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PubMed2006

Cytotoxic CD4+ T cells use granulysin to kill Cryptococcus neoformans, and activation of this pathway is defective in HIV patients.

An important mechanism of host defense to Cryptococcus neoformans involves the direct microbicidal activity of lymphocytes. The importance of CD4+ T cells is illustrated by the incidence of this infection in the acquired immunodeficiency syndrome (AIDS) patients; however, the relative activity of microbicidal CD4+ T cells compared with CD8+ T cells and natural killer (NK) cells has not been established. Further, although NK cells and CD8+ T cells use perforin or granulysin, respectively, to kill C neoformans, the effector molecule used by CD4+ T cells is not known. Experiments demonstrated that IL-2-activated peripheral blood lymphocytes from healthy adults acquire anticryptococcal activity, and surprisingly, that CD4+ T cells had the most profound effect on this activity. Using SrCl(2)induced degranulation and siRNA knockdown, granulysin was shown to be the effector molecule. Although activation by anti-CD3 + IL-2 resulted in the additional expression of perforin, this did not improve the anticryptococcal activity. Cryptococcal killing by CD4+ T cells was defective in human immunodeficiency virus (HIV)-infected patients due to dysregulated granulysin and perforin production in response to IL-2 or anti-CD3 + IL-2. In conclusion, CD4+ T cells are the major subset of cells responsible for killing C neoformans in peripheral blood. These cells use granulysin as the effector molecule, and priming is dysregulated in HIV-infected patients, which results in defective microbicidal activity.

Antibodies↗
PubMed2003

The polysaccharide capsule of Cryptococcus neoformans interferes with human dendritic cell maturation and activation.

The ability of encapsulated and acapsular strains of Cryptococcus neoformans to activate dendritic cells (DC) derived from monocytes stimulated with granulocyte macrophage-colony stimulating factor and interleukin-4 was evaluated. Profound differences in DC response to encapsulated and acapsular C. neoformans strains were observed. In particular, (i) the acapsular strain was easily phagocytosed by immature DC, and the process induced several molecular markers, such as major histocompatibility complex (MHC) class I and class II, CD40, and CD83, which are characteristic of mature DC; (ii) the encapsulated strain did not up-regulate MHC class I and class II and CD83 molecules; (iii) the soluble capsular polysaccharide glucuronoxylomannan (GXM) is unable to regulate MHC class I and class II molecules; (iv) the addition of monoclonal antibody to GXM (anti-GXM) to the encapsulated strain facilitated antigen-presenting cell maturation by promoting ingestion of C. neoformans via Fc receptor for immunoglobulin G (FcgammaR)II (CD32) and FcgammaRIII (CD16); (v) pertubation of FcRgammaII or FcgammaRIII was insufficient to promote DC maturation; and (vi) optimal DC maturation permitted efficient T cell activation and differentiation, as documented by the enhancement of lymphoproliferation and interferon-gamma production. These results indicate that the C. neoformans capsule interferes with DC activation and maturation, indicating a new pathway by which the fungus may avoid an efficient T cell response.

Cell Division↗
PubMed2006

Nitric oxide-dependent killing of Cryptococcus neoformans by B-1-derived mononuclear phagocyte.

The role of B lymphocytes in protecting the host against pulmonary Cryptococcus neoformans infection is until now, uncertain. A recent study using B lymphocyte-deficient mice suggests that B lymphocytes play a protective role in cryptococcal infection. It has been well established that at least three B cell subsets, B-1a, B-1b, and B-2, are present in the mouse periphery. B-1 cells constitute a minor fraction of the B cell population in the spleen and are not detected in lymph nodes of mice. We demonstrated that B-1 cells migrate to a nonspecific, inflammatory focus and differentiate into macrophage-like cells. However, the role these cells might play on the kinetics and evolution of the inflammatory response and on fungal infection has not yet been established. Based on these data, we decided to investigate the interaction of B-1-derived mononuclear phagocytes (BDMP) with C. neoformans to elucidate the possible influence of this cell in the progression of the disease. In this study, we demonstrated that the BDMP cell internalized C. neoformans and that this process was mediated by complement receptor 3. Thus, our results showed that the BDMP cell was more fungicidal than a macrophage and up-regulated major histocompatibility complex type II and the CD86 costimulatory molecule with the production of proinflammatory cytokines. The phagocytosis of C. neoformans results in the nitric oxide (NO)-mediated death of the fungus, as demonstrated by experiments using NO synthase 2 knockout and aminoguanidine-treated, wild-type mice.

Animals↗
PubMed1986

Ventriculoatrial shunt infection due to Cryptococcus neoformans: an ultrastructural and quantitative microbiological study.

A 28-year-old man presented with hydrocephalus due to an infection by Cryptococcus neoformans involving his ventriculoatrial shunt. Ultrastructural studies of the ventriculoatrial shunt demonstrated yeastlike organisms consistent with C. neoformans within a biofilm on the appliance. Quantitative microbiological studies of segments of the shunt demonstrated C. neoformans in a concentration gradient from 9 X 10(6) colony-forming units (CFU)/0.5-cm section involving the ventricular portion of the catheter to 1 X 10(2) CFU/0.5-cm section at the vascular tip. The clinical, microbiological, and ultrastructural data suggest that this cryptococcal infection started as a ventriculitis or encephalitis with extension to the meningeal surface. Future application of these methods may further elucidate the pathogenesis of ventriculoatrial shunt infections.

Adult↗
PubMed1992

Cloning and molecular analysis of cDNA encoding a carboxymethylcellulase of the yeast Cryptococcus flavus.

A cDNA copy for carboxymethylcellulase (CMCase 1) of the yeast Cryptococcus flavus was cloned by screening an expression cDNA library with anti-CMCase 1 antibody. The sequence of the cDNA had an open reading frame of 1023 bp that encoded a preprotein of 341 amino acids with a molecular weight of 35,698. The putative precursor begins with a hydrophobic segment that possibly acts as a signal sequence for secretion, which is followed by a presumed prosequence and a sequence consistent with the N-terminal amino acid sequence of secreted CMCase 1. No potential N-glycosylation site was found in the sequence of putative pro-CMCase 1. Comparison of the deduced protein sequence shows that the C. flavus CMCase 1 is partially homologous to the Trichoderma reesei endoglucanase EGIII. Alignment of the cDNA copy and the chromosomal DNA showed seven putative introns of 45 to 134 bp. When introduced into E. coli, the cDNA directed the synthesis of CMCase 1 as seen by CMCase activity and Western blotting using anti-CMCase 1 antibody.

Amino Acid Sequence↗
PubMed1996

Acid xylanase from yeast Cryptococcus sp. S-2: purification, characterization, cloning, and sequencing.

A xylan-degrading enzyme produced by yeast Cryptococcus sp. S-2 was isolated and purified, and characterized as an endoxylanase (1,4-beta-D-xylan xylanohydrolase [EC 3.2.1.8]). We estimated the molecular weight and isoelectric point of purified xylanase (xyn-CS2) to be 22,000 and 7.4, respectively. This low-molecular-weight xylanase had an unusual pH optimum of 2.0, and showed 75% of maximal activity even at pH 1.0. An open reading frame of the cDNA specified 209 amino acids, including a putative signal peptide of 25 amino acids. The deduced amino acid sequence of xyn-CS2 shared significant similarities with the family-G xylanases of B. pumilus, C. acetobutylicum, T. reesei, and A. kawachii. Xyn-CS2 included two unique cysteine residues in a putative catalytic region, raising the possibility that these residues are at least partially responsible for its acidophilic nature.

Amino Acid Sequence↗
PubMed2006

Characterization of cold- and high-pressure-active polygalacturonases from a deep-sea yeast, Cryptococcus liquefaciens strain N6.

A deep-sea yeast, Cryptococcus liquefaciens strain N6, produces two polygalacturonases, p36 and p40 (N6-PGases). These N6-PGases were highly active at 0-10 degrees C in comparison to a PGase from Aspergillus japonicus. The hydrolytic activity of these N6-PGases remained almost unchanged up to a hydrostatic pressure of 100 MPa at 24 degrees C with a very small activation volume of -1.1 ml/mol. At 10 degrees C, however, the activation volume increased to 3.3 or 5.4 ml/mol (p36 and p40, respectively), suggesting that the enzyme-substrate complexes can expand at their transition states. We speculate that such a volume expansion upon forming the enzyme-substrate complexes contributes to decreasing the activation energy for hydrolysis. This can account for the high activity of N6-PGases at low-temperature.

Cold Temperature↗
PubMed1990

Isolation of saprophytic Cryptococcus neoformans.

Isolation of Cryptococcus neoformans was carried out on sunflower seed agar medium (SFA) and Sabouraud dextrose agar (SDA). Out of 346 environmental substrates (133 fruits, 107 avian extreta, 91 vegetables and 15 wooden scrapings) tested, 3 specimens were positive for C. neoformans. The positive isolations came from the fruits of 2 banana (Musa sapientum) and a potato tuber (Solnum tuberosum). The pathogen could not be demonstrated in 107 samples of avian droppings and 15 of wooden materials. All the 3 isolates of the yeast were obtained on SFA, while they were not cultured on the plates of SDA with chloramphenicol which were badly contaminated with rapidly growing molds, yeasts and bacteria. To the present author's knowledge, this appears to be the first reports of the isolation of this pathogenic basidiomycetous yeast from contaminated fruits of banana. We suggest more comprehensive ecological surveys to search for environmental niche of C. neoformans var. neoformans and C. neoformans var. gattii as the latter variety is also implicated in the etiology of cryptococcosis.

Animals↗
PubMed1999

Cryptococcus neoformans and Mycobacterium bovis causing granulomatous pneumonia in a goat.

An adult Blanca-Celtibérica doe originating from a goat herd with a high prevalence of tuberculosis was presented with respiratory signs. At necropsy, this goat had a diffuse and severe mycotic pneumonia associated with the presence of Cryptococcus neoformans concomitant with pulmonary focal caseous nodules from which Mycobacterium bovis was isolated. Microscopically, the mycotic lesion was a granulomatous pneumonia with many large foamy macrophages containing intracellular yeast bodies. The extensive mycotic changes, their granulomatous nature, and the lack of positive response to different immunologic tests for mycobacterial infection suggested an impaired immune status in this animal.

Animals↗
PubMed2006

Iron regulation of the major virulence factors in the AIDS-associated pathogen Cryptococcus neoformans.

Iron overload is known to exacerbate many infectious diseases, and conversely, iron withholding is an important defense strategy for mammalian hosts. Iron is a critical cue for Cryptococcus neoformans because the fungus senses iron to regulate elaboration of the polysaccharide capsule that is the major virulence factor during infection. Excess iron exacerbates experimental cryptococcosis and the prevalence of this disease in Sub-Saharan Africa has been associated with nutritional and genetic aspects of iron loading in the background of the HIV/AIDS epidemic. We demonstrate that the iron-responsive transcription factor Cir1 in Cr. neoformans controls the regulon of genes for iron acquisition such that cir1 mutants are "blind" to changes in external iron levels. Cir1 also controls the known major virulence factors of the pathogen including the capsule, the formation of the anti-oxidant melanin in the cell wall, and the ability to grow at host body temperature. Thus, the fungus is remarkably tuned to perceive iron as part of the disease process, as confirmed by the avirulence of the cir1 mutant; this characteristic of the pathogen may provide opportunities for antifungal treatment.

AIDS-Related Opportunistic Infections↗
PubMed2025

Landscape of essential growth and fluconazole-resistance genes in the human fungal pathogen Cryptococcus neoformans.

Fungi can cause devastating invasive infections, typically in immunocompromised patients. Treatment is complicated both by the evolutionary similarity between humans and fungi and by the frequent emergence of drug resistance. Studies in fungal pathogens have long been slowed by a lack of high-throughput tools and community resources that are common in model organisms. Here we demonstrate a high-throughput transposon mutagenesis and sequencing (TN-seq) system in Cryptococcus neoformans that enables genome-wide determination of gene essentiality. We employed a random forest machine learning approach to classify the C. neoformans genome as essential or nonessential, predicting 1,465 essential genes, including 302 that lack human orthologs. These genes are ideal targets for new antifungal drug development. TN-seq also enables genome-wide measurement of the fitness contribution of genes to phenotypes of interest. As proof of principle, we demonstrate the genome-wide contribution of genes to growth in fluconazole, a clinically used antifungal. We show a novel role for the well-studied RIM101 pathway in fluconazole susceptibility. We also show that insertions of transposons into the 5' upstream region can drive sensitization of essential genes, enabling screenlike assays of both essential and nonessential components of the genome. Using this approach, we demonstrate a role for mitochondrial function in fluconazole sensitivity, such that tuning down many essential mitochondrial genes via 5' insertions can drive resistance to fluconazole. Our assay system will be valuable in future studies of C. neoformans, particularly in examining the consequences of genotypic diversity.

Cryptococcus neoformans↗
PubMed2006

Recombination hotspots flank the Cryptococcus mating-type locus: implications for the evolution of a fungal sex chromosome.

Recombination increases dramatically during meiosis to promote genetic exchange and generate recombinant progeny. Interestingly, meiotic recombination is unevenly distributed throughout genomes, and, as a consequence, genetic and physical map distances do not have a simple linear relationship. Recombination hotspots and coldspots have been described in many organisms and often reflect global features of chromosome structure. In particular, recombination frequencies are often distorted within or outside sex-determining regions of the genome. Here, we report that recombination is elevated adjacent to the mating-type locus (MAT) in the pathogenic basidiomycete Cryptococcus neoformans. Among fungi, C. neoformans has an unusually large MAT locus, and recombination is suppressed between the two >100-kilobase mating-type specific alleles. When genetic markers were introduced at defined physical distances from MAT, we found the meiotic recombination frequency to be approximately 20% between MAT and a flanking marker at 5, 10, 50, or 100 kilobases from the right border. As a result, the physical/genetic map ratio in the regions adjacent to MAT is distorted approximately 10- to 50-fold compared to the genome-wide average. Moreover, recombination frequently occurred on both sides of MAT and negative interference between crossovers was observed. MAT heterozygosity was not required for enhanced recombination, implying that this process is not due to a physical distortion from the two non-paired alleles and could also occur during same-sex mating. Sequence analysis revealed a correlation between high G + C content and these hotspot regions. We hypothesize that the presence of recombinational activators may have driven several key events during the assembly and reshaping of the MAT locus and may have played similar roles in the origins of both metabolic and biosynthetic gene clusters. Our findings suggest that during meiosis the MAT locus may be exchanged onto different genetic backgrounds and therefore have broad evolutionary implications with respect to mating-type switching in both model and pathogenic yeasts.

Base Composition↗
PubMed2006

Direct inhibition of T-cell responses by the Cryptococcus capsular polysaccharide glucuronoxylomannan.

The major virulence factor of the pathogenic fungi Cryptococcus neoformans and C. gattii is the capsule. Glucuronoxylomannan (GXM), the major component of the capsule, is a high-molecular-weight polysaccharide that is shed during cryptococcosis and can persist in patients after successful antifungal therapy. Due to the importance of T cells in the anticryptococcal response, we studied the effect of GXM on the ability of dendritic cells (DCs) to initiate a T-cell response. GXM inhibited the activation of cryptococcal mannoprotein-specific hybridoma T cells and the proliferation of OVA-specific OT-II T cells when murine bone marrow-derived DCs were used as antigen-presenting cells. Inhibition of OT-II T-cell proliferation was observed when either OVA protein or OVA323-339 peptide was used as antigen, indicating GXM did not merely prevent antigen uptake or processing. We found that DCs internalize GXM progressively over time; however, the suppressive effect did not require DCs, as GXM directly inhibited T-cell proliferation induced by anti-CD3 antibody, concanavalin A, or phorbol-12-myristate-13-acetate/ionomycin. Analysis of T-cell viability revealed that the reduced proliferation in the presence of GXM was not the result of increased cell death. GXM isolated from each of the four major cryptococcal serotypes inhibited the proliferation of human peripheral blood mononuclear cells stimulated with tetanus toxoid. Thus, we have defined a new mechanism by which GXM can impart virulence: direct inhibition of T-cell proliferation. In patients with cryptococcosis, this could impair optimal cell-mediated immune responses, thereby contributing to the persistence of cryptococcal infections.

Animals↗
PubMed2005

Immunology of infection caused by Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that may lead to life-threatening meningoencephalitis and pulmonary infections in immunosuppressed hosts. The lack of an effective fungicidal regimen and the development of antifungal resistant strains suggest that continued investigation is necessary to devise immunotherapeutic strategies and/or drug targets to combat C. neoformans infections. Studies to date involve investigating the host-pathogen interaction of cryptococcal infections through the genetic manipulation of the yeast, as well as the characterization of the host immune response. Macrophage phagocytosis and killing assays have proven to be invaluable tools in evaluating the putative effects of the genetic manipulation of C. neoformans strains on the virulence composite of the yeast. In addition, the assay is used to assess the efficacy of various immunotherapeutic agents (i.e., antibodies and cytokines) to enhance this cell-based antifungal activity. The purpose of this chapter is to provide a brief overview on host immunity to C. neoformans infection and, in addition, describe a protocol for performing macrophage phagocytosis and killing assays with C. neoformans and its mutants.

Animals↗
PubMed2006

Immune response and immunotherapy to Cryptococcus infections.

Cryptococcus neoformans is a ubiquitous fungus that can cause lifethreatening infections during immunosuppressive states such as acquired immunodeficiency syndrome (AIDS) and after bone marrow transplantation (BMT). Infected individuals normally succumb to meningitis and meningoencephalitis caused by dissemination of C. neoformans to the brain. In this review, we analyze the current understanding of the interaction between host immune response and C. neoformans as well as the current state of immunotherapeutic strategies for treating cryptococcosis.

Animals↗
PubMedDate not supplied

Effect of salt stress on the production and properties of extracellular polysaccharides produced by Cryptococcus laurentii.

The composition, main structural features and molecular properties of exopolysaccharides (EP) produced by Cryptococcus laurentii var. laurentii CCY 17-3-16 under optimal (EPo) and NaCI-stress conditions (EPs) as well as their subfractions isolated by gel chromatography were studied using chemical, FT-IR and NMR spectroscopy methods. The results showed that under stress conditions the yeast produced EP with a lower content of protein and phosphorus. In comparison to EPo, the EPs exhibited a substantially larger proportion of high molecular mass populations. NMR analysis of EPs revealed a higher degree of branching with single xylose side chains of the heteromannan components. The increase of the molecular mass and degree of branching of the macromolecular chains of the heteromannan components might in part be related to the function of EPs to protect the yeast cells from water loss and maintain growth conditions under the salt stress.

Chromatography, Gel↗
PubMed2004

A genetic linkage map of Cryptococcus neoformans variety neoformans serotype D (Filobasidiella neoformans).

To construct a genetic linkage map of the heterothallic yeast, Cryptococcus neoformans (Filobasidiella neoformans), we crossed two mating-compatible strains and analyzed 94 progeny for the segregation of 301 polymorphic markers, consisting of 228 restriction site polymorphisms, 63 microsatellites, two indels, and eight mating-type (MAT)-associated markers. All but six markers showed no significant (P < 0.05) segregation distortion. At a minimum LOD score of 6.0 and a maximum recombination frequency of 0.30, 20 linkage groups were resolved, resulting in a map length of approximately 1500 cM. Average marker density is 5.4 cM (range 1-28.7 cM). Hybridization of selected markers to blots of electrophoretic karyotypes unambiguously assigned all linkage groups to chromosomes and led us to conclude that the C. neoformans genome is approximately 20.2 Mb, comprising 14 chromosomes ranging in size from 0.8 to 2.3 Mb, with a ratio of approximately 13.2 kb/cM averaged across the genome. However, only 2 of 12 ungrouped markers hybridized to chromosome 10. The hybridizations revealed at least one possible reciprocal translocation involving chromosomes 8, 9, and 12. This map has been critical to genome sequence assembly and will be essential for future studies of quantitative trait inheritance.

Chromosome Mapping↗
PubMed2004

Genotype-environment interactions of spontaneous mutations for vegetative fitness in the human pathogenic fungus Cryptococcus neoformans.

Spontaneous mutation is the ultimate source of all genetic variation. By interacting with environmental factors, genetic variation determines the phenotype and fitness of individuals in natural populations. However, except in a few model organisms, relatively little is known about the patterns of genotype-environment interactions of spontaneous mutations. Here I examine the rates of spontaneous mutation and the patterns of genotype-environment interaction of mutations affecting vegetative growth in the human fungal pathogen Cryptococcus neoformans. Eight mutation accumulation (MA) lines were established from a single clone on the nutrient-rich medium YEPD for each of two temperatures, 25 degrees and 37 degrees. Cells from generations 100, 200, 400, and 600 for each of the 16 MA lines were stored and assayed for vegetative growth rates under each of four conditions: (i) 25 degrees on SD (a synthetic dextrose minimal medium); (ii) 25 degrees on YEPD; (iii) 37 degrees on SD; and (iv) 37 degrees on YEPD. Both MA conditions and assay environments for vegetative growth showed significant influence on the estimates of genomic mutation rates, average effect per mutation, and mutational heritability. Significant genotype-environment interactions were detected among the newly accumulated spontaneous mutations. Overall, clones from MA lines maintained at 37 degrees showed less decline in vegetative fitness than those maintained at 25 degrees. The result suggests that a high-temperature environment might be very important for the maintenance of the ability to grow at a high temperature. Results from comparisons between clinical and environmental samples of C. neoformans were consistent with laboratory experimental population analyses. This study calls into question our long-standing view that warm-blooded mammals were only occasional and accidental hosts of this human fungal pathogen.

Cryptococcus neoformans↗