Transcriptional Network of Multiple Capsule and Melanin Genes Governed by the Cryptococcus neoformans Cyclic AMP Cascade

ABSTRACT Cryptococcus neoformans is an opportunistic human fungal pathogen that elaborates several virulence attributes, including a polysaccharide capsule and melanin pigments. A conserved Gα protein/cyclic AMP (cAMP) pathway controls melanin and capsule production. To identify targets of this pathway, we used an expression profiling approach to define genes that are transcriptionally regulated by the Gα protein Gpa1. This approach revealed that Gpa1 transcriptionally regulates multiple genes involved in capsule assembly and identified two additional genes with a marked dependence on Gpa1 for transcription. The first is the LAC1 gene, encoding the laccase enzyme that catalyzes a rate-limiting step in diphenol oxidation and melanin production. The second gene identified (LAC2) is adjacent to the LAC1 gene and encodes a second laccase that shares 75% nucleotide identity with LAC1. Similar to the LAC1 gene, LAC2 is induced in response to glucose deprivation. However, LAC2 basal transcript levels are much lower than those for LAC1. Accordingly, a lac2 mutation results in only a modest delay in melanin formation. LAC2 overexpression suppresses the melanin defects of gpa1 and lac1 mutants and partially restores virulence of these strains. These studies provide mechanistic insights into the regulation of capsule and melanin production by the C. neoformans cAMP pathway and demonstrate that multiple laccases contribute to C. neoformans melanin production and pathogenesis.

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Genetic and environmental influences on the evolution of virulence in the HIV-associated opportunistic human fungal pathogen Cryptococcus neoformans

10.1101/2021.10.15.464490 ◽

2021 ◽

Author(s):

Yiwu Yu ◽

Yuanyuan Wang ◽

Linghua Li ◽

Xiaoqing Chen ◽

Xinhua Huang ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Natural Environment ◽

Large Scale ◽

Sequence Type ◽

Sequencing Analysis ◽

Uninfected Individual ◽

Human Fungal Pathogen ◽

Virulence Evolution ◽

Capsule Production ◽

Genetic And Environmental Factors

The fungus Cryptococcus neoformans is considered the leading cause of death in immunocompromised patients. Despite numerous investigations concerning its molecular epidemiology, there are only a few studies addressing the impacts of varying factors on genotype-phenotype correlations. It remains largely unknown whether genetic and environmental variabilities among isolates from different sources may have dramatic consequences on virulence. In this study, we analyzed 105 Chinese C. neoformans isolates, including 54 from HIV-infected patients, 44 from HIV-uninfected individuals and 7 from a natural environment, to investigate factors influencing the outcome of C. neoformans infection. MLST analysis clearly identified sequence type (ST) 5 as the prevalent sequence type in all clinical isolates and interestingly, genotypic diversities were observed in isolates from both HIV-uninfected individual and natural environment but not those from HIV-infected patients. Moreover, we found that compared to those from HIV-infected patients, the isolates from HIV-uninfected individuals exhibited enhanced virulence-associated traits including significantly elevated capsule production and melanin formation, increases in survival in human cerebrospinal fluid (CSF), less effective uptake by host phagocytes, and higher mortality in a mouse model of cryptococcosis. Consistently, pathogenic phenotypes were associated with CD4 counts of patients, implying environmental impact on within-host C. neoformans virulence. Importantly, a large-scale whole-genome sequencing analysis revealed that genomic variations within genes related to specific functions may act as a vital driving force of host intrinsic virulence evolution. Taken together, our results support a strong genotype-phenotype correlation suggesting that the pathogenic evolution of C. neoformans could be heavily affected by both genetic and environmental factors.

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Recapitulation of the Sexual Cycle of the Primary Fungal Pathogen Cryptococcus neoformans var. gattii: Implications for an Outbreak on Vancouver Island, Canada

Eukaryotic Cell ◽

10.1128/ec.2.5.1036-1045.2003 ◽

2003 ◽

Vol 2 (5) ◽

pp. 1036-1045 ◽

Cited By ~ 201

Author(s):

James A. Fraser ◽

Ryan L. Subaran ◽

Connie B. Nichols ◽

Joseph Heitman

Keyword(s):

Cryptococcus Neoformans ◽

Fungal Pathogen ◽

Large Scale ◽

Vancouver Island ◽

Sexual Cycle ◽

Gene Encoding ◽

Tropical Regions ◽

Human Fungal Pathogen ◽

Large Scale Analysis ◽

Serotype B

ABSTRACT Cryptococcus neoformans is a human fungal pathogen that exists as three distinct varieties or sibling species: the predominantly opportunistic pathogens C. neoformans var. neoformans (serotype D) and C. neoformans var. grubii (serotype A) and the primary pathogen C. neoformans var. gattii (serotypes B and C). While serotypes A and D are cosmopolitan, serotypes B and C are typically restricted to tropical regions. However, serotype B isolates of C. neoformans var. gattii have recently caused an outbreak on Vancouver Island in Canada, highlighting the threat of this fungus and its capacity to infect immunocompetent individuals. Here we report a large-scale analysis of the mating abilities of serotype B and C isolates from diverse sources and identify unusual strains that mate robustly and are suitable for further genetic analysis. Unlike most isolates, which are of both the a and α mating types but are predominantly sterile, the majority of the Vancouver outbreak strains are exclusively of the α mating type and the majority are fertile. In an effort to enhance mating of these isolates, we identified and disrupted the CRG1 gene encoding the GTPase-activating protein involved in attenuating pheromone response. crg1 mutations dramatically increased mating efficiency and enabled mating with otherwise sterile isolates. Our studies provide a genetic and molecular foundation for further studies of this primary pathogen and reveal that the Vancouver Island outbreak may be attributable to a recent recombination event.

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Secreted Acb1 Contributes to the Yeast-to-Hypha Transition in Cryptococcus neoformans

Applied and Environmental Microbiology ◽

10.1128/aem.03691-15 ◽

2015 ◽

Vol 82 (4) ◽

pp. 1069-1079 ◽

Cited By ~ 11

Author(s):

Xinping Xu ◽

Youbao Zhao ◽

Elyssa Kirkman ◽

Xiaorong Lin

Keyword(s):

Cryptococcus Neoformans ◽

Fungal Pathogens ◽

Matricellular Proteins ◽

Filamentous Form ◽

Content Type ◽

Human Fungal Pathogen ◽

Extracellular Secretion ◽

Macrophage Phagocytosis ◽

Capsule Production ◽

Acyl Coenzyme A

ABSTRACTAdaptation to stress by eukaryotic pathogens is often accompanied by a transition in cellular morphology. The human fungal pathogenCryptococcus neoformansis known to switch between the yeast and the filamentous form in response to amoebic predation or during mating. As in the classic dimorphic fungal pathogens, the morphotype is associated with the ability of cryptococci to infect various hosts. Many cryptococcal factors and environmental stimuli, including pheromones (small peptides) and nutrient limitation, are known to induce the yeast-to-hypha transition. We recently discovered that secreted matricellular proteins could also act as intercellular signals to promote the yeast-to-hypha transition. Here we show that the secreted acyl coenzyme A (acyl-CoA)-binding protein Acb1 plays an important role in enhancing this morphotype transition. Acb1 does not possess a signal peptide. Its extracellular secretion and, consequently, its function in filamentation are dependent on an unconventional GRASP (Golgi reassembly stacking protein)-dependent secretion pathway. Surprisingly, intracellular recruitment of Acb1 to the secretory vesicles is independent of Grasp. In addition to Acb1, Grasp possibly controls the secretion of other cargos, because thegraspΔ mutant, but not theacb1Δ mutant, is defective in capsule production and macrophage phagocytosis. Nonetheless, Acb1 is likely the major or the sole effector of Grasp in terms of filamentation. Furthermore, we found that the key residue of Acb1 for acyl binding, Y80, is critical for the proper subcellular localization and secretion of Acb1 and for cryptococcal morphogenesis.

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Induction of Capsule Growth in Cryptococcus neoformans by Mammalian Serum and CO2

Infection and Immunity ◽

10.1128/iai.71.11.6155-6164.2003 ◽

2003 ◽

Vol 71 (11) ◽

pp. 6155-6164 ◽

Cited By ~ 122

Author(s):

Oscar Zaragoza ◽

Bettina C. Fries ◽

Arturo Casadevall

Keyword(s):

Cyclic Amp ◽

Cryptococcus Neoformans ◽

Pathogenic Fungus ◽

Phenotypic Switching ◽

Considerable Variability ◽

Highly Efficient ◽

Camp Pathway ◽

Animal Infection ◽

Capsule Size

ABSTRACT The pathogenic fungus Cryptococcus neoformans has a polysaccharide capsule that is essential for virulence in vivo. Capsule size is known to increase during animal infection, and this phenomenon was recently associated with virulence. Although various conditions have been implicated in promoting capsule growth, including CO2 concentration, osmolarity, and phenotypic switching, it is difficult to reproduce the capsule enlargement effect in the laboratory. In this study, we report that serum can induce capsule growth, and we describe the conditions that induce this effect, not only by serum but also by CO2. Capsule enlargement was dependent on the medium used, and this determined whether the strain responded to serum or CO2 efficiently. Serum was most effective in inducing capsule growth under nutrient-limited conditions. There was considerable variability between strains in their response to either serum or CO2, with some strains requiring both stimuli. Sera from several animal sources were each highly efficient in inducing capsule growth. The cyclic AMP (cAMP) pathway and Ras1 were both necessary for serum-induced capsule growth. The lack of induction in the ras1 mutant was not complemented by exogenous cAMP, indicating that these pathways act in parallel. However, both cAMP and Ras1 were dispensable for inducing a partial capsule growth by CO2, suggesting that multiple pathways participate in this process. The ability of serum to induce capsule growth suggests a mechanism for the capsular enlargement observed during animal infection.

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The Cryptococcus neoformans Rho-GDP Dissociation Inhibitor Mediates Intracellular Survival and Virulence

Infection and Immunity ◽

10.1128/iai.00896-08 ◽

2008 ◽

Vol 76 (12) ◽

pp. 5729-5737 ◽

Cited By ~ 35

Author(s):

Michael S. Price ◽

Connie B. Nichols ◽

J. Andrew Alspaugh

Keyword(s):

Cryptococcus Neoformans ◽

Cell Morphology ◽

Culture Medium ◽

Intracellular Survival ◽

Murine Models ◽

Gene Encoding ◽

Cellular Processes ◽

Growth Defects ◽

Human Fungal Pathogen ◽

Gdp Dissociation Inhibitor

ABSTRACT Rho-GDP dissociation inhibitors (Rho-GDI) are repressors of Rho-type monomeric GTPases that control fundamental cellular processes, such as cytoskeletal arrangement, vesicle trafficking, and polarized growth. We identified and altered the expression of the gene encoding a Rho-GDI homolog in the human fungal pathogen Cryptococcus neoformans and investigated its impact on pathogenicity in animal models of cryptococcosis. Consistent with its predicted function to inhibit and sequester Rho-type GTPases, overexpression of RDI1 results in cytosolic localization of Cdc42. Likely as a result of this finding, RDI1-overexpressing strains exhibited altered morphology compared to that of the wild type, with apparent defects in maintaining proper cell polarity and cytokinesis. RDI1 deletion resulted in increased vacuole size in tissue culture medium and aberrant cell morphology at neutral pH. Maintenance of normal cell morphology is vital for C. neoformans pathogenicity. Accordingly, the rdi1Δ mutant strain also showed reduced intracellular survival in macrophages and severe attenuation of virulence in two murine models of cryptococcosis. This reduction in virulence of the rdi1Δ mutant occurs in the absence of major growth defects in rich medium and with classical virulence-associated phenotypes.

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A Rac Homolog Functions Downstream of Ras1 To Control Hyphal Differentiation and High-Temperature Growth in the Pathogenic Fungus Cryptococcus neoformans

Eukaryotic Cell ◽

10.1128/ec.4.6.1066-1078.2005 ◽

2005 ◽

Vol 4 (6) ◽

pp. 1066-1078 ◽

Cited By ~ 53

Author(s):

Marcelo A. Vallim ◽

Connie B. Nichols ◽

Larissa Fernandes ◽

Kari L. Cramer ◽

J. Andrew Alspaugh

Keyword(s):

High Temperature ◽

Cryptococcus Neoformans ◽

Pathogenic Fungus ◽

Ras Proteins ◽

Gene Encoding ◽

Temperature Growth ◽

Multicopy Suppressor ◽

Human Fungal Pathogen ◽

Hyphal Formation ◽

Mutant Phenotypes

ABSTRACT The Cryptococcus neoformans Ras1 protein serves as a central regulator for several signaling pathways. Ras1 controls the induction of the mating pheromone response cascade as well as a distinct signaling pathway that allows this pathogenic fungus to grow at human physiological temperature. To characterize elements of the Ras1-dependent high-temperature growth pathway, we performed a multicopy suppressor screen, identifying genes whose overexpression allows the ras1 mutant to grow at 37°C. Using this genetic technique, we identified a C. neoformans gene encoding a Rac homolog that suppresses multiple ras1 mutant phenotypes. Deletion of the RAC1 gene does not affect high-temperature growth. However, a rac1 mutant strain demonstrates a profound defect in haploid filamentation as well as attenuated mating. In a yeast two-hybrid assay, Rac1 physically interacts with the PAK kinase Ste20, which similarly regulates hyphal formation in this fungus. Similar to Rac1, overexpression of the STE20α gene also restores high-temperature growth to the ras1 mutant. These results support a model in which the small G protein Rac1 acts downstream of Ras proteins and coordinately with Ste20 to control high-temperature growth and cellular differentiation in this human fungal pathogen.

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Transcription Factor Nrg1 Mediates Capsule Formation, Stress Response, and Pathogenesis in Cryptococcus neoformans

Eukaryotic Cell ◽

10.1128/ec.00145-06 ◽

2006 ◽

Vol 5 (7) ◽

pp. 1147-1156 ◽

Cited By ~ 74

Author(s):

Kari L. Cramer ◽

Quincy D. Gerrald ◽

Connie B. Nichols ◽

Michael S. Price ◽

J. Andrew Alspaugh

Keyword(s):

Transcription Factor ◽

Cryptococcus Neoformans ◽

Transcriptional Control ◽

Glucose Dehydrogenase ◽

Phosphorylation Site ◽

Signal Transduction Pathway ◽

Site Mutation ◽

Gene Encoding ◽

Capsule Formation ◽

Capsule Production

ABSTRACT The Cryptococcus neoformans NRG1 gene was identified using gene microarrays to define putative transcription factor genes regulated by the cyclic AMP (cAMP) signal transduction pathway. Disruption of NRG1 results in delayed capsule formation and mating, two phenotypes that are directly controlled by cAMP signaling. Putative targets of the Nrg1 transcription factor were identified using a second genome microarray to define differences in the transcriptomes of the wild-type and nrg1 mutant strains. These experiments implicate Nrg1 in the transcriptional control of multiple genes involved in carbohydrate metabolism and substrate oxidation, as well as the UGD1 gene encoding a UDP-glucose dehydrogenase required for polysaccharide capsule production and cell wall integrity. In addition to being under transcriptional control of the cAMP pathway, Nrg1 contains a putative protein kinase A phosphorylation site; mutation of this motif results in reduced Nrg1 activity. Consistent with prior studies in hypocapsular mutants, the nrg1 mutant strain is attenuated in an animal model of disseminated cryptococcal disease.

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Comparative Transcriptome Analysis Reveals Novel Roles of the Ras and Cyclic AMP Signaling Pathways in Environmental Stress Response and Antifungal Drug Sensitivity in Cryptococcus neoformans

Eukaryotic Cell ◽

10.1128/ec.00309-09 ◽

2010 ◽

Vol 9 (3) ◽

pp. 360-378 ◽

Cited By ~ 52

Author(s):

Shinae Maeng ◽

Young-Joon Ko ◽

Gyu-Bum Kim ◽

Kwang-Woo Jung ◽

Anna Floyd ◽

...

Keyword(s):

Stress Response ◽

Cyclic Amp ◽

Environmental Stress ◽

Cryptococcus Neoformans ◽

Signaling Pathway ◽

Transcriptome Analysis ◽

Antifungal Drug ◽

Environmental Stress Response ◽

Content Type ◽

Camp Pathway

ABSTRACT The cyclic AMP (cAMP) pathway plays a central role in the growth, differentiation, and virulence of pathogenic fungi, including Cryptococcus neoformans. Three upstream signaling regulators of adenylyl cyclase (Cac1), Ras, Aca1, and Gpa1, have been demonstrated to control the cAMP pathway in C. neoformans, but their functional relationship remains elusive. We performed a genome-wide transcriptome analysis with a DNA microarray using the ras1Δ, gpa1Δ, cac1Δ, aca1Δ, and pka1Δ pka2Δ mutants. The aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka2Δ mutants displayed similar transcriptome patterns, whereas the ras1Δ mutant exhibited transcriptome patterns distinct from those of the wild type and the cAMP mutants. Interestingly, a number of environmental stress response genes are modulated differentially in the ras1Δ and cAMP mutants. In fact, the Ras signaling pathway was found to be involved in osmotic and genotoxic stress responses and the maintenance of cell wall integrity via the Cdc24-dependent signaling pathway. Notably, the Ras and cAMP mutants exhibited hypersensitivity to a polyene drug, amphotericin B, without showing effects on ergosterol biosynthesis, which suggested a novel method of antifungal combination therapy. Among the cAMP-dependent gene products that we characterized, two small heat shock proteins, Hsp12 and Hsp122, were found to be involved in the polyene antifungal drug susceptibility of C. neoformans.

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Cyclic AMP-Dependent Protein Kinase Controls Virulence of the Fungal Pathogen Cryptococcus neoformans

Molecular and Cellular Biology ◽

10.1128/mcb.21.9.3179-3191.2001 ◽

2001 ◽

Vol 21 (9) ◽

pp. 3179-3191 ◽

Cited By ~ 264

Author(s):

Cletus A. D'Souza ◽

J. Andrew Alspaugh ◽

Changli Yue ◽

Toshiaki Harashima ◽

Gary M. Cox ◽

...

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Cryptococcus Neoformans ◽

Fungal Pathogen ◽

Catalytic Subunit ◽

Regulatory Subunit ◽

Dependent Protein Kinase ◽

Gene Encoding ◽

Mutant Strains ◽

Dependent Protein

ABSTRACT Cryptococcus neoformans is an opportunistic fungal pathogen that infects the human central nervous system. This pathogen elaborates two specialized virulence factors: the antioxidant melanin and an antiphagocytic immunosuppressive polysaccharide capsule. A signaling cascade controlling mating and virulence was identified. ThePKA1 gene encoding the major cyclic AMP (cAMP)-dependent protein kinase catalytic subunit was identified and disrupted.pka1 mutant strains were sterile, failed to produce melanin or capsule, and were avirulent. The PKR1 gene encoding the protein kinase A (PKA) regulatory subunit was also identified and disrupted. pkr1 mutant strains overproduced capsule and were hypervirulent in animal models of cryptococcosis. pkr1 pka1 double mutant strains exhibited phenotypes similar to that of pka1 mutants, providing epistasis evidence that the Pka1 catalytic subunit functions downstream of the Pkr1 regulatory subunit. The PKA pathway was also shown to function downstream of the Gα protein Gpa1 and to regulate cAMP production by feedback inhibition. These findings define a Gα protein-cAMP-PKA signaling pathway regulating differentiation and virulence of a human fungal pathogen.

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Laccase Affects the Rate of Cryptococcus neoformans Nonlytic Exocytosis from Macrophages

mBio ◽

10.1128/mbio.02085-20 ◽

2020 ◽

Vol 11 (5) ◽

Cited By ~ 1

Author(s):

Stefânia de Oliveira Frazão ◽

Herdson Renney de Sousa ◽

Lenise Gonçalves da Silva ◽

Jéssica dos Santos Folha ◽

Kaio César de Melo Gorgonha ◽

...

Keyword(s):

Flow Cytometry ◽

Cryptococcus Neoformans ◽

Laccase Activity ◽

Severe Disease ◽

Cell Types ◽

Great Variability ◽

Melanin Production ◽

Content Type ◽

Fungal Melanin ◽

Laccase Enzyme

ABSTRACT Nonlytic exocytosis is a process in which previously ingested microbes are expelled from host phagocytes with the concomitant survival of both cell types. This process has been observed in the interaction of Cryptococcus spp. and other fungal cells with phagocytes as distant as mammalian, bird, and fish macrophages and ameboid predators. Despite a great amount of research dedicated to unraveling this process, there are still many questions about its regulation and its final benefits for host or fungal cells. During a study to characterize the virulence attributes of Brazilian clinical isolates of C. neoformans, we observed great variability in their rates of nonlytic exocytosis and noted a correlation between this process and fungal melanin production/laccase activity. Flow cytometry experiments using melanized cells, nonmelanized cells, and lac1Δ mutants revealed that laccase has a role in the process of nonlytic exocytosis that seems to be independent of melanin production. These results identify a role for laccase in virulence, independent of its role in pigment production, that represents a new variable in the regulation of nonlytic exocytosis. IMPORTANCE Cryptococcus neoformans is a yeast that causes severe disease, primarily in immunosuppressed people. It has many attributes that allow it to survive and cause disease, such as a polysaccharide capsule and the dark pigment melanin produced by the laccase enzyme. Upon infection, the yeast is ingested by cells called macrophages, whose function is to kill them. Instead, these fungal cells can exit from macrophages in a process called nonlytic exocytosis. We know that this process is controlled by both host and fungal factors, only some of which are known. As part of an ongoing study, we observed that C. neoformans isolates that produce melanin faster are more-frequent targets of nonlytic exocytosis. Further experiments showed that this is probably due to higher production of laccase, because fungi lacking this enzyme are nonlytically exocytosed less often. This shows that laccase is an important signal/regulator of nonlytic exocytosis of C. neoformans from macrophages.

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