Cryptococcus neoformans Hyperfilamentous Strain Is Hypervirulent in a Murine Model of Cryptococcal Meningoencephalitis

Cryptococcus neoformans is a human fungal pathogen that causes lethal infections of the lung and central nervous system in immunocompromised individuals. C. neoformans has a defined bipolar sexual life cycle with a and α mating types. During the sexual cycle, which can occur between cells of opposite mating types (bisexual reproduction) or cells of one mating type (unisexual reproduction), a dimorphic transition from yeast to hyphal growth occurs. Hyphal development and meiosis generate abundant spores that, following inhalation, penetrate deep into the lung to enter the alveoli, germinate, and establish a pulmonary infection growing as budding yeast cells. Unisexual reproduction has been directly observed only in the Cryptococcus var. neoformans (serotype D) lineage under laboratory conditions. However, hyphal development has been previously associated with reduced virulence and the serotype D lineage exhibits limited pathogenicity in the murine model. In this study we show that the serotype D hyperfilamentous strain XL280α is hypervirulent in an animal model. It can grow inside the lung of the host, establish a pulmonary infection, and then disseminate to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is usually observed in the highly virulent sibling species C. gattii, responsible for the Pacific Northwest outbreak. These studies provide a technological advance that will facilitate analysis of virulence genes and attributes in C. neoformans var. neoformans, and reveal the virulence potential of serotype D as broader and more dynamic than previously appreciated.     

Cryptococcus neoformans: A sugar-coated killer with designer genes

Cryptococcus neoformans has become a common central nervous system pathogen as the immunocompromised populations enlarge world-wide. This encapsulated yeast has significant advantages for the study of fungal pathogenesis and these include: (1) a clinically important human pathogen; (2) a tractable genetic system; (3) advanced molecular biology foundation; (4) understanding of several virulence phenotypes; (5) well-studied pathophysiology; and (6) robust animal models. With the use of a sequenced genome and site-directed mutagenesis to produce specific null mutants, the virulence composite of C. neoformans has begun to be identified one gene at a time. Studies into capsule production, melanin synthesis, high temperature growth, metabolic pathways and a variety of signaling pathways have led to understandings of what makes this yeast a pathogen at the molecular level. Multiple principles of molecular pathogenesis have been demonstrated in virulence studies with C. neoformans. These include evolutionary differences between the varieties of C. neoformans in their genes for virulence, quantitative impact of genes on the virulence composite, species and site-specific importance of a virulence gene, gene expression correlation with its functional importance or phenotype and the impact of a pathogenesis gene on the host immune response. C. neoformans has now become a primary model to study molecular fungal pathogenesis with the goal of identifying drug targets or vaccine strategies.     

Melanization of Cryptococcus neoformans in Murine Infection

Cryptococcus neoformans is a fungus that is pathogenic in humans and that can produce melanin in vitro. Melanization is associated with virulence, but there is no evidence that melanin is made during infection. Melanins are difficult to study because they are amorphous and insoluble. Melanin-binding peptides from a phage display library were used to demonstrate that C. neoformans makes melanin-like compounds in tissue. Melanin-binding peptides were characterized by a high proportion of positively charged and aromatic residues. Two other methods, demonstration of an antibody response to melanin in mice infected with C. neoformans and analysis of yeast cell walls in infected tissue by light microscopy, were used to support these findings. The demonstration that C. neoformans melanizes in tissue has important implications for pathogenesis and drug discovery.     

Extracellular DNase activity of Cryptococcus neoformans and Cryptococcus gattii

Extracellular DNase activity was studied in 73 strains of Cryptococcus neoformans and 12 strains of Cryptococcus gattii. DNase activity was measured by DNase agar clearance with and without Methyl Green. All strains tested showed extracellular DNase activity and no significant difference was found betweenC. neoformans and C. gattii strains. DNase production was higher in strains from clinical origin (average radius of 6.2 mm) than among environmental strains (average radius of 2.9 mm). The extracellular enzyme may be detected by DNA substrate PAGE assays and its molecular weight was estimated at 31 kD. These results suggest that extracellular DNase could be considered as a virulence factor involved in C. neoformans–C. gattii species complex pathogenicity.     

The outcome of Cryptococcus neoformans intracellular pathogenesis in human monocytes

Background  

Cryptococcus neoformans is an encapsulated yeast that is a facultative intracellular pathogen. The interaction between macrophages and C. neoformans is critical for extrapulmonary dissemination of this pathogenic yeast. C. neoformans can either lyse macrophages or escape from within them through a process known as phagosomal extrusion. However, most studies of intracellular pathogenesis have been made with mouse cells and their relevance to human infection is uncertain. In this study we extended studies of C. neoformans-macrophage cellular interaction/s to human peripheral blood monocytes.     

Two cyclophilin A homologs with shared and distinct functions important for growth and virulence of Cryptococcus neoformans

Cyclophilin A is the target of the immunosuppressant cyclosporin A (CsA) and is encoded by a single unique gene conserved from yeast to humans. In the pathogenic fungus Cryptococcus neoformans, two homologous linked genes, CPA1 and CPA2, were found to encode two conserved cyclophilin A proteins. In contrast to Saccharomyces cerevisiae, in which cyclophilin A mutations confer CsA resistance but few other phenotypes, cyclophilin A mutations conferred dramatic phenotypes in C. neoformans. The Cpa1 and Cpa2 cyclophilin A proteins play a shared role in cell growth, mating, virulence and CsA toxicity. The Cpa1 and Cpa2 proteins also have divergent functions. cpa1 mutants are inviable at 39°C and attenuated for virulence, whereas cpa2 mutants are viable at 39°C and fully virulent. cpa1 cpa2 double mutants exhibited synthetic defects in growth and virulence. Cyclophilin A active site mutants restored growth of cpa1 cpa2 mutants at ambient but not at higher temperatures, suggesting that the prolyl isomerase activity of cyclophilin A has an in vivo function.     

DNA Mutations Mediate Microevolution between Host-Adapted Forms of the Pathogenic Fungus Cryptococcus neoformans

The disease cryptococcosis, caused by the fungus Cryptococcus neoformans, is acquired directly from environmental exposure rather than transmitted person-to-person. One explanation for the pathogenicity of this species is that interactions with environmental predators select for virulence. However, co-incubation of C. neoformans with amoeba can cause a “switch” from the normal yeast morphology to a pseudohyphal form, enabling fungi to survive exposure to amoeba, yet conversely reducing virulence in mammalian models of cryptococcosis. Like other human pathogenic fungi, C. neoformans is capable of microevolutionary changes that influence the biology of the organism and outcome of the host-pathogen interaction. A yeast-pseudohyphal phenotypic switch also happens under in vitro conditions. Here, we demonstrate that this morphological switch, rather than being under epigenetic control, is controlled by DNA mutation since all pseudohyphal strains bear mutations within genes encoding components of the RAM pathway. High rates of isolation of pseudohyphal strains can be explained by the physical size of RAM pathway genes and a hypermutator phenotype of the strain used in phenotypic switching studies. Reversion to wild type yeast morphology in vitro or within a mammalian host can occur through different mechanisms, with one being counter-acting mutations. Infection of mice with RAM mutants reveals several outcomes: clearance of the infection, asymptomatic maintenance of the strains, or reversion to wild type forms and progression of disease. These findings demonstrate a key role of mutation events in microevolution to modulate the ability of a fungal pathogen to cause disease.     

Phagocytosis of Cryptococcus neoformans by,and Nonlytic Exocytosis from,Acanthamoeba castellanii

Cryptococcus neoformans, an encapsulated, pathogenic yeast, is endowed with a variety of virulence factors, including a polysaccharide capsule. During mammalian infection, the outcome of the interaction between C. neoformans and macrophages is central to determining the fate of the host. Previous studies have shown similarities between the interaction of C. neoformans with macrophages and with amoebae, resulting in the proposal that fungal virulence for mammals originated from selection by amoeboid predators. In this study, we investigated the interaction of C. neoformans with the soil amoeba Acanthamoeba castellanii. Comparison of phagocytic efficiency of the wild type, nonencapsulated mutants, and complemented strains showed that the capsule was antiphagocytic for amoebae. Capsular enlargement was associated with a significant reduction in phagocytosis, suggesting that this phenomenon protects against ingestion by phagocytic predators. C. neoformans var. neoformans cells were observed to exit amoebae several hours after ingestion, in a process similar to the recently described nonlytic exocytosis from macrophages. Cryptococcal exocytosis from amoebae was dependent on the strain and on actin and required fungal viability. Additionally, the presence of a capsule was inversely correlated with the likelihood of extrusion in certain strains. In summary, nonlytic exocytosis from amoebae provide another parallel to observations in fungus-macrophage interactions. These results provide additional support for the notion that some mechanisms of virulence observed during mammalian infection originated, and were selected for, by environmental interactions.The encapsulated yeast Cryptococcus neoformans is an environmental organism that is capable of causing human disease. This fungus is a facultative intracellular pathogen with a unique pathogenic strategy, despite no obvious need for replication in an animal host as part of its life cycle (10). C. neoformans is known to interact with protozoa, some of which have been shown to be effective predators for this fungus (6, 26), and amoebae appear to be important for the control of C. neoformans in the environment (28). Previously, we reported that the interaction of C. neoformans with Acanthamoeba castellanii directly paralleled the interaction with human macrophages (33). Similarities between C. neoformans interactions with amoebae and macrophages included intracellular replication in a phagosome and the release of polysaccharide-containing vesicles into the cytoplasm (33). Furthermore, passage of avirulent C. neoformans and Histoplasma capsulatum through slime mold and amoebae was shown to increase virulence in mice (31, 32). On the basis of these observations, it was proposed that the capacity for mammalian virulence emerged from interactions with phagocytic predators, such as amoebae and slime mold, in the environment (7, 17, 30). Consequently, single-cell protists have emerged as important systems for the study of C. neoformans virulence, and subsequent studies have investigated the interaction of this fungus with slime mold and paramecia (9, 31). Additional evidence for this concept comes from studies of insect fungal pathogens, which suggest that the capacity for insect pathogenicity may follow preadaptation from interactions with amoebae in the environment (4). Understanding the mechanisms by which virulence emerges in environmental microbes is important considering that global warming has been hypothesized to bring about new fungal diseases in the coming century (13).Recent work in our laboratory and in that of Robin May simultaneously uncovered a novel strategy of avoiding macrophage killing whereby yeast cells were expulsed without lysis of the host cell (2, 19). The process is remarkable in that extrusion of the C. neoformans-filled phagosome is accompanied by the survival of both the host cells and the yeast cells. Phagosome extrusion or fungal exocytosis appears to be a C. neoformans-dictated event that is dependent on both the presence of the polysaccharide capsule and on the depolymerization of actin. A corollary of the hypothesis that C. neoformans virulence emerged from interactions with environmental predators is that phenomena observed with mammalian cells are likely to have a counterpart in free-living phagocytic cells. Consequently, the observation of an apparently unique event such as phagosomal extrusion from mammalian macrophages suggested a need to search for similar events in C. neoformans interactions with environmental phagocytic predators.In this study, we investigated parallels between the intracellular pathogenic strategy of C. neoformans in both macrophages and A. castellanii, focusing on characterizing the impact of the capsule on protozoan phagocytosis and on ascertaining whether fungal cells could also exit amoebae, including the role of the capsule in that possible mechanism. Using time-lapse microscopy, we observed the exocytosis of C. neoformans from A. castellanii. While there are significant differences in the nonlytic exocytosis process when comparing amoebae and macrophages, the observation of this phenomenon in amoebae provides additional support for the idea that the virulence of C. neoformans was selected for, and is maintained, by interactions in the environment with other soil organisms.(This research was conducted by Cara Chrisman in partial fulfillment of the requirements for a Ph.D. from the Sue Golding Graduate Division of Medical Science, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY [awarded in 2010].)     

Capsular Material of Cryptococcus neoformans: Virulence and Much More

The capsule is generally considered one of the more powerful virulence factors of microorganisms, driving research in the field of microbial pathogenesis and in the development of vaccines. Cryptococcus neoformans is unique among the most common human fungal pathogens in that it possesses a complex polysaccharide capsule. This review focuses on the Cryptococcus neoformans capsule from the viewpoint of fungal pathogenesis, and the effective immune response target of the capsule’s main component, glucuronoxylomannan.     

Calcium: a central player in Cryptococcus biology

Adaptation to the host environment is crucial for fungal pathogenesis. Calcium (Ca²⁺) signals are essential for fungal cells to respond rapidly to stress stimuli. In eukaryotic cells, Ca²⁺ is the main intracellular secondary messenger and regulates a myriad of processes, including the cellular fitness of the fungal pathogen Cryptococcus neoformans. In this minireview, we highlight the main cryptococcal processes regulated by Ca²⁺. Moreover, we underline all the characterized proteins responsible for intracellular calcium homeostasis in this yeast, such as Ca²⁺ transporters and binding proteins. These elements, in general, are essential for C. neoformans’ growth and adaptation to the host environment, as well as to virulence mechanisms. We also revisit the specific traits of the calcineurin signaling pathway in C. neoformans, which is the major pathway regulated by calcium and is crucial for yeast pathogenesis, adaptation, and growth at 37 °C. Notably, several Ca²⁺-related functions are highly conserved throughout fungal cells. Moreover, C. neoformans exhibits exclusive, significant features that are required for disease progression, thus attracting attention as feasible targets for antifungal drug development. Collectively, all the available data related to Ca²⁺ processes clarify the complex role that Ca²⁺ plays within cryptococcal cells, participating in host adaptation, transmigration, antifungal resistance, cell growth, and more.     

Evidence of sexual recombination among Cryptococcus neoformans serotype A isolates in sub-Saharan Africa

The most common cause of fungal meningitis in humans, Cryptococcus neoformans serotype A, is a basidiomycetous yeast with a bipolar mating system. However, the vast majority (>99.9%) of C. neoformans serotype A isolates possess only one of the two mating type alleles (MATα). Isolates with the other allele (MATa) were recently discovered and proven to mate in the laboratory. It has been a mystery whether and where C. neoformans strains undergo sexual reproduction. Here, we applied population genetic approaches to demonstrate that a population of C. neoformans serotype A clinical isolates from Botswana contains an unprecedented proportion of fertile MATa isolates and exhibits evidence of both clonal expansion and recombination within two partially genetically isolated subgroups. Our findings provide evidence for sexual recombination among some populations of C. neoformans serotype A from sub-Saharan Africa, which may have a direct impact on their evolution.     

A Paracoccidioides brasiliensis glycan shares serologic and functional properties with cryptococcal glucuronoxylomannan

The cell wall of the yeast form of the dimorphic fungus Paracoccidioides brasiliensis is enriched with α1,3-glucans. In Cryptococcus neoformans, α1,3-glucans interact with glucuronoxylomannan (GXM), a heteropolysaccharide that is essential for fungal virulence. In this study, we investigated the occurrence of P. brasiliensis glycans sharing properties with cryptococcal GXM. Protein database searches in P. brasiliensis revealed the presence of sequences homologous to those coding for enzymes involved in the synthesis of GXM and capsular architecture in C. neoformans. In addition, monoclonal antibodies (mAbs) raised to cryptococcal GXM bound to P. brasiliensis cells. Using protocols that were previously established for extraction and analysis of C. neoformans GXM, we recovered a P. brasiliensis glycan fraction composed of mannose and galactose, in addition to small amounts of glucose, xylose and rhamnose. In comparison with the C. neoformans GXM, the P. brasiliensis glycan fraction components had smaller molecular dimensions. The P. brasiliensis components, nevertheless, reacted with different GXM-binding mAbs. Extracellular vesicle fractions of P. brasiliensis also reacted with a GXM-binding mAb, suggesting that the polysaccharide-like molecule is exported to the extracellular space in secretory vesicles. An acapsular mutant of C. neoformans incorporated molecules from the P. brasiliensis extract onto the cell wall, resulting in the formation of surface networks that resembled the cryptococcal capsule. Coating the C. neoformans acapsular mutant with the P. brasiliensis glycan fraction resulted in protection against phagocytosis by murine macrophages. These results suggest that P. brasiliensis and C. neoformans share metabolic pathways required for the synthesis of similar polysaccharides and that P. brasiliensis yeast cell walls have molecules that mimic certain aspects of C. neoformans GXM. These findings are important because they provide additional evidence for the sharing of antigenically similar components across phylogenetically distant fungal species. Since GXM has been shown to be important for the pathogenesis of C. neoformans and to elicit protective antibodies, the finding of similar molecules in P. brasiliensis raises the possibility that these glycans play similar functions in paracoccidiomycosis.     

A Small Protein Associated with Fungal Energy Metabolism Affects the Virulence of Cryptococcus neoformans in Mammals

The pathogenic yeast Cryptococcus neoformans causes cryptococcosis, a life-threatening fungal disease. C. neoformans has multiple virulence mechanisms that are non-host specific, induce damage and interfere with immune clearance. Microarray analysis of C. neoformans strains serially passaged in mice associated a small gene (CNAG_02591) with virulence. This gene, hereafter identified as HVA1 (hypervirulence-associated protein 1), encodes a protein that has homologs of unknown function in plant and animal fungi, consistent with a conserved mechanism. Expression of HVA1 was negatively correlated with virulence and was reduced in vitro and in vivo in both mouse- and Galleria-passaged strains of C. neoformans. Phenotypic analysis in hva1Δ and hva1Δ+HVA1 strains revealed no significant differences in established virulence factors. Mice infected intravenously with the hva1Δ strain had higher fungal burden in the spleen and brain, but lower fungal burden in the lungs, and died faster than mice infected with H99W or the hva1Δ+HVA1 strain. Metabolomics analysis demonstrated a general increase in all amino acids measured in the disrupted strain and a block in the TCA cycle at isocitrate dehydrogenase, possibly due to alterations in the nicotinamide cofactor pool. Macrophage fungal burden experiments recapitulated the mouse hypervirulent phenotype of the hva1Δ strain only in the presence of exogenous NADPH. The crystal structure of the Hva1 protein was solved, and a comparison of structurally similar proteins correlated with the metabolomics data and potential interactions with NADPH. We report a new gene that modulates virulence through a mechanism associated with changes in fungal metabolism.     

Antimicrobial activity of invertebrate-pathogenic fungi in the genera Akanthomyces and Gibellula

Infectious disease caused by antibiotic resistant microorganisms is a global public health problem. There is a need to search for new bioactive compounds from new sources. In this study, we focused on invertebrate-pathogenic fungi infecting spiders. One hundred and sixty-five crude extracts from Akanthomyces (n = 45) and Gibellula (n = 10) were screened for their antimicrobial activity against nine human pathogens. Twenty-one extracts out of 165 (12.73%) from 16 (29.09%) isolates exhibited antimicrobial activity against at least one test strain. The most activity was against Staphylococcus aureus American Type Culture Collection (ATCC 25923) (8.48%) followed by Cryptococcus neoformans ATCC 90112 (3.03%), C. neoformans ATCC 90113 (2.42%), methicillin-resistant Staphylococcus aureus (MRSA) SK-1 (2.42%), Penicillium marneffei (2.42%), Microsporum gypseum (1.21%), Candida albicans ATCC 90028 (1.21%), Pseudomonas aeruginosa ATCC 27853 (0.61%) and Escherichia coli ATCC 25922 (0.61%), respectively. The ethyl acetate extract of mycelia from Gibellula pulchra EPF083 had the strongest broad spectrum antimicrobial activity with a minimum inhibitory concentration (MIC) value of 16 μg/ml against S. aureus ATCC 25923, MRSA SK-1, C. neoformans (ATCC 90112 and ATCC 90113) and P. marneffei and exhibited fungicidal activity against C. neoformans ATCC 90112 and P. marneffei with minimum fungicidal concentration (MFC) values of 16 and 32 μg/ml, respectively. These preliminary data show that invertebrate-pathogenic fungi could be a potential source of antimicrobial agents.     

Cryptococcus neoformans I. Nonencapsulated Mutants

Seven nonencapsulated mutants of Cryptococcus neoformans were isolated from an encapsulated strain of human origin. Initially, the mutants were avirulent for mice. After several months of subculturing, six of the seven isolates reverted to the encapsulated state and possessed varying degrees of virulence. The results of these experiments suggest that a strong correlation exists between the presence of a capsule and the virulence of C. neoformans.     

Role of clathrin‐mediated endocytosis in the use of heme and hemoglobin by the fungal pathogen Cryptococcus neoformans

Heme is a major source of iron for pathogens of humans, and its use is critical in determining the outcome of infection and disease. Cryptococcus neoformans is an encapsulated fungal pathogen that causes life‐threatening infections in immunocompromised individuals. C. neoformans effectively uses heme as an iron source, but the underlying mechanisms are poorly defined. Non‐iron metalloporphyrins (MPPs) are toxic analogues of heme and are thought to enter microbial cells via endogenous heme acquisition systems. We therefore carried out a mutant screen for susceptibility against manganese MPP (MnMPP) to identify new components for heme uptake in C. neoformans. We identified several genes involved in signalling, DNA repair, sugar metabolism, and trafficking that play important roles in susceptibility to MnMPP and in the use of heme as an iron source. We focused on investigating the role of clathrin‐mediated endocytosis (CME) and found that several components of CME including Chc1, Las17, Rvs161, and Rvs167 are required for growth on heme and hemoglobin and for endocytosis and intracellular trafficking of these molecules. We show that the hemoglobin uptake process in C. neoformans involves clathrin heavy chain, Chc1, which appears to colocalise with hemoglobin‐containing vesicles and to potentially assist in proper delivery of hemoglobin to the vacuole. Additionally, C. neoformans strains lacking Chc1, Las17, Rvs161, or Rvs167 were defective in the elaboration of several key virulence factors, and a las17 mutant was avirulent in a mouse model of cryptococcosis. Overall, this study unveils crucial functions of CME in the use of heme iron by C. neoformans and reveals a role for CME in fungal pathogenesis.     

Induction by Klebsiella aerogenes of a Melanin-Like Pigment in Cryptococcus neoformans

While studying the interaction of Cryptococcus neoformans with Dictyostelium discoideum, we noticed that yeast colonies in agar with a feeder lawn of Klebsiella aerogenes were brown. This finding was intriguing because C. neoformans colonies are not pigmented unless they are provided with precursors for melanization. Strains of all C. neoformans serotypes produced brown pigment in response to K. aerogenes at 22, 30, and 37°C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when C. neoformans was exposed to live Escherichia coli or heat-killed K. aerogenes. Analysis of K. aerogenes supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by C. neoformans. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for C. neoformans melanization in the environment.