aneuploid, sterile, and display phenotypes intermediate to those of either parent, although self-fertile isolates and transgressive phenotypes have been observed. One important mechanism known to enforce species boundaries or lead to incipient speciation is the DNA mismatch repair system, which blocks recombination between divergent DNA sequences during meiosis. The aim of this study was to determine if genetically deleting the DNA mismatch repair component Msh2 would relax the species boundary between C. neoformans and C. deneoformans. Progeny derived from C. neoformans x C. deneoformans crosses in which both parental strains lacked Msh2 had higher viability, and unlike previous studies in Saccharomyces, these Cryptococcus hybrid progeny had higher levels of aneuploidy and no observable increase in meiotic recombination at the whole-genome level. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is a pathogenic basidiomycetous yeast that primarily infects immunocompromised individuals. C. neoformans can thrive during infections due to its three main virulence-related characteristics: the ability to grow at host temperature (37°C), formation of carbohydrate capsule, and its ability to produce melanin. C. neoformans strains lacking septin proteins Cdc3 or Cdc12 are viable at 25°C; however, they fail to proliferate at 37°C and are avirulent in the murine model of infection. The basis of septin contribution to growth at host temperature remains unknown. Septins are a family of conserved filament-forming GTPases with roles in cytokinesis and morphogenesis. In the model organism Saccharomyces cerevisiae septins are essential. S. cerevisiae septins form a higher order complex at the mother-bud neck to scaffold over 80 proteins, including those involved in cell wall organization, cell polarity, and cell cycle control. In C. neoformans, septins also form a complex at the mother-bud neck but the septin interacting proteome in this species remains largely unknown. Moreover, it remains possible that septins play other roles important for high temperature stress that are independent of their established role in cytokinesis. Therefore, we propose to perform a global analysis of septin Cdc10 binding partners in C. neoformans, including those that are specific to high temperature stress. This analysis will shed light on the underlying mechanism of survival of this pathogenic yeast during infection and can potentially lead to the discovery of novel drug targets. [ABSTRACT FROM AUTHOR]

Cryptococcosis has become a major global health problem since the advent of the HIV pandemic in 1980s. Although its molecular epidemiology is well-defined, using isolates recovered since then, no pre-HIV-pandemic era epidemiological data exist. We conducted a molecular epidemiological study using 228 isolates of the C. neoformans/C. gattii species complexes isolated before 1975. Genotypes were determined by URA5 restriction fragment length polymorphism analysis and multi-locus sequence typing. Population genetics were defined by nucleotide diversity measurements, neutrality tests, and recombination analysis. Growth at 37°C, melanin synthesis, capsule production, and urease activity as virulence factors were quantified. The pre-HIV-pandemic isolates consisted of 186 (81.5%) clinical, 35 (15.4%) environmental, and 7 (3.1%) veterinary isolates. Of those, 204 (89.5%) belonged to C. neoformans VNI (64.0%), VNII (14.9%) and VNIV (10.5%) while 24 (10.5%) belonged to C. gattii VGIII (7.5%), VGI (2.6%) and VGII (0.5%). Among the 47 sequence types (STs) identified, one of VNII and 8 of VNIV were novel. ST5/VNI (23.0%) in C. neoformans and ST75/VGIII (25.0%) in C. gattii were the most common STs in both species complexes. Among C. neoformans, VNIV had the highest genetic diversity (Hd = 0.926) and the minimum recombination events (Rm = 10), and clinical isolates had less genetic diversity (Hd = 0.866) than environmental (Hd = 0.889) and veterinary isolates (Hd = 0.900). Among C. gattii, VGI had a higher nucleotide diversity (π = 0.01436) than in VGIII (π = 0.00328). The high-virulence genotypes (ST5/VNI and VGIIIa/serotype B) did not produce higher virulence factors levels than other genotypes. Overall, high genetic variability and recombination rates were found for the pre-HIV-pandemic era among strains of the C. neoformans/C. gattii species complexes. Whole genome analysis and in vivo virulence studies would clarify the evolution of the genetic diversity and/or virulence of isolates of the C. neoformans/C. gattii species complexes during the pre- and post-HIV-pandemic eras. Author summary: Since the beginning of the HIV pandemic in 1980, infections due to isolates of the Cryptococcus neoformans/C. gattii species complexes have caused many deaths worldwide, especially in the HIV-infected population. Annually, approximately one-third, of all AIDS-related deaths,—representing more than 1,000,000 cases,—are caused by cryptococcosis. Since 1980, extensive molecular epidemiological surveys have been conducted, and the VNI molecular type has been found to be responsible for more than 90% of cryptococcosis in HIV patients. Whether the high VNI prevalence is associated with the HIV pandemic remains controversial as information on the isolates of the pre-HIV pandemic era is lacking. Therefore, this study of the molecular epidemiology and in vitro characteristics of the strains from the pre-HIV-pandemic era was undertaken. We found that only 64% of cryptococcosis was caused by VNI, and 9 sequence types existed only in the pre-HIV pandemic era. Unlike what was already known about the strains collected during the HIV pandemic era, ST5 and VGIIIa,—supposedly high virulence genotypes,—did not express higher virulence factors than other genotypes. These results implied that the HIV pandemic altered both the molecular epidemiology and virulence of Cryptococcus neoformans/C. gattii species complexes have been altered during HIV pandemic. However, detailed mechanism of these alteration remains to be deciphered further. [ABSTRACT FROM AUTHOR]

Qsp1 is a secreted quorum sensing peptide required for virulence of the fungal meningitis pathogen Cryptococcus neoformans. Qsp1 functions to control cell wall integrity in vegetatively growing cells and also functions in mating. Rather than acting on a cell surface receptor, Qsp1 is imported to act intracellularly via the predicted oligopeptide transporter Opt1. Here, we identify a transcription factor network as a target of Qsp1. Using whole-genome chromatin immunoprecipitation, we find Qsp1 controls the genomic associations of three transcription factors to genes whose outputs are regulated by Qsp1. One of these transcription factors, Cqs2, is also required for the action of Qsp1 during mating, indicating that it might be a shared proximal target of Qsp1. Consistent with this hypothesis, deletion of CQS2 impacts the binding of other network transcription factors specifically to Qsp1-regulated genes. These genetic and genomic studies illuminate mechanisms by which an imported peptide acts to modulate eukaryotic gene expression. Author summary: For many fungal pathogens, the ability to adapt to changing and diverse environments forms the basis for their ability to infect and survive inside macrophages and other niches in the human body, and these changes are accomplished by transcription factors. Many pathogenic microbes coordinate their gene expression as a function of cell density in a process known as quorum sensing. Here, in the human fungal meningitis pathogen Cryptococcus neoformans, we find that an imported eukaryotic quorum sensing peptide that is important for virulence, Qsp1, controls the binding of three different transcription factors to promoters, thereby modulating the expression of Qsp1-regulated genes. This discovery reveals the mechanism for how an imported peptide affects gene expression. [ABSTRACT FROM AUTHOR]

Macrophages serve as a first line of defense against infection with the facultative intracellular pathogen, Cryptococcus neoformans (Cn). However, the ability of these innate phagocytic cells to destroy ingested Cn is strongly influenced by polarization state with classically (M1) activated macrophages better able to control cryptococcal infections than alternatively (M2) activated cells. While earlier studies have demonstrated that intracellular Cn minimally affects the expression of M1 and M2 markers, the impact on the broader transcriptome associated with these states remains unclear. To investigate this, an in vitro cell culture model of intracellular infection together with RNA sequencing-based transcriptome profiling was used to measure the impact of Cn infection on gene expression in both polarization states. The gene expression profile of both M1 and M2 cells was extensively altered to become more like naive (M0) macrophages. Gene ontology analysis suggested that this involved changes in the activity of the Janus kinase-signal transducers and activators of transcription (JAK-STAT), p53, and nuclear factor-κB (NF-κB) pathways. Analyses of the principle polarization markers at the protein-level also revealed discrepancies between the RNA- and protein-level responses. In contrast to earlier studies, intracellular Cn was found to increase protein levels of the M1 marker iNos. In addition, common gene expression changes were identified that occurred post-Cn infection, independent of polarization state. This included upregulation of the transcriptional co-regulator Cited1, which was also apparent at the protein level in M1-polarized macrophages. These changes constitute a transcriptional signature of macrophage Cn infection and provide new insights into how Cn impacts gene expression and the phenotype of host phagocytes. [ABSTRACT FROM AUTHOR]

The emergence and spread of cryptococcosis caused by the Cryptococcus gattii species complex has become a major public concern worldwide. C. deuterogattii (VGIIa) outbreaks in the Pacific Northwest region demonstrate the expansion of this fungal infection to temperate climate regions. However, infections due to the C. gattii species complex in China have rarely been reported. In this study, we studied eleven clinical strains of the C. gattii species complex isolated from Guangxi, southern China. The genetic identity and variability of these isolates were analyzed via multi-locus sequence typing (MLST), and the phylogenetic relationships among these isolates and global isolates were evaluated. The mating type, physiological features and antifungal susceptibilities of these isolates were also characterized. Among the eleven isolates, six belonged to C. deuterogattii, while five belonged to C. gattii sensu stricto. The C. deuterogattii strains from Guangxi, southern China were genetically variable and clustered with different clinical isolates from Brazil. All strains were MATα, and three C. deuterogattii isolates (GX0104, GX0105 and GX0147) were able to undergo sexual reproduction. Moreover, most strains had capsule and were capable of melanin production when compared to the outbreak strain from Canada. Most isolates were susceptible to antifungal drugs; yet one of eleven immunocompetent patients died of cryptococcal meningitis caused by C. deuterogattii (GX0147). Our study indicated that the highly pathogenic C. deuterogattii may be emerging in southern China, and effective nationwide surveillance of C. gattii species complex infection is necessary. Author summary: Cryptococcosis is a fatal systemic fungal disease caused by Cryptococcus neoformans/gattii species complexes. As a former member of the C. neoformans, C. gattii had been easily neglected before being elevated to species level. Human C. gattii species complex infection was previously confined to the tropical and subtropical regions worldwide. However, in 1999, an outbreak of C. gattii species complex occurred on Vancouver Island in Canada then expanded to the Pacific Northwest in the USA, causing over 200 infections. The highly virulent, highly pathogenic and more resistant to antifungal drugs of this species have become a therapeutic problem. To initiate a better understanding of the infection characteristics and pathogenicity of C. gattii species complex in Guangxi, southern China, the current study aimed to characterize the C. gattii species complex isolates genetically and phenotypically. The ISHAM consensus MLST scheme was utilized to investigate the genetic structure of C. gattii species complex and to correlate their geographic origin, clinical source, virulence factors and antifungal susceptibility. The authors expect that this work can support surveillance and encourage more research and public health initiatives to prevent and control the cryptococcosis cause by C. gattii species complex. [ABSTRACT FROM AUTHOR]

Autophagy is a mechanism responsible for intracellular degradation and recycling of macromolecules and organelles, essential for cell survival in adverse conditions. More than 40 autophagy-related (ATG) genes have been identified and characterized in fungi, among them ATG4 and ATG8. ATG4 encodes a cysteine protease (Atg4) that plays an important role in autophagy by initially processing Atg8 at its C-terminus region. Atg8 is a ubiquitin-like protein essential for the synthesis of the double-layer membrane that constitutes the autophagosome vesicle, responsible for delivering the cargo from the cytoplasm to the vacuole lumen. The contributions of Atg-related proteins in the pathogenic yeast in the genus Cryptococcus remain to be explored, to elucidate the molecular basis of the autophagy pathway. In this context, we aimed to investigate the role of autophagy-related proteins 4 and 8 (Atg4 and Atg8) during autophagy induction and their contribution with non-autophagic events in C. neoformans. We found that Atg4 and Atg8 are conserved proteins and that they interact physically with each other. ATG gene deletions resulted in cells sensitive to nitrogen starvation. ATG4 gene disruption affects Atg8 degradation and its translocation to the vacuole lumen, after autophagy induction. Both atg4 and atg8 mutants are more resistant to oxidative stress, have an impaired growth in the presence of the cell wall-perturbing agent Congo Red, and are sensitive to the proteasome inhibitor bortezomib (BTZ). By that, we conclude that in C. neoformans the autophagy-related proteins Atg4 and Atg8 play an important role in the autophagy pathway; which are required for autophagy regulation, maintenance of amino acid levels and cell adaptation to stressful conditions. [ABSTRACT FROM AUTHOR]

Background: Fluconazole is used in combination with amphotericin B for induction treatment of cryptococcal meningitis and as monotherapy for consolidation and maintenance treatment. More than 90% of isolates from first episodes of cryptococcal disease had a fluconazole minimum inhibitory concentration (MIC) ≤4 μg/ml in a Gauteng population-based surveillance study of Cryptococcus neoformans in 2007–2008. We assessed whether fluconazole resistance had emerged in clinical cryptococcal isolates over a decade. Methodology and principal findings: We prospectively collected C. neoformans isolates from 1 January through 31 March 2017 from persons with a first episode of culture-confirmed cryptococcal disease at 37 South African hospitals. Isolates were phenotypically confirmed to C. neoformans species-complex level. We determined fluconazole MICs (range: 0.125 μg/ml to 64 μg/ml) of 229 C. neoformans isolates using custom-made broth microdilution panels prepared, inoculated and read according to Clinical and Laboratory Standards Institute M27-A3 and M60 recommendations. These MIC values were compared to MICs of 249 isolates from earlier surveillance (2007–2008). Clinical data were collected from patients during both surveillance periods. There were more males (61% vs 39%) and more participants on combination induction antifungal treatment (92% vs 32%) in 2017 compared to 2007–2008. The fluconazole MIC50, MIC90 and geometric mean MIC was 4 μg/ml, 8 μg/ml and 4.11 μg/ml in 2017 (n = 229) compared to 1 μg/ml, 2 μg/ml and 2.08 μg/ml in 2007–2008 (n = 249) respectively. Voriconazole, itraconazole and posaconazole Etests were performed on 16 of 229 (7%) C. neoformans isolates with a fluconazole MIC value of ≥16 μg/ml; only one had MIC values of >32 μg/ml for these three antifungal agents. Conclusions and significance: Fluconazole MIC50 and MIC90 values were two-fold higher in 2017 compared to 2007–2008. Although there are no breakpoints, higher fluconazole doses may be required to maintain efficacy of standard treatment regimens for cryptococcal meningitis. Author summary: Cryptococcus neoformans, a pathogenic fungal species-complex with an environmental niche, is the most common cause of meningitis among HIV-seropositive adults in sub-Saharan Africa. Fluconazole is recommended in combination with amphotericin B for induction treatment of cryptococcal meningitis and as monotherapy for consolidation and maintenance treatment. Fluconazole is also commonly prescribed to HIV-seropositive individuals for other indications; fluconazole exposure may result in secondary resistance if patients have concurrent active cryptococcal disease. Azole fungicides used in agriculture may potentially drive primary cryptococcal resistance when the fungus is exposed to these fungicides in the environment. We aimed to determine fluconazole MICs in 2017 and compare these values to those obtained in a 2007–2008 South African survey to assess whether fluconazole resistance had emerged in C. neoformans over a decade. We found that the proportion of isolates with an MIC of ≥16 μg/ml increased from 0% in 2007–2008 to 7% in 2017. MIC50 and MIC90 values were also two-fold higher in 2017 compared to 2007–2008. These study findings provided evidence for higher fluconazole dose recommendations (in combination with amphotericin B for the induction phase and as monotherapy for consolidation and maintenance phases) in the 2019 Southern African guideline for HIV-associated cryptococcosis. [ABSTRACT FROM AUTHOR]

Monocytes exist in two major populations, termed Ly6Chi and Ly6Clow monocytes. Compared to Ly6Chi monocytes, less is known about Ly6Clow monocyte recruitment and mechanisms involved in the recruitment of this subset. Furthermore, the role of Ly6Clow monocytes during infections is largely unknown. Here, using intravital microscopy, we demonstrate that Ly6Clow monocytes are predominantly recruited to the brain vasculature following intravenous infection with Cryptococcus neoformans, a fungal pathogen causing meningoencephalitis. The recruitment depends primarily on the interaction of VCAM1 expressed on the brain endothelium with VLA4 expressed on Ly6Clow monocytes. Furthermore, TNFR signaling is essential for the recruitment through enhancing VLA4 expression on Ly6Clow monocytes. Interestingly, the recruited Ly6Clow monocytes internalized C. neoformans and carried the organism while crawling on and adhering to the luminal wall of brain vasculature and migrating to the brain parenchyma. Our study reveals a substantial recruitment of Ly6Clow monocytes to the brain and highlights important properties of this subset during infection. Author summary: Monocytes are white blood cells, circulating in the bloodstream and playing important roles during infections. There are two subsets of monocytes in mice: Ly6Chi and Ly6Clow monocytes. In contrast to the recruitment of Ly6Chi monocytes shown in other infection models, we observed the predominant recruitment of Ly6Clow monocytes to the brain post-capillary venules during intravenous infection with C. neoformans, a fungal pathogen causing brain infection. The recruitment is mainly mediated by the interaction of VCAM1 and VLA4, which are expressed on the brain endothelium and monocytes, respectively. We further demonstrate that TNFR signaling plays an essential role during Ly6Clow monocyte recruitment through enhancing VLA4 expression on monocytes. We also observed that Ly6Clow monocytes internalize C. neoformans and, together with the ingested organism, crawl along the luminal wall of brain vasculatures and migrate to the brain parenchyma. Thus, VCAM1/VLA4 interaction mediates Ly6Clow monocyte recruitment to the brain in a TNFR signaling dependent manner during fungal infection. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is an opportunistic human pathogen, which causes serious disease in immunocompromised hosts. Infection with this pathogen is particularly relevant in HIV+ patients, where it leads to around 200,000 deaths per annum. A key feature of cryptococcal pathogenesis is the ability of the fungus to survive and replicate within the phagosome of macrophages, as well as its ability to be expelled from host cells via a novel non-lytic mechanism known as vomocytosis. Here we show that cryptococcal vomocytosis from macrophages is strongly enhanced by viral coinfection, without altering phagocytosis or intracellular proliferation of the fungus. This effect occurs with distinct, unrelated human viral pathogens and is recapitulated when macrophages are stimulated with the anti-viral cytokines interferon alpha or beta (IFNα or IFNβ). Importantly, the effect is abrogated when type-I interferon signalling is blocked, thus underscoring the importance of type-I interferons in this phenomenon. Lastly, our data help resolve previous, contradictory animal studies on the impact of type I interferons on cryptococcal pathogenesis and suggest that secondary viral stimuli may alter patterns of cryptococcal dissemination in the host. Author summary: Infectious diseases are typically studied in isolation, but in real life people often encounter multiple infections simultaneously. Here we investigate how the innate immune response to the fatal fungus Cryptococcus neoformans is influenced by viral coinfection. Whilst virally-infected macrophages retain a normal capacity to engulf and kill Cryptococci, they demonstrate a dramatically enhanced propensity to expel them through vomocytosis. Activation of vomocytosis is driven by type-I interferons, generic 'antiviral' molecules, which signal back to the infected macrophage, triggering expulsion of the fungus. We propose that this hitherto unobserved phenomenon represents a 'reprioritisation' pathway for innate immune cells, by which they can alter the frequency with which they expel one pathogen depending on the level of threat from a secondary infection. [ABSTRACT FROM AUTHOR]

Cryptococcal meningoencephalitis (CM) remains the most prevalent invasive fungal infection worldwide. The main objective of this study was to describe the prevalence of CM and cryptococcal infection in HIV-infected patients in Madagascar. The secondary objectives were to assess the adjusted prevalence of CM according to clinical presentation and patient characteristics, to determine crude 90-day survival according to cryptococcal antigen (CrAg) status and CM, and to identify the genotypes of Cryptococcus clinical isolates. This cross-sectional study was carried out at two urban hospitals in Antananarivo (central highlands) and Toamasina (east coast) between November 2014 and December 2016. Consecutive HIV-infected adults presenting with CD4 cell counts ≤200/μl were enrolled. Lateral flow immunoassays of CrAg were performed on serum for all patients, and on cerebrospinal fluid for patients with CM symptoms. MALDI-ToF MS, ITS sequencing, and determinations of the molecular and mating types of the isolates were performed. Fluconazole is the only drug for CM treatment available in Madagascar. Patients were treated orally, with high doses (1200 mg/day) for 10–12 weeks and then with 200 mg/day. Minimum inhibitory concentrations were determined for amphotericin B, flucytosine, voriconazole and fluconazole in E-tests. Overall prevalence was 13.2% (95% CI 7.9–20.3) for cryptococcal infection and 10.9% (95% CI 6.1–17.5) for CM, among the 129 HIV-infected patients studied. The 90-day mortality rate was 58.8% (10/17) in CrAg-positive patients and 17.9% (20/112) in CrAg-negative patients (p<0.001). The 13 Cryptococcus strains obtained at baseline were all Cryptococcus neoformans var. grubii, genotypes VNI-αA (3 isolates), VNII-αA (4 isolates) or hybrid VNI/VNII-αAAα (6 isolates), suggesting high diversity. Two strains acquired fluconazole resistance after four and five months of exposure, respectively. The prevalence of cryptococcosis is high in Madagascar and this serious condition is life-threatening in HIV-infected patients. These findings will be used to raise the awareness of national authorities to strengthen the national HIV/AIDS control program. Author summary: Cryptococcal meningoencephalitis (CM) remains the most prevalent invasive fungal infection worldwide, with an estimated mortality of 70% in sub-Saharan Africa. In this cross-sectional study, we investigated the prevalence, clinical features, case-management and outcome of CM in HIV-infected patients. We also molecularly characterized the Cryptococcus isolates from these patients. The study was conducted in the main hospitals of two geographically distant cities, one located at Antananarivo in the central highlands and the other at Toamasina on the east coast. The prevalence of cryptococcal infection was higher than previously reported global prevalence values, and most of the patients developed CM. Classical signs of meningoencephalitis, including headache, fever and neck pain, were observed, as reported in previous studies. Ninety-day mortality (58.8%) was higher in patients with Cryptococcus infections than in non-infected patients (17.9%). All patients diagnosed with infection were treated with high-dose fluconazole, the only treatment available in Madagascar, in accordance with WHO recommendations. Molecular analyses of isolates from both regions revealed high levels of genome diversity and suggested that further environmental and larger clinical studies would be worthwhile. In conclusion, the prevalence of cryptococcal diseases is high in Madagascar, which is currently faced with the challenges of prioritizing various diseases of public health concern, providing health centers with rapid diagnostic tools and facilitating access to more reliable molecules. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is a facultative intracellular pathogen responsible for the most common cause of fungal meningioencephalitis, occurring primarily in immunocompromised individuals. Antiphagocytic protein 1 (App1) is a virulence factor produced by C. neoformans that inhibits phagocytosis of the yeast by host macrophages. Treatment of cryptococcosis includes amphotericin B, fluconazole, and flucytosine. Virulence factors have been shown to affect the susceptibility of the pathogen to antifungal drugs. In this study, we aimed to examine the relationship between App1 and antifungal drugs. We found that short-term exposure to amphotericin B downregulates APP1 expression while exposure to fluconazole upregulates APP1. In addition, App1 was found to increase the susceptibility of the yeast to amphotericin B and fluconazole. This study provides evidence of an intricate relationship between App1 and antifungal drugs. [ABSTRACT FROM AUTHOR]

Genetic diversity analyses were performed by sero-genotyping and multi-locus sequence typing on 252 cryptococcal isolates from 13 HIV-positive Ivorian patients followed-up for cryptococcal meningitis. Antifungal susceptibility analyses were performed according to the CLSI M27A3 method. The majority (67.8%) of the isolates belonged to the Cryptococcus neoformans (serotype A) species complex, with 91.9% being VNI and 7.1% being VNII. Cryptococcus deuterogattii VGII (serotype B) represented 16.7% of the strains, while C. neoformans/C. deneoformans VNIII (serotype AD) hybrids accounted for 15.1% of the strains. One strain (0.4%) was not identifiable. Nine different sequence types (STs 5, 6, 23, 40, 93, 207, 311, and a new ST; 555) were identified in the C. neoformans population, while the C. deuterogattii population comprised the single ST 173. The distribution of the strains showed that, while the majority of patients (9/13) harboured a single sequence type, 4 patients showed mixed infections. These patients experienced up to 4 shifts in strain content either at the species and/or ST level during their follow-up. This evolution of diversity over time led to the co-existence of up to 3 different Cryptococcus species and 4 different ST within the same individual during the course of infection. Susceptibility testing showed that all strains were susceptible to amphotericin B while 3.6% of them had a none-wild type phenotype to 5-flucytosine. Concerning fluconazole, 2.9% of C.neoformans serotype A strains and 2.4% of C. deuterogattii had also respectively a none-wild type phenotype to this molecule. All C. neoformans x C. deneoformans serotype AD hybrids had however a wild type phenotype to fluconazole. The present study showed that mixed infections exist and could be of particular importance for care outcomes. Indeed, (i) the different Cryptococcus species are known to exhibit different virulence and different susceptibility patterns to antifungal drugs and (ii) the strains genetic diversity within the samples may influence the susceptibility to antifungal treatment. Author summary: Cryptococcal meningitis is a neglected fungal disease responsible for 181 000 deaths worldwide in 2014, with 75% of deaths occurring in sub-Saharan Africa. Cryptococcal meningitis is caused by environmental yeasts belonging to the Cryptococcus neoformans/Cryptococcus gattii species complexes. The evolution of the diversity of the yeast populations within the patients and during the course of treatment is poorly understood. Indeed, it was believed for a long time that infections were of a single strain type. It was only recently that the complexity of the yeast diversity during infection began to be assessed. Here, the researchers evaluated the diversity of the Cryptococcus population within Ivoirian patients. The purpose was to generate data about the overall diversity of such yeast in Western Africa where the data are scarce and to better understand the evolution of the pathogen populations during patient follow-up. This last point is particularly important because some species are more virulent or naturally more resistant to antifungal treatments and could be an issue in the case of relapses during care protocols. [ABSTRACT FROM AUTHOR]

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 protein splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced protein-splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 protein splicing can be modulated, with potential functional implications for the spliceosome. [ABSTRACT FROM AUTHOR]

Courtship is pivotal for successful mating. However, courtship is challenging for the Cryptococcus neoformans species complex, comprised of opportunistic fungal pathogens, as the majority of isolates are α mating type. In the absence of mating partners of the opposite mating type, C. deneoformans can undergo unisexual reproduction, during which a yeast-to-hyphal morphological transition occurs. Hyphal growth during unisexual reproduction is a quantitative trait, which reflects a strain’s ability to undergo unisexual reproduction. In this study, we determined whether unisexual reproduction confers an ecological benefit by promoting foraging for mating partners. Through competitive mating assays using strains with different abilities to produce hyphae, we showed that unisexual reproduction potential did not enhance competition for mating partners of the same mating type, but when cells of the opposite mating type were present, cells with enhanced hyphal growth were more competitive for mating partners of either the same or opposite mating type. Enhanced mating competition was also observed in a strain with increased hyphal production that lacks the mating repressor gene GPA3, which contributes to the pheromone response. Hyphal growth in unisexual strains also enables contact between adjacent colonies and enhances mating efficiency during mating confrontation assays. The pheromone response pathway activation positively correlated with unisexual reproduction hyphal growth during bisexual mating and exogenous pheromone promoted bisexual cell fusion. Despite the benefit in competing for mating partners, unisexual reproduction conferred a fitness cost. Taken together, these findings suggest C. deneoformans employs hyphal growth to facilitate contact between colonies at long distances and utilizes pheromone sensing to enhance mating competition. [ABSTRACT FROM AUTHOR]

Sexual development is a key evolutionary innovation of eukaryotes. In many species, mating involves interaction between compatible mating partners that can undergo cell and nuclear fusion and subsequent steps of development including meiosis. Mating compatibility in fungi is governed by the mating type (MAT) loci. In basidiomycetes, the ancestral state is hypothesized to be tetrapolar, with two genetically unlinked MAT loci containing homeodomain transcription factor genes (HD locus) and pheromone and pheromone receptor genes (P/R locus), respectively. Alleles at both loci must differ between mating partners for completion of sexual development. However, there are also basidiomycetes with bipolar mating systems, which can arise through genomic linkage of the HD and P/R loci. In the order Tremellales, bipolarity is found only in the pathogenic Cryptococcus species. Here, we describe the analysis of MAT loci from 24 species of the Trichosporonales, a sister order to the Tremellales. In all of the species analyzed, the MAT loci are fused and a single HD gene is present in each mating type, similar to the organization in the pathogenic Cryptococci. However, the HD and P/R allele combinations in the Trichosporonales are different from those in the pathogenic Cryptococci. This and the existence of tetrapolar species in the Tremellales suggest that fusion of the HD and P/R loci occurred independently in the Trichosporonales and pathogenic Cryptococci, supporting the hypothesis of convergent evolution towards fused MAT regions, similar to previous findings in other fungal groups. Unlike the fused MAT loci in several other basidiomycete lineages though, the gene content and gene order within the fused MAT loci are highly conserved in the Trichosporonales, and there is no apparent suppression of recombination extending from the MAT loci to adjacent chromosomal regions, suggesting different mechanisms for the evolution of physically linked MAT loci in these groups. [ABSTRACT FROM AUTHOR]

Background: The indications for lumbar puncture in non-HIV-infected, non-transplant (NHNT) patients with cryptococcosis without meningeal signs need to be more fully defined. Objectives: This study was designed to determine the optimal predictors of central nervous system (CNS) involvement in adult NHNT patients with cryptococcosis. Methods: The study population consisted of adult NHNT patients with culture-confirmed cryptococcosis who sought care at a university hospital in Taiwan from 2002 to 2016. We used a case-control method to identify the clinical characteristics and laboratory findings associated with CNS involvement in patients who underwent a diagnostic lumbar puncture. In the sensitivity analysis, we included additional control patients who did not undergo lumbar puncture, but were followed beyond 12 months without the development of CNS involvement in the absence of exposure to any fungicidal agents. Results: We entered 270 NHNT adult patients into the study during the 15-year period. CNS involvement was confirmed in 66 (71.0%) of 93 patients who underwent lumbar puncture. A multivariable analysis revealed that presence of neurological manifestations and elevated serum CRAG titers were independently associated with a 23.97-fold (95% confidence interval [CI], 4.37–182.23) and 1.53-fold (per 2-fold increment, 95% CI 1.26–1.92) increased odds ratio for CNS involvement, respectively. Headache and focal neurologic signs were independently associated with CNS involvement. A cut-off serum CRAG titer of ≥1:64 provided the highest diagnostic performance by Youden index (sensitivity 83% and specificity 65%). Similar findings were noted in the sensitivity analysis including 198 (73%) patients. Conclusion: Lumbar puncture is indicated for NHNT patients with cryptococcosis who have neurologic manifestations or a serum CRAG titer of ≥1:64. [ABSTRACT FROM AUTHOR]

Cryptococcus gattii is a capsular fungal pathogen, which causes life-threatening cryptococcosis in immunocompetent individuals. This emerging pathogen is less likely to be recognized by innate immunity compared to traditional Cryptococcus neoformans strains. Previous studies indicate that C-type lectin receptors (CLRs), including dectin-1 and dectin-2, play a role in recognizing cryptococcal cells; however, it remains to be elucidated whether the receptors physically associate with C. gattii yeast cell surfaces. Based on the previous findings, we hypothesized that culture conditions influence the expression or exposure of CLR ligands on C. gattii. Therefore, in the present study, we first investigated the culture conditions that induce exposure of CLR ligands on C. gattii yeast cells via the binding assay using recombinant fusion proteins of mouse CLR and IgG Fc, Fc dectin-1 and Fc dectin-2. Common fungal culture media, such as yeast extract–peptone–dextrose (YPD) broth, Sabouraud broth, and potato dextrose agar, did not induce the exposure of dectin-1 ligands, including β-1,3-glucan, on both capsular and acapsular C. gattii strains, in contrast to Fc dectin-1 and Fc dectin-2 bound to C. gattii cells growing in the conventional synthetic dextrose (SD) medium [may also be referred to as a yeast nitrogen base with glucose medium]. The medium also induced the exposure of dectin-1 ligands on C. neoformans, whereas all tested media induced dectin-1 and dectin-2 ligands in a control fungus Candida albicans. Notably, C. gattii did not expose dectin-1 ligands in SD medium supplemented with yeast extract or neutral buffer. In addition, compared to YPD medium-induced C. gattii, SD medium-induced C. gattii more efficiently induced the phosphorylation of Syk, Akt, and Erk1/2 in murine dendritic cells (DCs). Afterwards, the cells were considerably engulfed by DCs and remarkably induced DCs to secrete the inflammatory cytokines. Overall, the findings suggest that C. gattii alters its immunostimulatory potential in response to the environment. [ABSTRACT FROM AUTHOR]

Metabolically quiescent pathogens can persist in a viable non-replicating state for months or even years. For certain infectious diseases, such as tuberculosis, cryptococcosis, histoplasmosis, latent infection is a corollary of this dormant state, which has the risk for reactivation and clinical disease. During murine cryptococcosis and macrophage uptake, stress and host immunity induce Cryptococcus neoformans heterogeneity with the generation of a sub-population of yeasts that manifests a phenotype compatible with dormancy (low stress response, latency of growth). In this subpopulation, mitochondrial transcriptional activity is regulated and this phenotype has been considered as a hallmark of quiescence in stem cells. Based on these findings, we worked to reproduce this phenotype in vitro and then standardize the experimental conditions to consistently generate this dormancy in C. neoformans. We found that incubation of stationary phase yeasts (STAT) in nutriment limited conditions and hypoxia for 8 days (8D-HYPOx) was able to produced cells that mimic the phenotype obtained in vivo. In these conditions, mortality and/or apoptosis occurred in less than 5% of the yeasts compared to 30–40% of apoptotic or dead yeasts upon incubation in normoxia (8D-NORMOx). Yeasts in 8D-HYPOx harbored a lower stress response, delayed growth and less that 1% of culturability on agar plates, suggesting that these yeasts are viable but non culturable cells (VBNC). These VBNC were able to reactivate in the presence of pantothenic acid, a vitamin that is known to be involved in quorum sensing and a precursor of acetyl-CoA. Global metabolism of 8D-HYPOx cells showed some specific requirements and was globally shut down compared to 8D-NORMOx and STAT conditions. Mitochondrial analyses showed that the mitochondrial masse increased with mitochondria mostly depolarized in 8D-HYPOx compared to 8D-NORMox, with increased expression of mitochondrial genes. Proteomic and transcriptomic analyses of 8D-HYPOx revealed that the number of secreted proteins and transcripts detected also decreased compared to 8D-NORMOx and STAT, and the proteome, secretome and transcriptome harbored specific profiles that are engaged as soon as four days of incubation. Importantly, acetyl-CoA and the fatty acid pathway involving mitochondria are required for the generation and viability maintenance of VBNC. Altogether, these data show that we were able to generate for the first time VBNC phenotype in C. neoformans. This VBNC state is associated with a specific metabolism that should be further studied to understand dormancy/quiescence in this yeast. [ABSTRACT FROM AUTHOR]

Pathogens and hosts require rapid modulation of virulence and defense mechanisms at the infection axis, but monitoring such modulations is challenging. In studying the human fungal pathogen Cryptococcus neoformans, mouse and rabbit infection models are often employed to shed light on the disease mechanisms but that may not be clinically relevant. In this study, we developed an animal infection model using the non-human primate cynomolgus monkey Macaca fascicularis. In addition, we systematically profiled and compared transcriptional responses between the infected mice and the cynomolgus monkey, using simultaneous or dual RNA next-generation sequencing. We demonstrated that there are shared but distinct transcriptional profiles between the two models following C. neoformans infection. Specifically, genes involved in immune and inflammatory responses are all upregulated. Osteoclastogenesis and insulin signaling are also significantly co-regulated in both models and disrupting an osteoclastogenesis-associated gene (OC-STAMP) or the insulin-signaling process significantly altered the host tolerance to C. neoformans. Moreover, C. neoformans was shown to activate metal sequestration, dampen the sugar metabolism, and control cell morphology during infection. Taking together, we described the development of a non-human primate model of cryptococcosis that allowed us to perform an in-depth analysis and comparison of transcriptome profiles during infections of two animal models and conceptually identify host genes important in disease responses. This study provides new insights in understanding fungal pathogenesis mechanisms that potentially facilitate the identification of novel drug targets for the treatment of cryptococcal infection. [ABSTRACT FROM AUTHOR]

In this study, we report antifungal activity of auroramycin against Candida albicans, Candida tropicalis, and Cryptococcus neoformans. Auroramycin, a potent antimicrobial doubly glycosylated 24-membered polyene macrolactam, was previously isolated and characterized, following CRISPR-Cas9 mediated activation of a silent polyketide synthase biosynthetic gene cluster in Streptomyces rosesporous NRRL 15998. Chemogenomic profiling of auroramycin in yeast has linked its antifungal bioactivity to vacuolar transport and membrane organization. This was verified by disruption of vacuolar structure and membrane integrity of yeast cells with auroramycin treatment. Addition of salt but not sorbitol to the medium rescued the growth of auroramycin-treated yeast cells suggesting that auroramycin causes ionic stress. Furthermore, auroramycin caused hyperpolarization of the yeast plasma membrane and displayed a synergistic interaction with cationic hygromycin. Our data strongly suggest that auroramycin inhibits yeast cells by causing leakage of cations from the cytoplasm. Thus, auroramycin’s mode-of-action is distinct from known antifungal polyenes, reinforcing the importance of natural products in the discovery of new anti-infectives. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is one of the leading causes of invasive fungal infection in humans worldwide. C. neoformans uses macrophages as a proliferative niche to increase infective burden and avoid immune surveillance. However, the specific mechanisms by which C. neoformans manipulates host immunity to promote its growth during infection remain ill-defined. Here we demonstrate that eicosanoid lipid mediators manipulated and/or produced by C. neoformans play a key role in regulating pathogenesis. C. neoformans is known to secrete several eicosanoids that are highly similar to those found in vertebrate hosts. Using eicosanoid deficient cryptococcal mutants Δplb1 and Δlac1, we demonstrate that prostaglandin E2 is required by C. neoformans for proliferation within macrophages and in vivo during infection. Genetic and pharmacological disruption of host PGE2 synthesis is not required for promotion of cryptococcal growth by eicosanoid production. We find that PGE2 must be dehydrogenated into 15-keto-PGE2 to promote fungal growth, a finding that implicated the host nuclear receptor PPAR-γ. C. neoformans infection of macrophages activates host PPAR-γ and its inhibition is sufficient to abrogate the effect of 15-keto-PGE2 in promoting fungal growth during infection. Thus, we describe the first mechanism of reliance on pathogen-derived eicosanoids in fungal pathogenesis and the specific role of 15-keto-PGE2 and host PPAR-γ in cryptococcosis. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is a leading cause of invasive fungal infections among immunocompromised patients. However, the cellular constituents of the innate immune response that promote clearance versus progression of infection upon respiratory acquisition of C. neoformans remain poorly defined. In this study, we found that during acute C. neoformans infection, CCR2+ Ly6Chi inflammatory monocytes (IM) rapidly infiltrate the lungs and mediate fungal trafficking to lung-draining lymph nodes. Interestingly, this influx of IM is detrimental to the host, since ablating IM or impairing their recruitment to the lungs improves murine survival and reduces fungal proliferation and dissemination. Using a novel conditional gene deletion strategy, we determined that MHC class II expression by IM did not mediate their deleterious impact on the host. Furthermore, although ablation of IM reduced the number of lymphocytes, innate lymphoid cells, and eosinophils in the lungs, the effects of IM were not dependent on these cells. We ascertained that IM in the lungs upregulated transcripts associated with alternatively activated (M2) macrophages in response to C. neoformans, consistent with the model that IM assume a cellular phenotype that is permissive for fungal growth. We also determined that conditional knockout of the prototypical M2 marker arginase 1 in IM and deletion of the M2-associated transcription factor STAT6 were not sufficient to reverse the harmful effects of IM. Overall, our findings indicate that C. neoformans can subvert the fungicidal potential of IM to enable the progression of infection through a mechanism that is not dependent on lymphocyte priming, eosinophil recruitment, or downstream M2 macrophage polarization pathways. These results give us new insight into the plasticity of IM function during fungal infections and the level of control that C. neoformans can exert on host immune responses. [ABSTRACT FROM AUTHOR]

The phosphate sensing and acquisition (PHO) pathway of Cryptococcus neoformans is essential for growth in phosphate-limiting conditions and for dissemination of infection in a mouse model. Its key transcription factor, Pho4, regulates expression of genes controlling the acquisition of phosphate from both external and cellular sources. One such gene, BTA1, is highly up-regulated during phosphate starvation. Given that a significant proportion of cellular phosphate is incorporated into phospholipids, and that the Pho4-dependent BTA1 gene encodes an enzyme predicted to catalyse production of a phosphorus-free betaine lipid, we investigated whether phospholipids provide an accessible reservoir of phosphate during phosphate deficiency. By comparing lipid profiles of phosphate-starved WT C. neoformans, PHO4 (pho4Δ) and BTA1 (bta1Δ) deletion mutants using thin layer chromatography and liquid chromatography mass spectrometry, we showed that phosphatidylcholine (PC) is substituted by the phosphorus-free betaine lipids diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and diacylgyceryl hydroxymethyl-N,N,N-trimethyl-beta-alanine (DGTA) in a Pho4- and Bta1-dependent manner, and that BTA1 encodes a functional DGTS synthase. Synthesis of DGTA tightly correlated with that of DGTS, consistent with DGTS being the precursor of DGTA. Similar to pho4Δ, bta1Δ grew more slowly than WT in cell culture medium (RPMI) and was hypovirulent in a murine model of cryptococcosis. In contrast to pho4Δ, bta1Δ tolerated alkaline pH and disseminated to the brain. Our results demonstrate that Bta1-dependent substitution of PC by betaine lipids is tightly regulated in C. neoformans by the PHO pathway, to conserve phosphate and preserve membrane integrity and function. This phospholipid remodeling strategy may also contribute to cryptococcal virulence during host infection. [ABSTRACT FROM AUTHOR]