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PubMed2006

Evidence of mRNA-mediated intron loss in the human-pathogenic fungus Cryptococcus neoformans.

Introns are a defining feature of eukaryotic genomes, though the mechanism of intron gain or loss is not well understood. Reverse transcription of mRNA followed by homologous recombination with the genome has been posited as a mechanism of intron loss, though little direct evidence of recent loss events has been described to support this model. We find supporting evidence for an mRNA-mediated mechanism of loss through comparative genome analyses that revealed a recent loss of 10 adjacent introns in a 22-exon gene in the human-pathogenic fungus Cryptococcus neoformans. We surveyed the gene structures of the entire genomes of Cryptococcus gattii, which diverged from the C. neoformans lineage 37 million years ago (Mya), and C. neoformans var. grubii and var. neoformans, which diverged 18 Mya. Our comparison revealed greater than 99.9% intron conservation, with evidence from 20 genes showing evidence of intron loss, but no convincing evidence of intron gain. Our findings confirm that Cryptococcus introns have been quite stable over recent evolutionary time, with occasional mRNA-mediated intron loss events.

Biological Evolution↗
PubMed2004

Cryptococcus neoformans var. gattii can exploit Acanthamoeba castellanii for growth.

It has recently been proposed that the origin and maintenance of virulence in certain environmental fungi is influenced by their interactions with non-vertebrate hosts such as amoebae and nematodes. In prior studies we have shown that the interactions of the soil amoebae Acanthamoeba castellanii with Cryptococcus neoformans varieties neoformans and grubii resemble those with macrophages. Here we extend those studies to C. neoformans variety gattii and describe quantitative differences in the type and outcome of the interactions observed relative to the other varieties. C. neoformans var. gattii proliferated in the presence of A. castellanii but the interaction was primarily extracellular with a paucity of phagocytic events. Experiments with acapsular cells coated with polysaccharide suggest that differences in the capsule structure may be responsible for the different interactions between cells of varieties neoformans, grubii, and gattii with amoebae. The ability of C. neoformans var. gattii to exploit amoebae indicates that despite major biological differences between C. neoformans varieties, all retain the ability to be pathogenic for A. castellanii.

Acanthamoeba↗
PubMed2005

Cryptococcus neoformans mitochondrial superoxide dismutase: an essential link between antioxidant function and high-temperature growth.

Manganese superoxide dismutase is an essential component of the mitochondrial antioxidant defense system of most eukaryotes. In the present study, we used a reverse-genetics approach to assess the contribution of the Cryptococcus neoformans manganese superoxide dismutase (Sod2) for antioxidant defense. Strains with mutations in the SOD2 gene exhibited increased susceptibility to oxidative stress as well as poor growth at elevated temperatures compared to isogenic wild-type strains. The sod2Delta mutants were also avirulent in a murine model of inhaled cryptococcosis. Reconstitution of a sod2Delta mutant restored Sod2 activity, eliminated the oxidative stress and temperature-sensitive (ts) phenotypes, and complemented the virulence phenotype. Characterization of the ts phenotype revealed a dependency between Sod2 antioxidant activity and the ability of C. neoformans cells to adapt to growth at elevated temperatures. The ts phenotype could be suppressed by the addition of either ascorbic acid (10 mM) or Mn salen (200 muM) at 30 degrees C, but not at 37 degrees C. Furthermore, sod2Delta mutant cells that were incubated for 24 h at 37 degrees C under anaerobic, but not aerobic, conditions were viable when shifted to the permissive conditions of 25 degrees C in the presence of air. These data suggest that the C. neoformans Sod2 is a major component of the antioxidant defense system in this human fungal pathogen and that adaptation to growth at elevated temperatures is also dependent on Sod2 activity.

Animals↗
PubMed1998

Antibody-mediated modulation of Cryptococcus neoformans infection is dependent on distinct Fc receptor functions and IgG subclasses.

Coupling of an antibody response to effector cells through the Fc region of antibodies is a fundamental objective of effective vaccination. We have explored the role of the Fc receptor system in a murine model of Cryptococcus neoformans protection by infecting mice deleted for the common gamma chain of FcRs. Passive administration of an IgG1 mAb protects FcRgamma+/- mice infected with C. neoformans, but fails to protect FcRgamma-/- mice, indicating that the gamma chain acting through FcgammaRI and/or III is essential for IgG1-mediated protection. In contrast, passive administration of an IgG3 mAb with identical specificity resulted in enhanced pathogenicity in gamma chain-deficient and wild-type mice. In vitro studies with isolated macrophages demonstrate that IgG1-, IgG2a-, and IgG2b-opsonized C. neoformans are not phagocytosed or arrested in their growth in the absence of the FcRgamma chain. In contrast, opsonization of C. neoformans by IgG3 does not require the presence of the gamma chain or of FcRII, and the internalization of IgG3-treated organisms does not arrest fungal growth.

Animals↗
PubMed2005

Immunomodulator tuftsin increases the susceptibility of Cryptococcus neoformans to liposomal amphotericin B in immunocompetent BALB/c mice.

The co-administration of immunomodulators and antibiotics has been proved very successful for treatment of opportunistic infectious diseases. In the present study, we evaluated the combination of liposomal amphotericin B (lip-Amp B) and immunomodulator tuftsin to cure Cryptococcus neoformans infection in BALB/c mice. Mice infected with C. neoformans were treated with Amp B deoxycholate and tuftsin free or tuftsin-loaded Amp B liposomes. The results of the present study demonstrated higher efficacy of tuftsin-loaded Amp B liposomes against experimental murine cryptococcosis, in terms of enhanced survival rate and reduced fungal burden in organs (lungs and brain) of the treated mice. Interestingly, pre-treatment of mice with liposomal tuftsin before challenging them with the C. neoformans infection resulted in 100% survival of the treated animals followed by treatment with lip-Amp B. Immunomodulator-based therapy seems likely to be more beneficial for treatment of fungal infectious diseases.

Amphotericin B↗
PubMed2006

Heat killed cells of Cryptococcus neoformans var. grubii induces protective immunity in rats: immunological and histopathological parameters.

Different clinical parameters which included cell-mediated immune (CMI) response, were evaluated in a model of disseminated cryptococcosis in rats. The experimental animals were pretreated four days prior to their exposure to Cryptococcus neoformans var. grubii with either heat killed cells of this yeastlike pathogen (HKC) or capsular polysaccharide (CPS) emulsified in complete Freund adjuvant (CFA). Rats treated with HKC-CFA and intraperitoneally infected with C. neoformans var. grubii had significantly better clearance of yeasts from tissues, a lower concentration of the cryptococcal capsular polysaccharide, glucuronoxylomannan (GXM), in serum and tissues, and better histopathological parameters compared to unpretreated infected rats. In contrast, rats treated with CPS-CFA presented an exacerbation of infection with a significantly higher fungal burden in tissues, a higher concentration of GXM in serum, and worse histopathological parameters compared to similar unpretreated infected rats. In addition, HKC-CFA treatment produced a T helper 1 (Th1) profile with improvements in the spleen cell proliferative response, in the level of INFgamma production by CD4 T cells, and in the nitric oxide (NO) production by peritoneal cells. On the other hand, rats treated with CPS-CFA showed an increased level of the immunoregulatory cytokine IL10 production by CD4 T cells, but no modification in the NO production by peritoneal cells.

Animals↗
PubMed2004

The RAM1 gene encoding a protein-farnesyltransferase beta-subunit homologue is essential in Cryptococcus neoformans.

Many small G proteins require post-translational modification to allow functional association to the cell membrane. This process often involves the enzymic addition of hydrophobic prenyl groups to a conserved cysteine residue near the C-terminus of the protein. The enzymes that catalyse these reactions include protein farnesyltransferase and protein geranylgeranyltransferases. The human fungal pathogen Cryptococcus neoformans requires functional Ras and Rho proteins in order to undergo normal growth and differentiation. Since farnesylation and geranylgeranylation are likely required for the proper function of these small G proteins, we hypothesized that inhibition of these prenylation events would alter the growth and cellular morphogenesis of this fungus. We cloned the RAM1 gene encoding the single protein-farnesyltransferase beta-chain homologue in C. neoformans. Using a gene-disruption strategy in a diploid C. neoformans strain, we demonstrated that this gene encodes an essential function, in contrast to the case in Saccharomyces cerevisiae, in which the homologous RAM1 gene is not essential for growth. Pharmacological inhibition of farnesyltransferase activity resulted in dose-dependent cytostasis of C. neoformans, as well as prevention of hyphal differentiation. Simultaneous inhibition of farnesylation and calcineurin signalling results in a synthetic effect on growth. Protein farnesylation is required for the growth and cellular differentiation of C. neoformans and may provide novel targets for antifungal therapy.

Alkyl and Aryl Transferases↗
PubMed2004

Cloning and characterization of the lanosterol 14alpha-demethylase (ERG11) gene in Cryptococcus neoformans.

The ergosterol pathway in fungal pathogens is an attractive antimicrobial target because it is unique from the major sterol (cholesterol) producing pathway in humans. Lanosterol 14alpha-demethylase is the target for a major class of antifungals, the azoles. In this study we have isolated the gene for this enzyme from Cryptococcus neoformans. The gene, ERG11, was recovered using degenerate PCR with primers designed with a novel algorithm called CODEHOP. Sequence analysis of Erg11p identified a highly conserved region typical of the cytochrome P450 class of mono-oxygenases. The gene was present in single copy in the genome and mapped to one end of the largest chromosome. Comparison of the protein sequence to a number of major human fungal pathogen Erg11p homologs revealed that the C. neoformans protein was highly conserved, and most closely related to the Erg11p homologs from other basidiomycetes. Functional studies demonstrated that the gene could complement a Saccharomyces cerevisiae erg11 mutant, which confirmed the identity of the C. neoformans gene.

Algorithms↗
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↗
PubMed1997

The immunosuppressant FK506 and its nonimmunosuppressive analog L-685,818 are toxic to Cryptococcus neoformans by inhibition of a common target protein.

The immunosuppressant FK506 (tacrolimus) is an antifungal natural product macrolide that suppresses the immune system by blocking T-cell activation. In complex with the intracellular protein FKBP12, FK506 inhibits calcineurin, a Ca(2+)-calmodulin-dependent serine-threonine protein phosphatase. We recently reported that growth of the opportunistic fungal pathogen Cryptococcus neoformans is resistant to FK506 at 24 degrees C but sensitive at 37 degrees C and that calcineurin, the target of FKBP12-FK506, is required for growth at 37 degrees C in vitro and pathogenicity in vivo. These findings identify calcineurin as a potential antifungal drug target. In previous studies the calcineurin inhibitor cyclosporin A (CsA) was effective against murine pulmonary infections but exacerbated cryptococcal meningitis in rabbits and mice, likely because CsA does not cross the blood-brain barrier. Although we find that FK506 penetrates the CNS, FK506 also exacerbates cryptococcal meningitis in rabbits. Thus, FK506 immunosuppression outweighs antifungal action in vivo. Like FK506, the nonimmunosuppressive FK506 analog L-685,818 is toxic to C. neoformans in vitro at 37 degrees C but not at 24 degrees C, and FK506-resistant mutants are resistant to L-685,818, indicating a similar mechanism of action. Fluconazole-resistant C. neoformans clinical isolates were also found to be susceptible to both FK506 and L-685,818. Our findings identify calcineurin as a novel antifungal drug target and suggest the nonimmunosuppressive FK506 analog L-685,818 or other congeners warrant further consideration as antifungal drugs for C. neoformans.

Animals↗
PubMed2004

Identification of Cryptococcus neoformans temperature-regulated genes with a genomic-DNA microarray.

The ability to survive and proliferate at 37 degrees C is an essential virulence attribute of pathogenic microorganisms. A partial-genome microarray was used to profile gene expression in the human-pathogenic fungus Cryptococcus neoformans during growth at 37 degrees C. Genes with orthologs involved in stress responses were induced during growth at 37 degrees C, suggesting that a conserved transcriptional program is used by C. neoformans to alter gene expression during stressful conditions. A gene encoding the transcription factor homolog Mga2 was induced at 37 degrees C and found to be important for high-temperature growth. Genes encoding fatty acid biosynthetic enzymes were identified as potential targets of Mga2, suggesting that membrane remodeling is an important component of adaptation to high growth temperatures. mga2Delta mutants were extremely sensitive to the ergosterol synthesis inhibitor fluconazole, indicating a coordination of the synthesis of membrane component precursors. Unexpectedly, genes involved in amino acid and pyrimidine biosynthesis were repressed at 37 degrees C, but components of these pathways were found to be required for high-temperature growth. Our findings demonstrate the utility of even partial-genome microarrays for delineating regulatory cascades that contribute to microbial pathogenesis.

Cryptococcus neoformans↗
PubMed2005

Interaction between genetic background and the mating-type locus in Cryptococcus neoformans virulence potential.

The study of quantitative traits provides a window on the interactions between multiple unlinked genetic loci. The interaction between hosts and pathogenic microbes, such as fungi, involves aspects of quantitative genetics for both partners in this dynamic equilibrium. One important pathogenic fungus is Cryptococcus neoformans, a basidiomycete yeast that can infect the human brain and whose mating system has two mating type alleles, a and alpha. The alpha mating-type allele has previously been linked to increased virulence potential. Here congenic C. neoformans strains were generated in the two well-characterized genetic backgrounds B3501alpha and NIH433a to examine the potential influence of genes outside of the mating-type locus on the virulence potential of mating type. The congenic nature of these new strain pairs was established by karyotyping, amplified fragment length polymorphism genotyping, and whole-genome molecular allele mapping (congenicity mapping). Virulence studies revealed that virulence was equivalent between the B3501 a and alpha congenic strains but the alpha strain was more virulent than its a counterpart in the NIH433 genetic background. These results demonstrate that genomic regions outside the mating type locus contribute to differences in virulence between a and alpha cells. The congenic strains described here provide a foundation upon which to elucidate at genetic and molecular levels how mating-type and other unlinked loci interact to enable microbial pathogenesis.

Animals↗
PubMed2000

Biological correlates of capsular (quellung) reactions of Cryptococcus neoformans.

The capsular swelling or quellung reaction was reported almost 100 years ago and described the effect of Abs on the appearance of microbial capsules. Despite widespread use to assess Ab binding to capsules, relatively little is known as to the mechanism of this effect or its biological consequences. The fungus Cryptococcus neoformans is an attractive system to study capsule reactions because it has a large polysaccharide capsule that is readily visible by light microscopy. When viewed by differential interference contrast microscopy, binding of mAb to C. neoformans cells produced two distinct capsular reactions that depended on the Ab epitope specificity and the yeast serotype. In the first pattern, termed "rim," the capsule appears transparent with a highly refractive outer edge. In the second pattern, termed "puffy," the capsule appears opaque and lacks a highly refractive outer rim. mAbs that bind with a rim pattern suppress the overall rate of C3 deposition on the yeast via the classical and alternative complement pathways. In contrast, mAbs that bind with a puffy pattern do not affect C3 deposition. Protective and nonprotective IgM mAbs produce rim and puffy patterns, respectively. These results indicate that: 1) capsule reactions are a consequence of Ab-induced changes in capsular refractive index; 2) the type of capsule reaction depends on the Ab specificity; and 3) Ab-induced changes in refractive index correlate with biological activities important for host defense against C. neoformans. Our results provide the first evidence associating distinct capsule reaction patterns with Ab biological activity.

Antibodies, Monoclonal↗
PubMed1999

In vitro susceptibilities of Candida and Cryptococcus neoformans isolates from blood cultures of neutropenic patients.

Fluconazole-resistant Candida albicans and intrinsically fluconazole-resistant Candida species have been reported as bloodstream isolates. However, an association between the isolation of fluconazole-resistant Candida from the bloodstream and patient risk factors for fungemia has not been established. The purpose of this study was to determine the prevalence of fluconazole resistance in bloodstream isolates of Candida species and Cryptococcus neoformans collected from patients with neutropenia, one of the most important risk factors for fungemia. MICs of voriconazole, fluconazole, itraconazole, ketoconazole, amphotericin B, and flucytosine were determined by the National Committee for Clinical Laboratory Standards M27-A method (1997). Voriconazole, on a per-weight basis, was the most active azole tested. Fluconazole resistance (MIC >/= 64 microg/ml) was not identified in any of the C. albicans (n = 513), Candida parapsilosis (n = 78), Candida tropicalis (n = 62), or C. neoformans (n = 38) isolates tested.

Candida↗
PubMed1996

Effects of interleukin-10 on human peripheral blood mononuclear cell responses to Cryptococcus neoformans, Candida albicans, and lipopolysaccharide.

Deactivation of mononuclear phagocytes is critical to limit the inflammatory response but can be detrimental in the face of progressive infection. We compared the effects of the deactivating cytokine interleukin 10 (IL-10) on human peripheral blood mononuclear cell (PBMC) responses to lipopolysaccharide (LPS), Cryptococcus neoformans, and Candida albicans. IL-10 effected dose-dependent inhibition of tumor necrosis factor alpha (TNF-alpha) release in PBMC stimulated by LPS and C. neoformans, with significant inhibition seen with 0.1 U/ml and greater than 90% inhibition noted with 10 U/ml. In contrast, even at doses as high as 100 U/ml, IL-10 inhibited TNF-alpha release in response to C. albicans by only 50%. IL-10 profoundly inhibited release of IL-1beta from PBMC stimulated by all three stimuli. TNF-alpha mRNA and release was inhibited even if IL-10 was added up to 8 h after cryptococcal stimulation. In contrast, inhibition of IL-1 beta mRNA was of lesser magnitude and occurred only when IL-10 was added within 2 h of cryptococcal stimulation. IL-10 inhibited translocation of NF-kappaB in response to LPS but not the fungal stimuli. All three stimuli induced IL-10 production in PBMC, although over 10-fold less IL-10 was released in response to C. neoformans compared with LPS and C. albicans. Thus, while IL-10 has deactivating effects on PBMC responses to all three stimuli, disparate stimulus- and response-specific patterns of deactivation are seen. Inhibition by IL-10 of proinflammatory cytokine release appears to occur at the level of gene transcription for TNF-alpha and both transcriptionally and posttranscriptionally for IL-1beta.

Candida albicans↗
PubMed1999

The yeast Cryptococcus neoformans uses 'mammalian' enhancer sites in the regulation of the virulence gene, CNLAC1.

Transcriptional regulation in mammalian and plant cells is distinguished from fungi by the presence of blocks of multiple interacting DNA binding sites distributed over a relatively large upstream region of genes and the ability to use glutamine-rich enhancers such as Sp1. We offer evidence that the haploid yeast Cryptococcus neoformans contains a virulence gene, CNLAC1, having regulatory properties more similar to mammalian systems than to that of yeast. We used a novel promoter plasmid, pVEW, and electromobility shift assay techniques adapted for the fungus for the first systematic structural and functional study of a 5'-enhancer region of a basidiomycete fungus using the upstream region of CNLAC1. Two groups of interactive enhancer regions, located over a range of 1.5kb from the mRNA start site are involved in CNLAC1 regulation (region 2: -1721 to -1615 and region 7) in addition to a TATA promoter at position -539. Region 2 contains a consensus Sp1 site and region 7 contains a consensus E2F site, each of which shows significant binding to nuclear proteins under derepressed conditions; cooperative binding was also suggested between DNA-binding protein of these sites and those binding nearby CCAAT sequences in each region. Two regions of repression were also evident under derepressed conditions (region 5: -1351 to -1207 and region 8: -991 to -971). Identification of functional Sp1 binding sites and the presence of multiple interactive enhancer sites over a fairly large upstream range suggests that cryptococcal transcriptional regulation contains features often associated with higher eukaryotic regulation. C. neoformans thus may provide a unique system for the study of certain aspects of higher eukaryotic transcription, using yeast genetic approaches. In addition, properties of basidiomycete yeast such as Cryptococcus exemplified in the present study suggest an evolutionary progression in gene regulation within fungi toward properties exhibited in the kingdoms Animalia and Plantae.

Animals↗
PubMed1990

Opsonization of Cryptococcus neoformans by a family of isotype-switch variant antibodies specific for the capsular polysaccharide.

A family of immunoglobulin isotype-switch variants was isolated by sib selection from a murine hybridoma which produced an immunoglobulin G subclass 1 (IgG1) antibody specific for the capsular polysaccharide of Cryptococcus neoformans. Antibodies of the IgG1, IgG2a, and IgG2b isotypes had similar serotype specificity patterns in double immunodiffusion assays which used polysaccharides of the four cryptococcal serotypes as antigens. A quantitative difference in the ability of the isotypes to form a precipitate with the polysaccharide was observed in a double immunodiffusion assay and confirmed in a quantitative precipitin assay. The relative precipitating activity of the antibodies was IgG2a greater than IgG1 much greater than IgG2b. Analysis by enzyme-linked immunosorbent assay of the reactivity of the three isotypes with cryptococcal polysaccharide showed identical titers and slopes, suggesting that the variable region of the class-switch antibodies was unaltered. This system allowed us to examine the effect of the Fc portion of the antibody on opsonization of encapsulated cryptococci. Yeast cells were precoated with antibodies of each isotype and incubated with murine macrophages or cultured human monocytes. Antibodies of all three isotypes exhibited a dose-dependent opsonization for phagocytosis by both human and murine phagocytes. The relative opsonic activity of the antibodies was IgG2a greater than IgG1 greater than IgG2b.

Animals↗