Fungicidal activity of IFN-gamma-activated macrophages. Extracellular killing of Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast-form fungus which causes pulmonary and meningeal infections preferentially in the immunocompromised host. It is thought that cell-mediated immunity is important for acquired resistance against cryptococcosis with activated macrophages as the final effector cells. However, specific polysaccharides in the capsule of C. neoformans protect the fungus from adherence to phagocytes and from subsequent phagocytosis. We have studied extracellular killing of C. neoformans by IFN-gamma-activated macrophages and their products. Murine bone marrow-derived macrophages stimulated with rIFN-gamma for 24 h were able to effectively suppress the growth of C. neoformans and the effect of IFN-gamma was augmented by LPS. Killing of C. neoformans was also achieved by cell-free supernatants from bone marrow-derived macrophages stimulated with IFN-gamma plus LPS. Our results indicate that killing of C. neoformans by activated macrophages is independent from toxic oxygen radicals and mediated by secreted protein(s) of apparent molecular mass of 15 and 30 kDa. These findings indicate that activated macrophages play a major role in host defense, although the fungus resists phagocytosis and remains in the extracellular milieu.     

Study of the role of pigeons in the dissemination of Cryptococcus neoformans in nature.

Cryptococcus neoformans was recovered from droppings collected within the first 24 h from pigeons experimentally fed with a dose of 5 X 10(6) cells. The fungus proved to multiply well though differently in the sterilized pigeon and chicken excreta seeded with the organism. In both unsterile types of droppings no viable cells of C. neoformans were detected after 4 weeks incubation. Isolated bacterial flora from the intestinal contents of apparently healthy pigeons showed a complete inhibitory effect on the growth of C. neoforms in vitro. It has been concluded that pigeons do not favor multiplication of the fungus in their gut and consequently they do not seem to play an active biological role in dissemination of C. neoformans in nature.     

Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells requires protein kinase C-alpha activation

Pathogenic fungus Cryptococcus neoformans has a predilection for the central nervous system causing devastating meningoencephalitis. Traversal of C. neoformans across the blood–brain barrier (BBB) is a crucial step in the pathogenesis of C. neoformans . Our previous studies have shown that the CPS1 gene is required for C. neoformans adherence to the surface protein CD44 of human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In this report, we demonstrated that C. neoformans invasion of HBMEC was blocked in the presence of G109203X, a protein kinase C (PKC) inhibitor, and by overexpression of a dominant-negative form of PKCα in HBMEC. During C. neoformans infection, phosphorylation of PKCα was induced and the PKC enzymatic activity was detected in the HBMEC membrane fraction. Our results suggested that the PKCα isoform might play a crucial role during C. neoformans invasion. Immunofluorescence microscopic images showed that induced phospho-PKCα colocalized with β-actin on the membrane of HBMEC. In addition, cytochalasin D (an F-filament-disrupting agent) inhibited fungus invasion into HBMEC in a dose-dependent manner. Furthermore, blockage of PKCα function attenuated actin filament activity during C. neoformans invasion. These results suggest a significant role of PKCα and downstream actin filament activity during the fungal invasion into HBMEC.     

The origin and maintenance of virulence for the human pathogenic fungus Cryptococcus neoformans

The origin of virulence in environmental fungi that have no requirement for animal hosts in their life cycle is enigmatic. Cryptococcus neoformans is a human pathogenic fungus with virulence factors for mammalian pathogenesis that also contribute to environmental survival. C. neoformans virulence may originate from selection pressures imposed by environmental predators.     

Glucuronoxylomannan exhibits potent immunosuppressive properties

Cryptococcus neoformans is an opportunistic fungus responsible for life-threatening infections in immunocompromised and occasionally in immunocompetent hosts. The fungus is endowed with several virulence factors such as its capsular polysaccharide, which plays a key role in virulence. The major component of capsular material of C. neoformans is glucuronoxylomannan, a polysaccharide that exhibits potent immunosuppressive properties in vitro and in vivo. Here we describe a new aspect relative to glucuronoxylomannan-induced immunosuppression. In this review we analyze the suppressive effects of glucuronoxylomannan and the mechanisms leading to induction of apoptosis in T cells.     

The capsular dynamics of Cryptococcus neoformans

Cryptococcus neoformans is a soil-dwelling fungus that causes life-threatening illness in immunocompromised individuals and latently infects many healthy individuals. C. neoformans, unlike other human pathogenic fungi, is surrounded by a polysaccharide capsule that is essential for survival and enables C. neoformans to thwart the mammalian immune system. The capsule is a dynamic structure that undergoes changes in size and rearranges during budding. Here, the latest information and unresolved questions regarding capsule synthesis, structure, assembly, growth and rearrangements are discussed along with the concept that self-assembly is important in capsular dynamics.     

Survey of Cryptococcus neoformans in the respiratory tract of patients with bronchopulmonary disorders and in the air.

Cryptococcus neoformans was cultured from 9 (1%) of 835 clinical specimens examined from the respiratory tract of patients. These isolations came from 3 (0.4%) of the760 patients; 8 isolates were from sputum and one from urine. The fungus was not demonstrable in the air at a selected site during a 2-year study although other species of Cryptococcus, namely, C. albidus, C. ater, C. flavus, C. laurentii, C. magnus, C. terreus and C. uniguttulatus were isolated. The three C. neoformans positive patients were males, with pulmonary tuberculosis as the primary disease and history of repeated exposure to pigeon excreta in two. None of these patients manifested any overt signs and symptoms specificially attributable to cryptococcosis, nor did they receive any antifungal therapy. Repeated isolations of C. neoformans from sputum, a positive urine culture and demonstration of cryptococcal antibodies in a serum specimen, followed by negative cultures and serology, suggested that patient 1 had spontaneously recovered from an episode of benign, minimal pulmonary cryptococcosis. Patients 2 and 3 probably carried the fungus as a transient resident of the respiratory tract. The results suggest that C. neoformans is of uncommon occurrence in the respiratory tract of patients with bronchopulmonary disorders and that the isolation of the fungus from this site may not necessarily imply an etiologic relationship.     

Pathogenicity of Cryptococcus neoformans: virulence factors and immunological mechanisms

Cryptococcus neoformans is the causative agent of cryptococcosis and cryptococcal meningitis, which are serious pathological conditions affecting up to 10% of patients with AIDS. Mechanisms of pathogenicity of C. neoformans and the host defenses against this fungus are reviewed, incorporating recent data and perspectives.     

Peroxisomal and Mitochondrial β-Oxidation Pathways Influence the Virulence of the Pathogenic Fungus Cryptococcus neoformans

An understanding of the connections between metabolism and elaboration of virulence factors during host colonization by the human-pathogenic fungus Cryptococcus neoformans is important for developing antifungal therapies. Lipids are abundant in host tissues, and fungal pathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. In addition, lipids are important signaling molecules in both fungi and mammals. In this report, we demonstrate that defects in the peroxisomal and mitochondrial β-oxidation pathways influence the growth of C. neoformans on fatty acids as well as the virulence of the fungus in a mouse inhalation model of cryptococcosis. Disease attenuation may be due to the cumulative influence of altered carbon source acquisition or processing, interference with secretion, changes in cell wall integrity, and an observed defect in capsule production for the peroxisomal mutant. Altered capsule elaboration in the context of a β-oxidation defect was unexpected but is particularly important because this trait is a major virulence factor for C. neoformans. Additionally, analysis of mutants in the peroxisomal pathway revealed a growth-promoting activity for C. neoformans, and subsequent work identified oleic acid and biotin as candidates for such factors. Overall, this study reveals that β-oxidation influences virulence in C. neoformans by multiple mechanisms that likely include contributions to carbon source acquisition and virulence factor elaboration.     

'Ready made' virulence and 'dual use' virulence factors in pathogenic environmental fungi--the Cryptococcus neoformans paradigm

Environmental pathogenic fungi present a paradox in that they are virulent in animals without requiring animal hosts for replication or survival, a phenomenon we call 'ready-made' virulence. In the human pathogenic fungus Cryptococcus neoformans, the capacity for virulence in animals may originate from environmental selective pressures imposed by such organisms as amoeboid and nematode predators. Many C. neoformans virulence factors appear to have 'dual use' capabilities that confer survival advantages in both animal hosts and in the environment. The findings with C. neoformans may provide a paradigm for understanding the origin and maintenance of virulence in other pathogenic environmental fungi.     

Cryptococcus neoformans carried by Odontomachus bauri ants

Cryptococcus neoformans is the most common causative agent of cryptococcosis worldwide. Although this fungus has been isolated from a variety of organic substrates, several studies suggest that hollow trees constitute an important natural niche for C. neoformans. A previously surveyed hollow of a living pink shower tree (Cassia grandis) positive for C. neoformans in the city of Rio de Janeiro, Brazil, was chosen for further investigation. Odontomachus bauri ants (trap-jaw ants) found inside the hollow were collected for evaluation as possible carriers of Cryptococcus spp. Two out of 10 ants were found to carry phenoloxidase-positive colonies identified as C. neoformans molecular types VNI and VNII. The ants may have acted as a mechanical vector of C. neoformans and possibly contributed to the dispersal of the fungi from one substrate to another. To the best of our knowledge, this is the first report on the association of C. neoformans with ants of the genus Odontomachus.     

Characterization of Phenotypic Switching in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> Biofilms

Cryptococcus neoformans is an encapsulated yeast-like fungus that is a relatively frequent cause of meningoencephalitis in immunocompromised patients and also occasionally causes disease in apparently healthy individuals. This fungus collectively forms biofilms on polystyrene plates and medical devices, whereas individually can undergo phenotypic switching. Both events have profound consequences in the establishment of fungal infection and are associated with persistent infection due to increase resistance to antimicrobial therapy. In this study, we characterized switch phenotypes in C. neoformans biofilms. Smooth, mucoid, and wrinkled switch phenotypes of various switching C. neoformans strains were examined for their adhering and biofilm-forming ability on 96-well plates using cell counts and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay, respectively. Both assays showed that C. neoformans strains with the parent smooth phenotype adhered and formed stronger biofilms than their mucoid and wrinkled counterparts. Furthermore, the phenotypic switching frequencies of the individual colony types grown in biofilms or as planktonic cells were investigated. For the parent smooth variant of most strains, we found enhanced phenotypic switching in cryptococcal biofilms when compared to switching rates of planktonic cells. In contrast, the back-switching rate of mucoid to smooth variant was significantly higher in planktonic cells of seven strains of C. neoformans strains. These results suggested that phenotypic switching can occur in cryptococcal biofilms and extend our understanding of the relationship of both phenomena.     

对两种新生隐球菌选择性培养基的比较研究

比较鸟籽琼脂（GASA,Guizotia abyssinica seed agar）和咖啡酸玉米琼脂（CACA,Caffeic acidcommeal agar）对新生变种和格特变种的培养效果。再同时用两种培养基分离命粪和澳洲赤桉标本中的新生隐球菌。结果表明,CACA对新生隐球菌的培养和选择性分离效果与GASA相同,能够用于新生隐球菌的选择性分离。     

Deciphering the model pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is a basidiomycete fungal pathogen of humans that has diverged considerably from other model fungi such as Neurospora crassa, Aspergillus nidulans, Saccharomyces cerevisiae and the common human fungal pathogen Candida albicans. The recent completion of the genome sequences of two related C. neoformans strains and the ongoing genome sequencing of three other divergent Cryptococcus strains with different virulence phenotypes and environmental distributions should improve our understanding of this important pathogen. We discuss the biology of C. neoformans in light of this genomic data, with a special emphasis on the role that evolution and sexual reproduction have in the complex relationships of the fungus with the environment and the host.     

Isolation and characterization of the Cryptococcus neoformans MATa pheromone gene

Cryptococcus neoformans is a heterothallic basidiomycete with two mating types, MATa and MATalpha. The mating pathway of this fungus has a number of conserved genes, including a MATalpha-specific pheromone (MFalpha1). A modified differential display strategy was used to identify a gene encoding the MATa pheromone. The gene, designated MFa1, is 42 amino acids in length and contains a conserved farnesylation motif. MFa1 is present in three linked copies that span a 20-kb fragment of MATa-specific DNA and maps to the MAT-containing chromosome. Transformation studies showed that MFa1 induced filament formation only in MATalpha cells, demonstrating that MFa1 is functionally conserved. Sequence analysis of the predicted Mfa1 and Mfalpha1 proteins revealed that, in contrast to other fungi such as Saccharomyces cerevisiae, the C. neoformans pheromone genes are structurally and functionally conserved. However, unlike the MFalpha1 gene, which is found in MATalpha strains of both varieties of C. neoformans, MFa1 is specific for the neoformans variety of C. neoformans.     

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 degrees 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.     

A Ras1-Cdc24 signal transduction pathway mediates thermotolerance in the fungal pathogen Cryptococcus neoformans

Pathogenic microorganisms must precisely regulate morphogenesis to survive and proliferate within an infected host. This regulation is often controlled by conserved signal transduction pathways that direct morphological changes in varied species. One such pathway, whose components include Ras proteins and the PAK kinase Ste20, allows the human fungal pathogen Cryptococcus neoformans to grow at high temperature. Previously, we found that Ras1 signalling is required for differentiation, thermotolerance and pathogenesis in C. neoformans. We show here that the guanine nucleotide exchange factor Cdc24 is a Ras1 effector in C. neoformans to mediate the ability of this fungus to grow at high temperature and to cause disease. In addition, we provide evidence that the Ras1-Cdc24 signalling cascade functions specifically through one of the three Cdc42/Rac1 homologues in C. neoformans. In conclusion, our studies illustrate how components of conserved signalling cascades can be specialized for different downstream functions, such as pathogenesis.     

The Cryptococcus neoformans STE12α gene: a putative Saccharomyces cerevisiae STE12 homologue that is mating type specific

Cryptococcus neoformans possesses two mating types, MAT α and MATa . α-Cells are more virulent than a -cells and are also, unlike a -cells, capable of producing extensive hyphae in the haploid phase. The molecular analysis of hyphae production in C. neoformans has resulted in the identification of a gene which displays substantial similarity to other fungal STE12 genes, including the presence of a highly conserved homeodomain. Overexpression of the C. neoformans gene resulted in poor growth, altered morphology and the presence of hyphal projections, phenotypes reported in similar studies of the Saccharomyces cerevisiae STE12 gene. Overexpression was also found to induce MF α, a pheromone, and CNLAC1 , a confirmed C. neoformans virulence gene. The C. neoformans STE12 α gene, however, has one striking difference from other fungal STE12 genes; it is found only in α-cells. The existence of STE12 α in C. neoformans suggests that this fungus has elements of a conserved MAP kinase cascade, which may be organized in a novel manner.     

Decreased susceptibility of melanized Cryptococcus neoformans to UV light.

Melanized Cryptococcus neoformans cells were less susceptible than nonmelanized cells to the fungicidal effects of UV light. Phenoloxidase-catalyzed production of melanin-like pigments may serve to protect the fungus against ionizing radiation.