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PubMed1996

Purified capsular polysaccharide of Cryptococcus neoformans induces interleukin-10 secretion by human monocytes.

In this study, we demonstrated that purified capsular polysaccharide of Cryptococcus neoformans is a potent inducer of interleukin-10 (IL-10) secretion by human monocytes. Endogenous IL-10 was involved in regulating tumor necrosis factor alpha and IL-1beta secretion by human monocytes in response to encapsulated C. neoformans strains. Our results suggest a new immunosuppressive effect exerted by glucuronoxylomannan through the induction of IL-10, a potent downregulator of proinflammatory cytokines.

Anti-Bacterial Agents↗
PubMed2000

Geographic grouping of Cryptococcus neoformans var. gattii by random amplified polymorphic DNA fingerprint patterns and ITS sequence divergence.

Eleven reference strains of Cryptococcus neoformans var. gattii were analyzed by random amplified polymorphic DNA (RAPD) patterns using three oligo primers. Three major RAPD pattern profiles (profiles I, II and III) were identified with A-1 oligo primer. Each profile was found to relate to a geographic region. Since the strains which belong to profiles I and II were mainly the isolates from America, and profile III from Asia with A-1 primer, these three profiles were assigned to the geographic grouping of America-1, America-2 and Asia, respectively. Analysis of rDNA sequences coding an internal transcribed spacer (ITS) region of C. neformans var. gattii revealed that the fungus with each RAPD profile has characteristic base sequences at the four positions (10 and 15 positions in ITS1 and 8 and 56 positions at ITS2) of the ITS regions. On the basis of the combinations of the four bases specific for the ITS regions, four ITS types, AAGG (America-1), AAAC (America-2), GGGC (Asia-1) and AGGC (Asia-2) were identified: the geographic group of Asia was further classified into two subgroups of Asia-1 and Asia-2 based on the ITS typing. Clinical isolates from Thailand (6 strains) and Brazil (7 strains) were found to belong to the geographic group of America-1 nd America-2, respectively. Five reference strains of C. neoformans var. gattii from the CBS culture collection were classified into two America-2, one Asia-1 and two Asia-2 groups. This ITS region analysis allowed us to distinguish all isolates of C. neoformans var. gattii into four geographic groups based on the ITS base sequence, and further molecular epidemiological and ecological research on this fungus is recommended.

Base Sequence↗
PubMed2003

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 (MATalpha). 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.

AIDS-Related Opportunistic Infections↗
PubMed1994

Enhancement of HIV-1 replication in peripheral blood mononuclear cells by Cryptococcus neoformans is monocyte-dependent but tumour necrosis factor-independent.

OBJECTIVE: To investigate the possible role of Cryptococcus neoformans in HIV-1 pathogenesis. DESIGN: An in vitro system was developed to study HIV-1 replication in freshly HIV-1-infected peripheral blood mononuclear cells (PBMC) incubated with whole azide-killed C. neoformans. METHODS: Human PBMC or peripheral blood lymphocytes were infected with lymphocytotropic HIV-1 and incubated with azide-killed encapsulated or non-encapsulated C. neoformans for 10 days. Viral replication was followed by HIV-1 p24 enzyme-linked immunosorbent assay and median tissue culture infective dose determination. Tumour necrosis factor (TNF) release by PBMC, induced by C. neoformans, was measured. Anti-TNF monoclonal antibodies or pentoxifylline were used to inhibit TNF bioactivity. RESULTS: Both encapsulated and non-encapsulated C. neoformans enhanced HIV-1 replication in PBMC but not in peripheral blood lymphocytes. C. neoformans induced TNF release by PBMC. Inhibition of TNF bioactivity did not block C. neoformans-enhanced HIV-1 replication in PBMC. CONCLUSIONS: C. neoformans can enhance HIV-1 replication in T cells only in the presence of monocytic cells. This enhancement is not dependent on encapsulation nor can it be attributed to TNF release.

Antibodies, Monoclonal↗
PubMed1999

A glucan synthase FKS1 homolog in cryptococcus neoformans is single copy and encodes an essential function.

Cryptococcal meningitis is a fungal infection, caused by Cryptococcus neoformans, which is prevalent in immunocompromised patient populations. Treatment failures of this disease are emerging in the clinic, usually associated with long-term treatment with existing antifungal agents. The fungal cell wall is an attractive target for drug therapy because the syntheses of cell wall glucan and chitin are processes that are absent in mammalian cells. Echinocandins comprise a class of lipopeptide compounds known to inhibit 1,3-beta-glucan synthesis, and at least two compounds belonging to this class are currently in clinical trials as therapy for life-threatening fungal infections. Studies of Saccharomyces cerevisiae and Candida albicans mutants identify the membrane-spanning subunit of glucan synthase, encoded by the FKS genes, as the molecular target of echinocandins. In vitro, the echinocandins show potent antifungal activity against Candida and Aspergillus species but are much less potent against C. neoformans. In order to examine why C. neoformans cells are less susceptible to echinocandin treatment, we have cloned a homolog of S. cerevisiae FKS1 from C. neoformans. We have developed a generalized method to evaluate the essentiality of genes in Cryptococcus and applied it to the FKS1 gene. The method relies on homologous integrative transformation with a plasmid that can integrate in two orientations, only one of which will disrupt the target gene function. The results of this analysis suggest that the C. neoformans FKS1 gene is essential for viability. The C. neoformans FKS1 sequence is closely related to the FKS1 sequences from other fungal species and appears to be single copy in C. neoformans. Furthermore, amino acid residues known to be critical for echinocandin susceptibility in Saccharomyces are conserved in the C. neoformans FKS1 sequence.

Amino Acid Sequence↗
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↗
PubMed2006

Calcineurin, Mpk1 and Hog1 MAPK pathways independently control fludioxonil antifungal sensitivity in Cryptococcus neoformans.

Fludioxonil is employed as an agricultural fungicide to control plant-pathogenic fungi such as Botrytis cinerea. Cryptococcus neoformans is a basidiomycetous human fungal pathogen that causes fatal disease in immunocompromised hosts. This paper demonstrates that three different signalling cascades regulate sensitivity of C. neoformans to fludioxonil. Fludioxonil inhibited growth of the serotype A sequence reference strain H99 but not that of the sequenced serotype D strain JEC21. In the drug-sensitive wild-type strain, fludioxonil exposure activated the Hog1 osmosensing pathway, and hog1Delta mutations conferred fludioxonil resistance. Fludioxonil treatment caused cell growth inhibition following cell swelling and cytokinesis defects in the sensitive wild-type but not in a hog1Delta mutant strain, suggesting that Hog1 activation results in morphological cellular defects. Fludioxonil exerted a fungistatic effect on the wild-type strain H99, but exhibited fungicidal activity against calcineurin mutant strains, indicating that the calcineurin pathway contributes to drug resistance in this fungus. Combination of fludioxonil and the calcineurin inhibitor FK506 synergistically inhibited C. neoformans growth. mpk1Delta MAPK mutant strains exhibited fludioxonil hypersensitivity, indicating that this pathway also contributes to drug resistance. These studies provide evidence that the broad-spectrum antifungal drug fludioxonil exerts its action via activation of the Hog1 MAPK pathway and provide insight into novel targets for synergistic antifungal drug combinations.

Antifungal Agents↗
PubMed1991

Inhibition of I-A expression in rat peritoneal macrophages due to T-suppressor cells induced by Cryptococcus neoformans.

The expression of I-A antigen in rat peritoneal cells was significantly reduced during infection with Cryptococcus neoformans. When studying the in vitro action of T-suppressor cells induced by the fungus, or a soluble factor from the T-suppressor cells, a significant decrease in I-A expression by the peritoneal cells was observed. This expression was partially restored by indomethacin.

Animals↗
PubMed1998

Multi-centre evaluation of the Etest method for antifungal drug susceptibility testing of Candida spp. and Cryptococcus neoformans. BSAC Working Party on Antifungal Chemotherapy.

Ten laboratories tested 18 isolates of Candida spp. and two of Cryptococcus neoformans against amphotericin B, flucytosine, fluconazole and itraconazole on two occasions by the Etest method. Two individuals read each set of results. Of the 18 isolates of Candida spp., five were duplicated, but the participants were not told this. In 40 of the 60 drug-organism combinations studied, at least 80% of the Etest MICs fell within a five-concentration range corresponding to the modal MIC +/- 1 log2 dilution. In five combinations, >50% of the Etest MICs fell outside this five-concentration range. In 17 (85%) of the 20 drug-organism combinations tested in duplicate, at least 80% of the paired Etest results fell within two concentrations of each other (corresponding to one log2 dilution). Overall, 88.5% of the paired Etest results for amphotericin B agreed to within two concentrations, as did 94% of results for flucytosine, 92% for fluconazole and 79% for itraconazole. The broth microdilution MICs of the four antifungal agents for the 15 isolates were measured on five occasions in the Mycology Reference Laboratory, Bristol. In each case, the results fell within a three log2 concentration range. In 24 (40%) of the 60 drug-organism combinations tested, at least 80% of the Etest results fell within the broth microdilution test MIC range, but 27 (45%) showed <50% exact agreement. In 33 (73%) of 45 drug-organism combinations involving flucytosine, fluconazole or itraconazole, at least 80% of the Etest results fell within the same class (susceptible, resistant, or susceptible dependent upon dose) as the broth microdilution results. With fluconazole, the Etest method misclassified three susceptible isolates of Candida spp. as resistant in 1.5-15% of tests. With itraconazole, the Etest misclassified seven susceptible isolates of Candida spp. as resistant in 5-62.5% of tests. The Etest also misclassified both C. neoformans isolates as resistant to flucytosine, fluconazole and itraconazole in 7.5-65% of tests. Our results suggest that the Etest is suitable for routine use with Candida spp. and amphotericin B or flucytosine. It is less reliable for the azoles, and isolates that appear to demonstrate acquired resistance should be retested with well-established reference methods.

Amphotericin B↗
PubMed2004

Determination of Cryptococcus neoformans var. neoformans mating type by multiplex PCR.

Mating type plays an important role in the epidemiology and virulence of Cryptococcus neoformans. The present study designed a multiplex PCR method to distinguish the six mating type patterns (Aa, Da, Aalpha, Dalpha, Aa/Dalpha, and Aalpha/Da) of C. neoformans var. neoformans. PCR amplification identified one fragment for Aa (860 bp), Dalpha (413 bp) and Da (645 bp) strains, two fragments for Aalpha (320 and 400 bp) and Aa/Dalpha (860 and 413 bp) strains, and three fragments (645, 400, 320 bp) for an Aalpha/Da strain. The method appears to be a valid, simple and relatively inexpensive tool for epidemiological and virulence studies.

Cryptococcus neoformans↗
PubMed2007

Protection against cryptococcosis by using a murine gamma interferon-producing Cryptococcus neoformans strain.

We evaluated cell-mediated immune (CMI) responses in mice given a pulmonary infection with a Cryptococcus neoformans strain engineered to produce the Th1-type cytokine gamma interferon (IFN-gamma). Mice given a pulmonary infection with an IFN-gamma-producing C. neoformans strain were able to resolve the primary infection and demonstrated complete (100%) protection against a second pulmonary challenge with a pathogenic C. neoformans strain. Pulmonary cytokine analyses showed that Th1-type/proinflammatory cytokine and chemokine expression were significantly higher and Th2-type cytokine expression was significantly lower in mice infected with the IFN-gamma-producing C. neoformans strain compared to wild-type-infected mice. This increased pulmonary Th1-type cytokine expression was also associated with significantly lower pulmonary fungal burden and significantly higher pulmonary leukocyte and T-lymphocyte recruitment in mice infected with the IFN-gamma-producing C. neoformans strain compared to wild-type-infected mice. Our results demonstrate that pulmonary infection of mice with a C. neoformans strain expressing IFN-gamma results in the stimulation of local Th1-type anti-cryptococcal CMI responses and the development of protective host immunity against future pulmonary cryptococcal infections. The use of fungi engineered to produce host cytokines is a novel method to study immune responses to infection and may be useful in developing vaccine strategies in humans.

Animals↗
PubMed1996

Combination of 5-flucytosine and capsule-binding monoclonal antibody in the treatment of murine Cryptococcus neoformans infections and in vitro.

The efficacy of combination therapy with 5-flucytosine and an IgG1 monoclonal antibody to Cryptococcus neoformans capsular glucuronoxylomannan was studied in vitro with J774. 16 murine macrophage-like cells and in vivo in murine cryptococcal infection. The combination of 5-flucytosine and IgG1 was more effective in reducing the numbers of C. neoformans colony-forming units in vitro and in vivo than either agent alone.

Animals↗
PubMed2006

Role of AFR1, an ABC transporter-encoding gene, in the in vivo response to fluconazole and virulence of Cryptococcus neoformans.

We have recently demonstrated that upregulation of the ATP binding cassette (ABC) transporter-encoding gene AFR1 in Cryptococcus neoformans is involved in the in vitro resistance to fluconazole of this yeast. In the present study, we investigated the role of AFR1 in the in vivo response to fluconazole in a mouse model of systemic cryptococcosis. Mice were infected with a wild-type fluconazole-susceptible strain of C. neoformans, strain BPY22; an afr1 mutant, BPY444, which displayed hypersusceptibility to fluconazole in vitro; or an AFR1-overexpressing strain, BPY445, which exhibited in vitro resistance to the drug. In each of the three groups, infected animals were randomly assigned to fluconazole treatment or untreated-control subgroups. As expected, fluconazole prolonged survival and reduced fungal tissue burdens (compared with no treatment) in BPY22- and BPY444-infected mice, whereas it had no significant effects in mice infected with BPY445. When the pathogenicities of these strains in mice were investigated, strain BPY445 was significantly more virulent than BPY22 following inhalational or intravenous inoculation, but mice infected with BPY444 survived significantly longer than BPY22-infected animals only when infection was acquired via the respiratory tract. In in vitro macrophage infection studies, strain BPY445 also displayed enhanced intracellular survival compared with strains BPY22 and BPY444, suggesting that its increased virulence may be due to its reduced vulnerability to the antimicrobial factors produced by phagocytic cells. These findings indicate that the upregulation of the AFR1 gene is an important factor in either determining the in vivo resistance to fluconazole or influencing the virulence of C. neoformans.

ATP-Binding Cassette Transporters↗
PubMed2002

Mating-type locus of Cryptococcus neoformans: a step in the evolution of sex chromosomes.

The sexual development and virulence of the fungal pathogen Cryptococcus neoformans is controlled by a bipolar mating system determined by a single locus that exists in two alleles, alpha and a. The alpha and a mating-type alleles from two divergent varieties were cloned and sequenced. The C. neoformans mating-type locus is unique, spans >100 kb, and contains more than 20 genes. MAT-encoded products include homologs of regulators of sexual development in other fungi, pheromone and pheromone receptors, divergent components of a MAP kinase cascade, and other proteins with no obvious function in mating. The alpha and a alleles of the mating-type locus have extensively rearranged during evolution and strain divergence but are stable during genetic crosses and in the population. The C. neoformans mating-type locus is strikingly different from the other known fungal mating-type loci, sharing features with the self-incompatibility systems and sex chromosomes of algae, plants, and animals. Our study establishes a new paradigm for mating-type loci in fungi with implications for the evolution of cell identity and self/nonself recognition.

Alleles↗
PubMed2000

Human antibodies against a purified glucosylceramide from Cryptococcus neoformans inhibit cell budding and fungal growth.

A major ceramide monohexoside (CMH) was purified from lipidic extracts of Cryptococcus neoformans. This molecule was analyzed by high-performance thin-layer chromatography (HPTLC), gas chromatography coupled with mass spectrometry, and fast atom bombardment-mass spectrometry. The cryptococcal CMH is a beta-glucosylceramide, with the carbohydrate residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic acid. Sera from patients with cryptococcosis and a few other mycoses reacted with the cryptococcal CMH. Specific antibodies were purified from patients' sera by immunoadsorption on the purified glycolipid followed by protein G affinity chromatography. The purified antibodies to CMH (mainly immunoglobulin G1) bound to different strains and serological types of C. neoformans, as shown by flow cytofluorimetry and immunofluorescence labeling. Transmission electron microscopy of yeasts labeled with immunogold-antibodies to CMH and immunostaining of isolated cell wall lipid extracts separated by HPTLC showed that the cryptococcal CMH predominantly localizes to the fungal cell wall. Confocal microscopy revealed that the beta-glucosylceramide accumulates mostly at the budding sites of dividing cells with a more disperse distribution at the cell surface of nondividing cells. The increased density of sphingolipid molecules seems to correlate with thickening of the cell wall, hence with its biosynthesis. The addition of human antibodies to CMH to cryptococcal cultures of both acapsular and encapsulated strains of C. neoformans inhibited cell budding and cell growth. This process was complement-independent and reversible upon removal of the antibodies. The present data suggest that the cryptococcal beta-glucosylceramide is a fungal antigen that plays a role on the cell wall synthesis and yeast budding and that antibodies raised against this component are inhibitory in vitro.

Antibodies, Fungal↗
PubMed1986

Poorly encapsulated Cryptococcus neoformans from patients with AIDS. II. Correlation of capsule size observed directly in cerebrospinal fluid with that after animal passage.

Cryptococcus neoformans recovered from the cerebrospinal fluid (CSF) of eight patients, seven with the acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC), were studied to assess the relationship between degree of encapsulation noted in fresh CSF with that observed after animal passage. We further correlated encapsulation with extent of immunodeficiency in these patients. Results of these studies showed poor encapsulation (mean capsule plus cell diameter less than 10 micron) in six patients, intermediate in one (mean 15.5 micron), and full encapsulation in one (mean 24.4 micron). The last isolate was observed in the CSF from the only patient without convincing clinical evidence for AIDS. Mouse passage of cryptococci from 5 AIDS patients and one with ARC resulted in a statistically significant (P less than 0.05) increase in capsule size over that observed directly in fresh cerebrospinal fluid. Cryptococci derived from the non-AIDS patient did not show an increase in encapsulation after mouse passage. These studies suggest that the immune deficiency state associated with AIDS exerts little selective pressure on inhaled poorly encapsulated C. neoformans.

AIDS-Related Complex↗
PubMed1994

In vitro susceptibility of the opportunistic fungus Cryptococcus neoformans to anthelmintic benzimidazoles.

Ten benzimidazole derivatives and amphotericin B were tested in vitro against three isolates of Cryptococcus neoformans. Drug concentrations inhibiting 50% of growth (IC50s) were determined. Four derivatives, including mebendazole and albendazole, had moderately high activities (IC50 = 0.1 to 0.3 microgram/ml). Fenbendazole, however, was 10-fold more active (IC50 = 0.01 to 0.02 microgram/ml) and also 2-fold more active than amphotericin B. Ten additional clinical isolates of C. neoformans were tested against fenbendazole, mebendazole, and albendazole; similar susceptibilities were observed. Drug concentrations lethal to 90% of the cells (LC90s) were determined for two isolates. The LC90s of albendazole and mebendazole were 0.92 to 2.1 micrograms/ml, and those of fenbendazole were 0.06 to 0.07 microgram/ml; the latter are eight to ninefold lower than the LC90s of amphotericin B that were obtained. Spontaneously arising mutants displaying partial resistance to fenbendazole arose at a low frequency (5 x 10(-9).

Amphotericin B↗
PubMed1998

Molecular epidemiology and antifungal susceptibility of Cryptococcus neoformans isolates from Ugandan AIDS patients.

Little is known of the antifungal susceptibility patterns and molecular epidemiology of Cryptococcus neoformans from tropical regions. We studied 164 clinical isolates of C. neofomans from 120 Ugandan AIDS patients with cryptococcal meningitis by analyzing their electrophoretic karyotypes and antifungal susceptibility profiles. Computer-assisted analysis of karyotype patterns was performed to generate dendrograms. MICs of fluconazole and flucytosine were determined by reference methods. A total of 43 distinguishable DNA types were identified among the 164 isolates. Only 30 patients (25%) were infected with their own unique strain of c. neoformans, whereas 75% of the patients shared their infecting strain with at least one other patient. Among 17 patients with more than one CSF isolate of C. neoformans, sequential isolates were identical or highly related in 12 (71%) and were different in five patients (29%). The isolates were susceptible to both fluconazole and flucytosine and there were no instances in which a stepwise increase in either fluconazole or flucytosine MICs was observed among serial isolates. These findings suggest that the epidemiology of cryptococcal disease in AIDS patients from tropical regions may be somewhat different from that observed in more temperate climates.

Acquired Immunodeficiency Syndrome↗