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1.
Molecules ; 28(17)2023 Aug 24.
Article in English | MEDLINE | ID: mdl-37687052

ABSTRACT

Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of -10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future.


Subject(s)
Antifungal Agents , Biofilms , Cryptococcus neoformans , Fungal Proteins , Lysophospholipase , Macrophages, Alveolar , Propolis , Humans , Biofilms/drug effects , Cell Line, Tumor , Cryptococcosis/prevention & control , Cryptococcosis/therapy , Cryptococcus neoformans/drug effects , Cryptococcus neoformans/enzymology , Cryptococcus neoformans/pathogenicity , Ethanol/chemistry , Fungal Proteins/antagonists & inhibitors , Liposomes , Lung Diseases, Fungal/prevention & control , Lung Diseases, Fungal/therapy , Lysophospholipase/antagonists & inhibitors , Macrophages, Alveolar/microbiology , Propolis/chemistry , Propolis/pharmacology , Virulence/drug effects , Virulence Factors/antagonists & inhibitors , Antifungal Agents/chemistry , Antifungal Agents/pharmacology
2.
Nat Commun ; 13(1): 7938, 2022 12 24.
Article in English | MEDLINE | ID: mdl-36566249

ABSTRACT

Pathogenic fungi of the genus Cryptococcus can undergo two sexual cycles, involving either bisexual diploidization (after fusion of haploid cells of different mating type) or unisexual diploidization (by autodiploidization of a single cell). Here, we construct a gene-deletion library for 111 transcription factor genes in Cryptococcus deneoformans, and explore the roles of these regulatory networks in the two reproductive modes. We show that transcription factors crucial for bisexual syngamy induce the expression of known mating determinants as well as other conserved genes of unknown function. Deletion of one of these genes, which we term FMP1, leads to defects in bisexual reproduction in C. deneoformans, its sister species Cryptococcus neoformans, and the ascomycete Neurospora crassa. Furthermore, we show that a recently evolved regulatory cascade mediates pre-meiotic unisexual autodiploidization, supporting that this reproductive process is a recent evolutionary innovation. Our findings indicate that genetic circuits with different evolutionary ages govern hallmark events distinguishing unisexual and bisexual reproduction in Cryptococcus.


Subject(s)
Cryptococcus neoformans , Fungal Proteins , Meningitis, Cryptococcal , Cryptococcus neoformans/growth & development , Cryptococcus neoformans/pathogenicity , Fungal Proteins/genetics , Fungal Proteins/metabolism , Genes, Mating Type, Fungal/genetics , Reproduction, Asexual/genetics , Meningitis, Cryptococcal/parasitology
3.
mBio ; 13(6): e0294422, 2022 12 20.
Article in English | MEDLINE | ID: mdl-36377896

ABSTRACT

The KEOPS (kinase, putative endopeptidase, and other proteins of small size) complex has critical functions in eukaryotes; however, its role in fungal pathogens remains elusive. Herein, we comprehensively analyzed the pathobiological functions of the fungal KEOPS complex in Cryptococcus neoformans (Cn), which causes fatal meningoencephalitis in humans. We identified four CnKEOPS components: Pcc1, Kae1, Bud32, and Cgi121. Deletion of PCC1, KAE1, or BUD32 caused severe defects in vegetative growth, cell cycle control, sexual development, general stress responses, and virulence factor production, whereas deletion of CGI121 led to similar but less severe defects. This suggests that Pcc1, Kae1, and Bud32 are the core KEOPS components, and Cgi121 may play auxiliary roles. Nevertheless, all KEOPS components were essential for C. neoformans pathogenicity. Although the CnKEOPS complex appeared to have a conserved linear arrangement of Pcc1-Kae1-Bud32-Cgi121, as supported by physical interaction between Pcc1-Kae1 and Kae1-Bud32, CnBud32 was found to have a unique extended loop region that was critical for the KEOPS functions. Interestingly, CnBud32 exhibited both kinase activity-dependent and -independent functions. Supporting its pleiotropic roles, the CnKEOPS complex not only played conserved roles in t6A modification of ANN codon-recognizing tRNAs but also acted as a major transcriptional regulator, thus controlling hundreds of genes involved in various cellular processes, particularly ergosterol biosynthesis. In conclusion, the KEOPS complex plays both evolutionarily conserved and divergent roles in controlling the pathobiological features of C. neoformans and could be an anticryptococcal drug target. IMPORTANCE The cellular function and structural configuration of the KEOPS complex have been elucidated in some eukaryotes and archaea but have never been fully characterized in fungal pathogens. Here, we comprehensively analyzed the pathobiological roles of the KEOPS complex in the globally prevalent fungal meningitis-causing pathogen C. neoformans. The CnKEOPS complex, composed of a linear arrangement of Pcc1-Kae1-Bud32-Cgi121, not only played evolutionarily conserved roles in growth, sexual development, stress responses, and tRNA modification but also had unique roles in controlling virulence factor production and pathogenicity. Notably, a unique extended loop structure in CnBud32 is critical for the KEOPS complex in C. neoformans. Supporting its pleiotropic roles, transcriptome analysis revealed that the CnKEOPS complex governs several hundreds of genes involved in carbon and amino acid metabolism, pheromone response, and ergosterol biosynthesis. Therefore, this study provides novel insights into the fungal KEOPS complex that could be exploited as a potential antifungal drug target.


Subject(s)
Cryptococcus neoformans , Fungal Proteins , Humans , Cryptococcus neoformans/enzymology , Cryptococcus neoformans/metabolism , Cryptococcus neoformans/pathogenicity , Ergosterol , Fungal Proteins/genetics , Fungal Proteins/metabolism , Phosphotransferases/metabolism , Endopeptidases/metabolism
4.
Nature ; 608(7921): 161-167, 2022 08.
Article in English | MEDLINE | ID: mdl-35896747

ABSTRACT

Invasive fungal pathogens are major causes of human mortality and morbidity1,2. Although numerous secreted effector proteins that reprogram innate immunity to promote virulence have been identified in pathogenic bacteria, so far, there are no examples of analogous secreted effector proteins produced by human fungal pathogens. Cryptococcus neoformans, the most common cause of fungal meningitis and a major pathogen in AIDS, induces a pathogenic type 2 response characterized by pulmonary eosinophilia and alternatively activated macrophages3-8. Here, we identify CPL1 as an effector protein secreted by C. neoformans that drives alternative activation (also known as M2 polarization) of macrophages to enable pulmonary infection in mice. We observed that CPL1-enhanced macrophage polarization requires Toll-like receptor 4, which is best known as a receptor for bacterial endotoxin but is also a poorly understood mediator of allergen-induced type 2 responses9-12. We show that this effect is caused by CPL1 itself and not by contaminating lipopolysaccharide. CPL1 is essential for virulence, drives polarization of interstitial macrophages in vivo, and requires type 2 cytokine signalling for its effect on infectivity. Notably, C. neoformans associates selectively with polarized interstitial macrophages during infection, suggesting a mechanism by which C. neoformans generates its own intracellular replication niche within the host. This work identifies a circuit whereby a secreted effector protein produced by a human fungal pathogen reprograms innate immunity, revealing an unexpected role for Toll-like receptor 4 in promoting the pathogenesis of infectious disease.


Subject(s)
Cryptococcosis , Cryptococcus neoformans , Fungal Proteins , Hypersensitivity , Inflammation , Toll-Like Receptor 4 , Virulence Factors , Animals , Cryptococcosis/immunology , Cryptococcosis/microbiology , Cryptococcosis/pathology , Cryptococcus neoformans/immunology , Cryptococcus neoformans/pathogenicity , Cytokines/immunology , Fungal Proteins/immunology , Fungal Proteins/metabolism , Hypersensitivity/immunology , Hypersensitivity/microbiology , Immunity, Innate , Inflammation/immunology , Inflammation/microbiology , Lipopolysaccharides/immunology , Lung/immunology , Lung/microbiology , Macrophages/cytology , Macrophages/immunology , Macrophages/microbiology , Mice , Toll-Like Receptor 4/immunology , Toll-Like Receptor 4/metabolism , Virulence , Virulence Factors/immunology
5.
BMC Microbiol ; 22(1): 162, 2022 06 22.
Article in English | MEDLINE | ID: mdl-35733100

ABSTRACT

BACKGROUND: Cryptococcosis is a life-threatening infection is primarily caused by two sibling species Cryptococcus neoformans and Cryptococcus gattii. Several virulence-related factors of these cryptococci have been widely investigated in Caenorhabditis elegans, representing a facile in vivo model of host-pathogen interaction. While recent studies elucidated cryptococcal virulence factors, intrinsic host factors that affect susceptibility to infections by cryptococci remain unclear and poorly investigated. RESULTS: Here, we showed that defects in C. elegans insulin/insulin-like growth factor-1 (IGF-1) signaling (IIS) pathway influenced animal lifespan and mechanisms of host resistance in cryptococcal infections, which required the activation of aging regulator DAF-16/Forkhead box O transcription factor. Moreover, accumulation of lipofuscin, DAF-16 nuclear localization, and expression of superoxide dismutase (SOD-3) were elevated in C. elegans due to host defenses during cryptococcal infections. CONCLUSION: The present study demonstrated the relationship between longevity and immunity, which may provide a possibility for novel therapeutic intervention to improve host resistance against cryptococcal infections.


Subject(s)
Caenorhabditis elegans Proteins , Cryptococcosis , Cryptococcus gattii , Cryptococcus neoformans , Forkhead Transcription Factors , Animals , Caenorhabditis elegans/microbiology , Caenorhabditis elegans/physiology , Caenorhabditis elegans Proteins/genetics , Cryptococcus gattii/pathogenicity , Cryptococcus neoformans/pathogenicity , Forkhead Transcription Factors/genetics , Immunity , Longevity , Virulence Factors/metabolism
6.
Fungal Genet Biol ; 160: 103697, 2022 05.
Article in English | MEDLINE | ID: mdl-35472450

ABSTRACT

Cryptococcus neoformans, a basidiomycete yeast, causes lethal meningitis in immunocompromised individuals. The ability of C. neoformans to proliferate at 37°C is essential for virulence. We identified anillin-like protein, CnBud4, as essential for proliferation of C. neoformans at 37°C and for virulence in a heterologous host Galleria mellonella at 25°C. C. neoformans cells lacking CnBud4 were inviable at 25°C in the absence of active calcineurin and were hypersensitive to membrane stress and an anti-fungal agent fluconazole, phenotypes previously described for C. neoformans mutants lacking septins. CnBud4 localized to the mother-bud neck during cytokinesis in a septin-dependent manner. In the absence of CnBud4, septin complex failed to transition from a collar-like single ring to the double ring during cytokinesis. In an ascomycete yeast, Saccharomyces cerevisiae, the anillin-like homologue ScBud4 participates in the organization of the septin ring at the mother-bud neck and plays an important role in specifying location for new bud emergence, known as axial budding pattern. In contrast to their role in S. cerevisiae, neither septins nor CnBud4 were needed to direct the position of the new bud in C. neoformans, suggesting that this function is not conserved in basidiomycetous yeasts. Our data suggest that the requirement of CnBud4 for growth at 37°C and pathogenicity in C. neoformans is based on its conserved role in septin complex organization.


Subject(s)
Body Temperature , Contractile Proteins , Cryptococcus neoformans , Cryptococcosis/microbiology , Cryptococcus neoformans/growth & development , Cryptococcus neoformans/pathogenicity , Host Microbial Interactions , Humans , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins , Septins/metabolism
7.
BMC Microbiol ; 21(1): 341, 2021 12 13.
Article in English | MEDLINE | ID: mdl-34903172

ABSTRACT

BACKGROUND: Fungal infections impact over 25% of the global population. For the opportunistic fungal pathogen, Cryptococcus neoformans, infection leads to cryptococcosis. In the presence of the host, disease is enabled by elaboration of sophisticated virulence determinants, including polysaccharide capsule, melanin, thermotolerance, and extracellular enzymes. Conversely, the host protects itself from fungal invasion by regulating and sequestering transition metals (e.g., iron, zinc, copper) important for microbial growth and survival. RESULTS: Here, we explore the intricate relationship between zinc availability and fungal virulence via mass spectrometry-based quantitative proteomics. We observe a core proteome along with a distinct zinc-regulated protein-level signature demonstrating a shift away from transport and ion binding under zinc-replete conditions towards transcription and metal acquisition under zinc-limited conditions. In addition, we revealed a novel connection among zinc availability, thermotolerance, as well as capsule and melanin production through the detection of a Wos2 ortholog in the secretome under replete conditions. CONCLUSIONS: Overall, we provide new biological insight into cellular remodeling at the protein level of C. neoformans under regulated zinc conditions and uncover a novel connection between zinc homeostasis and fungal virulence determinants.


Subject(s)
Cryptococcus neoformans/pathogenicity , Molecular Chaperones/metabolism , Proteome/metabolism , Secretome/metabolism , Zinc/metabolism , Cryptococcus neoformans/metabolism , Fungal Capsules/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Melanins/metabolism , Molecular Chaperones/genetics , Mutation , Proteomics , Thermotolerance , Virulence/genetics
8.
Int J Mol Sci ; 22(22)2021 Nov 17.
Article in English | MEDLINE | ID: mdl-34830272

ABSTRACT

Fungal pathogens cause an array of diseases by targeting both immunocompromised and immunocompetent hosts. Fungi overcome our current arsenal of antifungals through the emergence and evolution of resistance. In particular, the human fungal pathogen, Cryptococcus neoformans is found ubiquitously within the environment and causes severe disease in immunocompromised individuals around the globe with limited treatment options available. To uncover fundamental knowledge about this fungal pathogen, as well as investigate new detection and treatment strategies, mass spectrometry-based proteomics provides a plethora of tools and applications, as well as bioinformatics platforms. In this review, we highlight proteomics approaches within the laboratory to investigate changes in the cellular proteome, secretome, and extracellular vesicles. We also explore regulation by post-translational modifications and the impact of protein-protein interactions. Further, we present the development and comprehensive assessment of murine models of cryptococcal infection, which provide valuable tools to define the dynamic relationship between the host and pathogen during disease. Finally, we explore recent quantitative proteomics studies that begin to extrapolate the findings from the bench to the clinic for improved methods of fungal detection and monitoring. Such studies support a framework for personalized medical approaches to eradicate diseases caused by C. neoformans.


Subject(s)
Cryptococcosis/metabolism , Cryptococcus neoformans/metabolism , Fungal Proteins/metabolism , Proteome/metabolism , Proteomics/methods , Animals , Antifungal Agents/therapeutic use , Cryptococcosis/drug therapy , Cryptococcosis/microbiology , Cryptococcus neoformans/genetics , Cryptococcus neoformans/pathogenicity , Disease Models, Animal , Drug Resistance, Fungal/genetics , Extracellular Vesicles/metabolism , Fungal Proteins/genetics , Host-Parasite Interactions/genetics , Humans , Mice , Precision Medicine/methods , Protein Interaction Maps/genetics , Protein Processing, Post-Translational/genetics , Proteome/genetics , Secretome/metabolism , Transcriptome , Treatment Outcome , Virulence Factors/metabolism
9.
mBio ; 12(6): e0279021, 2021 12 21.
Article in English | MEDLINE | ID: mdl-34724824

ABSTRACT

The environmental yeast Cryptococcus neoformans is the most common cause of deadly fungal meningitis in primarily immunocompromised populations. A number of factors contribute to cryptococcal pathogenesis. Among them, inositol utilization has been shown to promote C. neoformans development in nature and invasion of central nervous system during dissemination. The mechanisms of the inositol regulation of fungal virulence remain incompletely understood. In this study, we analyzed inositol-induced capsule growth and the contribution of a unique inositol catabolic pathway in fungal development and virulence. We found that genes involved in the inositol catabolic pathway are highly induced by inositol, and they are also highly expressed in the cerebrospinal fluid of patients with meningoencephalitis. This pathway in C. neoformans contains three genes encoding myo-inositol oxygenases that convert myo-inositol into d-glucuronic acid, a substrate of the pentose phosphate cycle and a component of the polysaccharide capsule. Our mutagenesis analysis demonstrates that inositol catabolism is required for C. neoformans virulence and deletion mutants of myo-inositol oxygenases result in altered capsule growth as well as the polysaccharide structure, including O-acetylation. Our study indicates that the ability to utilize the abundant inositol in the brain may contribute to fungal pathogenesis in this neurotropic fungal pathogen. IMPORTANCE The human pathogen Cryptococcus neoformans is the leading cause of fungal meningitis in primarily immunocompromised populations. Understanding how this environmental organism adapts to the human host to cause deadly infection will guide our development of novel disease control strategies. Our recent studies revealed that inositol utilization by the fungus promotes C. neoformans development in nature and invasion of the central nervous system during infection. The mechanisms of the inositol regulation in fungal virulence remain incompletely understood. In this study, we found that C. neoformans has three genes encoding myo-inositol oxygenase, a key enzyme in the inositol catabolic pathway. Expression of these genes is highly induced by inositol, and they are highly expressed in the cerebrospinal fluid of patients with meningoencephalitis. Our mutagenesis analysis indeed demonstrates that inositol catabolism is required for C. neoformans virulence by altering the growth and structure of polysaccharide capsule, a major virulence factor. Considering the abundance of free inositol and inositol-related metabolites in the brain, our study reveals an important mechanism of host inositol-mediated fungal pathogenesis for this neurotropic fungal pathogen.


Subject(s)
Cryptococcus neoformans/metabolism , Cryptococcus neoformans/pathogenicity , Fungal Capsules/chemistry , Inositol/metabolism , Meningitis, Cryptococcal/microbiology , Animals , Brain/metabolism , Brain/microbiology , Cryptococcus neoformans/chemistry , Cryptococcus neoformans/genetics , Female , Fungal Capsules/genetics , Fungal Capsules/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Humans , Male , Meningitis, Cryptococcal/metabolism , Mice , Oxygenases/genetics , Oxygenases/metabolism , Rabbits , Virulence
10.
mBio ; 12(6): e0231321, 2021 12 21.
Article in English | MEDLINE | ID: mdl-34724829

ABSTRACT

Cryptococcus neoformans is a major human central nervous system (CNS) fungal pathogen causing considerable morbidity and mortality. In this study, we provide the widest view to date of the yeast transcriptome directly from the human subarachnoid space and within cerebrospinal fluid (CSF). We captured yeast transcriptomes from C. neoformans of various genotypes in 31 patients with cryptococcal meningoencephalitis as well as several Cryptococcus gattii infections. Using transcriptome sequencing (RNA-seq) analyses, we compared the in vivo yeast transcriptomes to those from other environmental conditions, including in vitro growth on nutritious media or artificial CSF as well as samples collected from rabbit CSF at two time points. We ranked gene expressions and identified genetic patterns and networks across these diverse isolates that reveal an emphasis on carbon metabolism, fatty acid synthesis, transport, cell wall structure, and stress-related gene functions during growth in CSF. The most highly expressed yeast genes in human CSF included those known to be associated with survival or virulence and highlighted several genes encoding hypothetical proteins. From that group, a gene encoding the CMP1 putative glycoprotein (CNAG_06000) was selected for functional studies. This gene was found to impact the virulence of Cryptococcus in both mice and the CNS rabbit model, in agreement with a recent study also showing a role in virulence. This transcriptional analysis strategy provides a view of regulated yeast genes across genetic backgrounds important for human CNS infection and a relevant resource for the study of cryptococcal genes, pathways, and networks linked to human disease. IMPORTANCE Cryptococcus is the most common fungus causing high-morbidity and -mortality human meningitis. This encapsulated yeast has a unique propensity to travel to the central nervous system to produce disease. In this study, we captured transcriptomes of yeasts directly out of the human cerebrospinal fluid, the most concerning site of infection. By comparing the RNA transcript levels with other conditions, we gained insights into how the basic machinery involved in metabolism and environmental responses enable this fungus to cause disease at this body site. This approach was applied to clinical isolates with diverse genotypes to begin to establish a genotype-agnostic understanding of how the yeast responds to stress. Based on these results, future studies can focus on how these genes and their pathways and networks can be targeted with new therapeutics and possibly classify yeasts with bad infection outcomes.


Subject(s)
Cryptococcosis/microbiology , Cryptococcus neoformans/genetics , Meningoencephalitis/microbiology , Animals , Central Nervous System/microbiology , Cryptococcosis/cerebrospinal fluid , Cryptococcus neoformans/classification , Cryptococcus neoformans/isolation & purification , Cryptococcus neoformans/pathogenicity , Disease Models, Animal , Female , Fungal Proteins/genetics , Fungal Proteins/metabolism , Genotype , Humans , Male , Meningoencephalitis/diagnosis , Mice , RNA-Seq , Rabbits , Transcriptome , Virulence
11.
mBio ; 12(5): e0250921, 2021 10 26.
Article in English | MEDLINE | ID: mdl-34634930

ABSTRACT

The pathogenic yeast Cryptococcus neoformans produces polyploid titan cells in response to the host lung environment that are critical for host adaptation and subsequent disease. We analyzed the in vivo and in vitro cell cycles to identify key aspects of the C. neoformans cell cycle that are important for the formation of titan cells. We identified unbudded 2C cells, referred to as a G2 arrest, produced both in vivo and in vitro in response to various stresses. Deletion of the nonessential cyclin Cln1 resulted in overproduction of titan cells in vivo and transient morphology defects upon release from stationary phase in vitro. Using a copper-repressible promoter PCTR4-CLN1 strain and a two-step in vitro titan cell formation assay, our in vitro studies revealed Cln1 functions after the G2 arrest. These studies highlight unique cell cycle alterations in C. neoformans that ultimately promote genomic diversity and virulence in this important fungal pathogen. IMPORTANCE Dysregulation of the cell cycle underlies many human genetic diseases and cancers, yet numerous organisms, including microbes, also manipulate the cell cycle to generate both morphologic and genetic diversity as a natural mechanism to enhance their chances for survival. The eukaryotic pathogen Cryptococcus neoformans generates morphologically distinct polyploid titan cells critical for host adaptation and subsequent disease. We analyzed the C. neoformans in vivo and in vitro cell cycles to identify changes required to generate the polyploid titan cells. C. neoformans paused cell cycle progression in response to various environmental stresses after DNA replication and before morphological changes associated with cell division, referred to as a G2 arrest. Release from this G2 arrest was coordinated by the cyclin Cln1. Reduced CLN1 expression after the G2 arrest was associated with polyploid titan cell production. These results demonstrate a mechanism to generate genomic diversity in eukaryotic cells through manipulation of the cell cycle that has broad disease implications.


Subject(s)
Cell Cycle/genetics , Cryptococcus neoformans/genetics , Cyclins/genetics , Fungal Proteins/genetics , G2 Phase Cell Cycle Checkpoints/genetics , Stress, Physiological/genetics , Animals , Cell Cycle/physiology , Cryptococcosis/microbiology , Cryptococcus neoformans/pathogenicity , Cryptococcus neoformans/physiology , Cyclins/metabolism , Disease Models, Animal , Female , Fungal Proteins/metabolism , Host-Pathogen Interactions , Stress, Physiological/physiology , Virulence
12.
J Microbiol ; 59(7): 658-665, 2021 Jul.
Article in English | MEDLINE | ID: mdl-34212289

ABSTRACT

Yvh1 is a dual-specificity phosphatase (DUSP) that is evolutionarily conserved in eukaryotes, including yeasts and humans. Yvh1 is involved in the vegetative growth, differentiation, and virulence of animal and plant fungal pathogens. All Yvh1 orthologs have a conserved DUSP catalytic domain at the N-terminus and a zinc-binding (ZB) domain with two zinc fingers (ZFs) at the C-terminus. Although the DUSP domain is implicated in the regulation of MAPK signaling in humans, only the ZB domain is essential for most cellular functions of Yvh1 in fungi. This study aimed to analyze the functions of the DUSP and ZB domains of Yvh1 in the human fungal pathogen Cryptococcus neoformans, whose Yvh1 (CnYvh1) contains a DUSP domain at the C-terminus and a ZB domain at the N-terminus. Notably, CnYvh1 has an extended internal domain between the two ZF motifs in the ZB domain. To elucidate the function of each domain, we constructed individual domain deletions and swapping strains by complementing the yvh1Δ mutant with wild-type (WT) or mutated YVH1 alleles and examined their Yvh1-dependent phenotypes, including growth under varying stress conditions, mating, and virulence factor production. Here, we found that the complementation of the yvh1Δ mutant with the mutated YVH1 alleles having two ZFs of the ZB domain, but not the DUSP and extended internal domains, restored the WT phenotypic traits in the yvh1Δ mutant. In conclusion, the ZB domain, but not the N-terminal DUSP domain, plays a pivotal role in the pathobiological functions of cryptococcal Yvh1.


Subject(s)
Cryptococcus neoformans/enzymology , Dual-Specificity Phosphatases/chemistry , Dual-Specificity Phosphatases/metabolism , Protein Domains , Zinc/metabolism , Catalytic Domain , Cryptococcus neoformans/cytology , Cryptococcus neoformans/genetics , Cryptococcus neoformans/pathogenicity , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Genetic Complementation Test , Melanins/biosynthesis , Mutation , Protein Binding , Urease/biosynthesis , Virulence Factors/biosynthesis , Zinc Fingers
13.
Int J Mol Sci ; 22(12)2021 Jun 11.
Article in English | MEDLINE | ID: mdl-34208294

ABSTRACT

Cryptococcus neoformans is a facultative intracellular pathogen responsible for fungal meningoencephalitis primarily in immunocompromised individuals. It has become evident the pathogenicity of C. neoformans is dependent on the fungal cell's environment. The differential expression of virulence factors, based on the cell's environmental conditions, is one mechanism allowing for the environmental control of the pathogenic ability of C. neoformans. Here, we discuss how these virulence factors (including melanin, the polysaccharide capsule, and Antiphagocytic protein 1) have been shown to be differentially expressed dependent on the cell's environment. The genetics and signaling pathways leading to the environmental-dependent regulation of virulence factors will also be examined. Susceptibility to antifungal therapeutics is also regulated by the environment, and thus affects the pathogenic abilities of C. neoformans and disease outcomes. This review will also examine the role of the C. neoformans's environment on antifungal susceptibilities, and the genetics and signaling pathways responsible for these susceptibility alterations. By examining the complex interplay between the environment and the pathogenicity of C. neoformans, we have a better understanding of the intricacies of the pathogen-environment interaction and how to exploit this interaction to develop the most effective treatment protocols.


Subject(s)
Antifungal Agents/pharmacology , Cryptococcus neoformans/drug effects , Cryptococcus neoformans/pathogenicity , Environment , Virulence Factors/metabolism , Animals , Humans , Lung/microbiology , Microbial Sensitivity Tests
14.
Infect Immun ; 89(10): e0033021, 2021 09 16.
Article in English | MEDLINE | ID: mdl-34251289

ABSTRACT

The cell walls and capsules of Cryptococcus neoformans, a yeast-type fungal pathogen, are rich in polysaccharides. Dectin-2 is a C-type lectin receptor (CLR) that recognizes high-mannose polysaccharides. Previously, we demonstrated that Dectin-2 is involved in cytokine production by bone marrow-derived dendritic cells (BM-DCs) in response to stimulation with C. neoformans. In the present study, we analyzed the role of Dectin-2 in the phagocytosis of C. neoformans by BM-DCs. The engulfment of this fungus by BM-DCs was significantly decreased in mice lacking Dectin-2 (Dectin-2 knockout [Dectin-2KO]) or caspase recruitment domain-containing protein 9 (CARD9KO), a common adapter molecule that delivers signals triggered by CLRs, compared to wild-type (WT) mice. Phagocytosis was likewise inhibited, to a similar degree, by the inhibition of Syk, a signaling molecule involved in CLR-triggered activation. A PI3K inhibitor, in contrast, completely abrogated the phagocytosis of C. neoformans. Actin polymerization, i.e., conformational changes in cytoskeletons detected at sites of contact with C. neoformans, was also decreased in BM-DCs of Dectin-2KO and CARD9KO mice. Finally, the engulfment of C. neoformans by macrophages was significantly decreased in the lungs of Dectin-2KO mice compared to WT mice. These results suggest that Dectin-2 may play an important role in the actin polymerization and phagocytosis of C. neoformans by DCs, possibly through signaling via CARD9 and a signaling pathway mediated by Syk and PI3K.


Subject(s)
Cryptococcosis/microbiology , Cryptococcus neoformans/pathogenicity , Dendritic Cells/metabolism , Lectins, C-Type/metabolism , Phagocytosis/physiology , Animals , Bone Marrow Cells/metabolism , Bone Marrow Cells/microbiology , CARD Signaling Adaptor Proteins/metabolism , Cryptococcosis/metabolism , Cytokines/metabolism , Dendritic Cells/microbiology , Female , Lung/metabolism , Lung/microbiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphatidylinositol 3-Kinases/metabolism
15.
Med Microbiol Immunol ; 210(4): 221-233, 2021 Aug.
Article in English | MEDLINE | ID: mdl-34228244

ABSTRACT

In the airways, the adhesion of Cryptococcus neoformans with airway epithelial cells is crucial for the establishment of cryptococcosis. Tobacco smoke is considered a risk factor for cryptococcosis. Here, we evaluated the effects of cigarette smoke extract (CSE) on human bronchial epithelial cells (BEAS-2B) stimulated with C. neoformans. Multiplicities of infection (MOIs) of 1-100 of C. neoformans per cell led to increased IL-8 production and no cytotoxic effects when compared to those of controls. C. neoformans (MOI 100) also significantly increased the concentration of IL-6. In cells stimulated with CSE doses (1.0, 2.5 and 5.0%) from one or five cigarettes, increased IL-1ß production was observed only in doses from one (1.0%) and five (2.5%) cigarettes when compared to that of controls. However, only 1.0% CSE failed to show cytotoxic effects. In addition, CSE significantly increased the concentration of IL-8. Cells stimulated with both CSE and C. neoformans demonstrated a reduction in IL-6/STAT3 signalling compared to that in cells stimulated by C. neoformans. In addition, a significant increase in IL-10 production was also observed. No alterations in NF-kB or ICAM-1 expression were observed among the groups. The combination of CSE and C. neoformans favoured the increase of fungal numbers and extracellular adhering of C. neoformans on BEAS-2B cells. In addition, the internalization of C. neoformans on BEAS-2B cells was reduced after CSE stimulation. In conclusion, the association of CSE and C. neoformans induced an anti-inflammatory effect in bronchial epithelial cells, which might favour the development of C. neoformans infection in the airways.


Subject(s)
Cryptococcosis/pathology , Cryptococcus neoformans/pathogenicity , Cytokines/metabolism , Epithelial Cells/drug effects , Epithelial Cells/microbiology , Smoke/adverse effects , Tobacco Products/adverse effects , Bronchi/cytology , Bronchi/drug effects , Bronchi/microbiology , Cell Line , Cell Survival , Cryptococcosis/microbiology , Humans , Intercellular Adhesion Molecule-1/metabolism , Interleukin-10/metabolism , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Interleukin-8/metabolism , NF-kappa B/metabolism , Phagocytosis/drug effects , Risk Factors , STAT3 Transcription Factor/metabolism , Signal Transduction
16.
mBio ; 12(4): e0167221, 2021 08 31.
Article in English | MEDLINE | ID: mdl-34311572

ABSTRACT

There is a critical need for new antifungal drugs; however, the lack of available fungus-specific targets is a major hurdle in the development of antifungal therapeutics. Spore germination is a differentiation process absent in humans that could harbor uncharacterized fungus-specific targets. To capitalize on this possibility, we developed novel phenotypic assays to identify and characterize inhibitors of spore germination of the human fungal pathogen Cryptococcus. Using these assays, we carried out a high-throughput screen of ∼75,000 drug-like small molecules and identified and characterized 191 novel inhibitors of spore germination, many of which also inhibited yeast replication and demonstrated low cytotoxicity against mammalian cells. Using an automated, microscopy-based, quantitative germination assay (QGA), we discovered that germinating spore populations can exhibit unique phenotypes in response to chemical inhibitors. Through the characterization of these spore population dynamics in the presence of the newly identified inhibitors, we classified 6 distinct phenotypes based on differences in germination synchronicity, germination rates, and overall population behavior. Similar chemical phenotypes were induced by inhibitors that targeted the same cellular function or had shared substructures. Leveraging these features, we used QGAs to identify outliers among compounds that fell into similar structural groups and thus refined relevant structural moieties, facilitating target identification. This approach led to the identification of complex II of the electron transport chain as the putative target of a promising structural cluster of germination inhibitory compounds. These inhibitors showed high potency against Cryptococcus spore germination while maintaining low cytotoxicity against mammalian cells, making them prime candidates for development into novel antifungal therapeutics. IMPORTANCE Fungal pathogens cause 1.5 million deaths annually, and there is a critical need for new antifungal drugs. However, humans and fungi are very similar on a molecular level, and so many drugs that kill fungi also damage human cells, leading to extreme side effects, including death. The lack of fungus-specific targets is a major hurdle in the development of antifungal therapeutics. Spore germination is a process absent in humans that could harbor fungus-specific targets. To capitalize on this possibility, we developed new assays to identify and characterize inhibitors of spore germination of the human fungal pathogen Cryptococcus. Using these assays, we identified and characterized 191 novel inhibitors of spore germination. These inhibitors showed high potency against Cryptococcus spore germination while maintaining low cytotoxicity against mammalian cells, making them prime candidates for development into novel antifungal therapeutics.


Subject(s)
Antifungal Agents/pharmacology , Cryptococcus neoformans/drug effects , Spores, Fungal/drug effects , Spores, Fungal/growth & development , Cryptococcosis/drug therapy , Cryptococcus neoformans/growth & development , Cryptococcus neoformans/pathogenicity , Drug Discovery , High-Throughput Screening Assays , Humans , Phenotype , Spores, Fungal/classification , Spores, Fungal/pathogenicity
17.
PLoS Biol ; 19(5): e3001182, 2021 05.
Article in English | MEDLINE | ID: mdl-33979323

ABSTRACT

Melanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanin's broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate-the most widely used herbicide globally-inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in 2 evolutionary distant species, Galleria mellonella (Lepidoptera: Pyralidae) and Anopheles gambiae (Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival of G. mellonella caterpillars following infection with the fungus Cryptococcus neoformans and decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria-causing parasite Plasmodium falciparum in A. gambiae mosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosate's mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation-reduction balance. Overall, these findings suggest that glyphosate's environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations.


Subject(s)
Anopheles/drug effects , Glycine/analogs & derivatives , Melanins/metabolism , Moths/drug effects , Animals , Anopheles/immunology , Cryptococcus neoformans/pathogenicity , Diptera/drug effects , Diptera/immunology , Glycine/metabolism , Glycine/pharmacology , Immunity, Innate/drug effects , Immunity, Innate/immunology , Infections/immunology , Infections/metabolism , Infections/physiopathology , Insecta/drug effects , Insecta/immunology , Lepidoptera/drug effects , Lepidoptera/immunology , Moths/immunology , Plasmodium falciparum/pathogenicity , Virulence , Glyphosate
18.
Diagn Cytopathol ; 49(9): E348-E351, 2021 Sep.
Article in English | MEDLINE | ID: mdl-34021719

ABSTRACT

Cryptococcosis is caused by Cryptococcus neoformans and is fatal in children. The fungus is known to enter respiratory tract by inhalation and localizes in lungs in immunocompetent host. Patients with immunocompromised state facilitate dissemination of disease. However, disseminated cases have been described in immunocompetent HIV-negative individuals. CSF rhinorrhoea as a predisposing cause of cryptococcal meningitis has been rarely reported. We hereby describe C. neoformans directly spreading to the meninges in 1 year child due to CSF rhinorrhoea and the fungus was detected on fluid cytology.


Subject(s)
Cerebrospinal Fluid Rhinorrhea/pathology , Cryptococcosis/pathology , Cerebrospinal Fluid Rhinorrhea/microbiology , Cryptococcosis/microbiology , Cryptococcus neoformans/isolation & purification , Cryptococcus neoformans/pathogenicity , Humans , Infant , Male
19.
mBio ; 12(2)2021 04 27.
Article in English | MEDLINE | ID: mdl-33906924

ABSTRACT

Amoeboid predators, such as amoebae, are proposed to select for survival traits in soil microbes such as Cryptococcus neoformans; these traits can also function in animal virulence by defeating phagocytic immune cells, such as macrophages. Consistent with this notion, incubation of various fungal species with amoebae enhanced their virulence, but the mechanisms involved are unknown. In this study, we exposed three strains of C. neoformans (1 clinical and 2 environmental) to predation by Acanthamoeba castellanii for prolonged times and then analyzed surviving colonies phenotypically and genetically. Surviving colonies comprised cells that expressed either pseudohyphal or yeast phenotypes, which demonstrated variable expression of traits associated with virulence, such as capsule size, urease production, and melanization. Phenotypic changes were associated with aneuploidy and DNA sequence mutations in some amoeba-passaged isolates, but not in others. Mutations in the gene encoding the oligopeptide transporter (CNAG_03013; OPT1) were observed among amoeba-passaged isolates from each of the three strains. Isolates derived from environmental strains gained the capacity for enhanced macrophage toxicity after amoeba selection and carried mutations on the CNAG_00570 gene encoding Pkr1 (AMP-dependent protein kinase regulator) but manifested reduced virulence in mice because they elicited more effective fungal-clearing immune responses. Our results indicate that C. neoformans survival under constant amoeba predation involves the generation of strains expressing pleiotropic phenotypic and genetic changes. Given the myriad potential predators in soils, the diversity observed among amoeba-selected strains suggests a bet-hedging strategy whereby variant diversity increases the likelihood that some will survive predation.IMPORTANCECryptococcus neoformans is a ubiquitous environmental fungus that is also a leading cause of fatal fungal infection in humans, especially among immunocompromised patients. A major question in the field is how an environmental yeast such as C. neoformans becomes a human pathogen when it has no need for an animal host in its life cycle. Previous studies showed that C. neoformans increases its pathogenicity after interacting with its environmental predator amoebae. Amoebae, like macrophages, are phagocytic cells that are considered an environmental training ground for pathogens to resist macrophages, but the mechanism by which C. neoformans changes its virulence through interactions with protozoa is unknown. Our study indicates that fungal survival in the face of amoeba predation is associated with the emergence of pleiotropic phenotypic and genomic changes that increase the chance of fungal survival, with this diversity suggesting a bet-hedging strategy to ensure that some forms survive.


Subject(s)
Acanthamoeba castellanii/physiology , Cryptococcosis/microbiology , Cryptococcus neoformans/pathogenicity , Phagocytosis , Acanthamoeba castellanii/microbiology , Animals , Cryptococcosis/immunology , Cryptococcus neoformans/classification , Cryptococcus neoformans/genetics , Cytokines/immunology , Female , Humans , Larva/microbiology , Macrophages/microbiology , Mice, Inbred C57BL , Moths/microbiology , Phagocytes/microbiology , Phenotype , Virulence
20.
mSphere ; 6(2)2021 03 24.
Article in English | MEDLINE | ID: mdl-33762317

ABSTRACT

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


Subject(s)
Coinfection/microbiology , Coinfection/virology , Cryptococcus neoformans/immunology , HIV-1/immunology , Macrophages/immunology , Macrophages/virology , Tumor Necrosis Factor-alpha/analysis , Coinfection/immunology , Cryptococcus neoformans/pathogenicity , HIV-1/pathogenicity , Humans , Lung , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/immunology , THP-1 Cells , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Up-Regulation , NF-kappaB-Inducing Kinase
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