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PubMed2006

Monoclonal antibodies can affect complement deposition on the capsule of the pathogenic fungus Cryptococcus neoformans by both classical pathway activation and steric hindrance.

The capsule of the human pathogenic fungus Cryptococcus neoformans presents the immune system with a formidable problem for phagocytosis. Capsule-mediated activation of the alternative complement (C) pathway results in component 3 (particularly, C3) binding to the capsule near the cell wall surface. Hence, for cells with large capsule, C3 cannot interact with the complement receptor (CR) and is not opsonic. However, C activation in either immune serum or in the presence of monoclonal antibody (mAb) to capsular polysaccharide localizes C3 to the capsular edge. When C. neoformans cells were coated with both C and antibody (Ab) opsonins, Ab bound first and promoted C3 deposition at the edge of the capsule. The mechanism for the Ab-mediated change in C3 localization to the capsule edge involved both classical C pathway activation and steric hindrance preventing C3 penetration into the capsule. The change in C3 localization changed the mode of phagocytosis in macrophages, such that localizing C3 at the edge of the capsule allowed phagocytosis through C3-CR3 and C3-CR4 interactions, which did not occur in serum without Ab. These findings reveal a new mechanism of Ab action whereby Abs affect the location of C3 and its interaction with its receptor in macrophages depending on the immunoglobulin concentration.

Antibodies, Fungal↗
PubMed2005

Synthesis and immunological studies of glycoconjugates of Cryptococcus neoformans capsular glucuronoxylomannan oligosaccharide structures.

Antibodies to the glucuronoxylomannan (GXM) component of the polysaccharide capsule of Cryptococcus neoformans are protective and GXM-protein conjugate vaccines can elicit protective immune responses. We report the synthesis of a heptasaccharide oligosaccharide representing the putative dominant motif of serotype A GXM and demonstrate that it is recognized by some monoclonal antibodies (mAbs) generated to GXM. Conjugation of the heptasaccharide to human serum albumin (HSA) resulted in an immunogenic compound that elicited high-titer IgG responses in mice when given with complete Freund's adjuvant. The antibody response elicited by the oligosaccharide conjugate vaccine had characteristics of a T-cell-dependent response. The availability of an immunogenic oligosaccharide representing a structural motif of GXM will prove useful in studies of antibody epitope specificity and represents a potential synthetic oligosaccharide vaccine against this fungal pathogen.

Animals↗
PubMed2004

Serotype distribution of Cryptococcus neoformans in patients in a tertiary care center in India.

The prevalence of specific serotypes of Cryptococcus neoformans in a given area bears on regional epidemiological patterns, the expected spectrum of clinical disease, and predicted response to therapy. In this retrospective study we analyzed the serotypes of 45 degrees C neoformans isolates from 36 North Indian patients with varied clinical presentations. The majority of the isolates were serotype A (87%), and surprisingly, a significant number were serotype B (five isolates, 11%), which caused infection in patients both positive and negative for HIV. One unusual isolate was not typable with factor sera. Study of serotype distribution in patients showed serotypes A and B to be present, respectively, in 92% and 8% of 36 patients. In one apparently immunocompetent patient two serotypes, A and B, were isolated simultaneously from two different sites, lung and scalp abscess. This is the first reported case in which an individual was infected with two serotypes at the same time. In one HIV-infected child serotype A was isolated from blood. Our results suggest that the distribution of serotypes in Indian clinical isolates is different than that found in other regions.

Cryptococcosis↗
PubMed1976

Nature of the effector cells responsible for antibody-dependent cell-mediated killing of Cryptococcus neoformans.

Studies were performed to identify the types of human peripheral blood leukocytes capable of killing Cryptococcus neoformans in the presence of anticryptococcal antibody in vitro. A total of 24.1 +/- 2.7% (mean +/- standard error of the mean of four experiments) of the original cryptococcal inoculum survived in a mixed mononuclear, cell preparation (approximately 30% monocytes) after 4 h of incubation at 37 degrees C with rabbit anticryptococcal antibody. When phagocytic cells were removed, there was 36.4 +/- 4.6% survival in six experiments, compared with 52.8% survival in the presence of purified granulocytes (mean of two experiments) and 96.9 +/- 1% survival in the presence of purified T cells. There was never any significant killing in control mixtures that contained leukocytes with normal rabbit serum nor in those that contained anticryptococcal antibody without effector leukocytes. Significant antibody-dependent fungicidal activity was seen with ratios of effector to target cells as low as 6.25:1. These observations indicate that multiple types of peripheral blood leukocytes, excluding T cells, are capable of antibody-dependent fungicidal activity.

Antibodies, Fungal↗
PubMed2005

Rates of antifungal resistance among Spanish clinical isolates of Cryptococcus neoformans var. neoformans.

OBJECTIVES: Activities in vitro of six antifungal agents were tested against a collection of 317 Cryptococcus neoformans var. neoformans clinical isolates. METHODS: The procedure described in document 7.1 by the European Committee on Antibiotic Susceptibility Testing with minor modifications was employed. RESULTS: Amphotericin B, itraconazole, voriconazole and ravuconazole exhibited a potent activity with geometric mean (GM) MICs under 0.26 mg/L. The GM MIC of flucytosine was 7.33 mg/L and that of fluconazole was 4.16 mg/L. The rates of antifungal resistance were 5.3% for amphotericin B, 0.9% for voriconazole and 3.1% for ravuconazole. Fifteen point eight per cent of strains had itraconazole MICs > or = 1 mg/L, and 46% of strains had flucytosine MICs > or = 8 mg/L. Fluconazole susceptibility (MIC < or = 8 mg/L) stood at 53.4%. CONCLUSIONS: The percentage of fluconazole susceptibility was significantly lower than that in other surveys. Cross-resistance to itraconazole was common (33.8%) but almost the whole collection was susceptible to voriconazole and ravuconazole. These results should be confirmed with prospective and population-based surveillance programmes.

Antifungal Agents↗
PubMed2001

Inhibition of H(+)-ATPase-mediated proton pumping in Cryptococcus neoformans by a novel conjugated styryl ketone.

We investigated the in vitro susceptibility of clinical isolates of Cryptococcus neoformans to the novel conjugated styryl ketone NC1175 by broth microdilution. The MIC(90) and the MFC of NC1175 for C. neoformans were 1 and 2 mg/L, respectively. NC1175 at low concentrations (1-4 mg/L) completely inhibited the glucose-induced acidification of the external medium caused by the extrusion of intracellular protons mediated by the plasma membrane located H(+)-ATPase. These data suggest that NC1175 is a fungicidal agent for C. neoformans and its possible cellular target(s) include the H(+)-ATPase.

Antifungal Agents↗
PubMed1995

Further analysis of the CAP59 locus of Cryptococcus neoformans: structure defined by forced expression and description of a new ribosomal protein-encoding gene.

Cryptococcus neoformans (Cn) produces an extracellular polysaccharide capsule that is an essential factor for virulence. We previously isolated a gene, CAP59, which is necessary for capsule formation. To dissect the functional region of CAP59, we placed it under control of the Cn GAL7 promoter (pGAL7). Among the several pGAL7::CAP59 fusion constructs, only the one containing the entire open reading frame of CAP59 was able to complement the acapsular phenotype under galactose induction. A missense mutation in the coding region abolished complementation by the fusion construct. We also found that the CAP59 locus is contiguous to a convergently transcribed L27 ribosomal protein-encoding gene (CL27). The distance between the cDNA ends of these two genes is only 25 bp. CL27 has two introns near its N terminus. The translated CL27 protein is 183 amino acids (aa) in length with an estimated molecular mass of 20 kDa, and the first 34 aa at the N terminus may be a targeting peptide for mitochondria. A high degree of restriction-fragment-length polymorphism was detected in the DNA sequence containing CAP59 and CL27.

Amino Acid Sequence↗
PubMed2001

Cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages.

Cryptococcus neoformans (Cn) is a soil fungus that causes life-threatening meningitis in immunocompromised patients and is a facultative intracellular pathogen capable of replication inside macrophages. The mechanism by which environmental fungi acquire and maintain virulence for mammalian hosts is unknown. We hypothesized that the survival strategies for Cn after ingestion by macrophages and amoebae were similar. Microscopy, fungal and amoebae killing assays, and phagocytosis assays revealed that Cn is phagocytosed by and replicates in Acanthamoeba castellanii, which leads to death of amoebae. An acapsular strain of Cn did not survive when incubated with amoebae, but melanization protected these cells against killing by amoebae. A phospholipase mutant had a decreased replication rate in amoebae compared with isogenic strains. These observations suggest that cryptococcal characteristics that contribute to mammalian virulence also promote fungal survival in amoebae. Intracellular replication was accompanied by the accumulation of polysaccharide containing vesicles similar to those described in Cn-infected macrophages. The results suggest that the virulence of Cn for mammalian cells is a consequence of adaptations that have evolved for protection against environmental predators such as amoebae and provide an explanation for the broad host range of this pathogenic fungus.

Acanthamoeba↗
PubMed1995

Antibodies to the Cryptococcus neoformans capsular glucuronoxylomannan are ubiquitous in serum from HIV+ and HIV- individuals.

Murine MoAbs to the Cryptococcus neoformans capsular glucuronoxylomannan (GXM) polysaccharide are protective in mice in vivo and in vitro. The prevalence of protective anti-GXM antibodies in human serum is unknown. To provide further insight into the human antibody response to C. neoformans we determined the prevalence, isotype, and IgG subclass utilization of human anti-GXM antibodies in HIV+ and HIV- sera by a sensitive antigen capture FLISA assay. One hundred and twenty-three sera from the Bronx Municipal Hospital Centre serum bank were studied retrospectively. Seventy were from HIV+ individuals, 10 with a history of cryptococcal meningitis (CM), and 53 were from HIV- individuals. Serum GXM determinations were also performed on 61 HIV+ sera. Our results demonstrated that anti-GXM IgG, IgA, and IgM are ubiquitous in both HIV+ (including those with CM), and HIV- sera. Anti-GXM IgA titres and total serum IgA concentration were elevated in HIV+ sera. Anti-GXM IgG antibodies were almost exclusively isotype-restricted to the IgG2 subclass. Our data also demonstrated elevations of anti-bovine serum albumin (BSA) titres in HIV+ sera. Taken together, our findings confirm hypergammaglobulinaemia and expansion of anti-protein (BSA) antibodies in HIV+ individuals and isotype restriction of human anti-carbohydrate (GXM) antibodies to the IgG2 subclass. Our report of ubiquitous anti-GXM antibodies of the IgG and IgA isotypes suggests that anti-GXM antibodies exist before HIV infection.

Antibodies, Fungal↗
PubMed1992

Iron assimilation in Cryptococcus neoformans.

We studied the effects of iron chelators and of a thallium salt on growth of Cryptococcus neoformans in defined medium. An oxidant-sensitive mutant strain was found to require exogenous ferric iron for growth. Using this strain, we found that the synthetic iron chelator, N-hydroxyethylenediamine triacetate (HEDTA), in several saturation states, stimulated growth as well as the comparably saturated siderophore deferoxamine. This non-specific result makes the existence of a cryptococcal ferrihydroxamate receptor doubtful. The catechols, caffeic acid, L-3, 4-dihydroxyphenylalanine, epinephrine, gallic acid, 3-hydroxytyramine (dopamine) and norepinephrine, were tested for growth stimulation in iron deprivation, under conditions in which deferoxamine was stimulatory. Catechols were found to be either neutral or inhibitory. The ferrous iron chelator, bathophenanthroline disulfonate (BPDS), inhibited growth strongly in the absence of exogenous iron, suggesting that ferric ion must be reduced before it can be internalized. Direct evidence of extracellular reduction was provided by accumulation of red-coloured ferrous-BPDS complex. The inhibition caused by BPDS was relieved by ferric HEDTA, even in the presence of 10-fold increased BPDS, suggesting a second, low-affinity, non-reductive iron uptake pathway. This inference was further supported by the observation that toxicity of the non-reducible ferric analogue, thallium (III), is relieved by iron repletion.

Biological Transport↗
PubMed1998

Isolation of the third capsule-associated gene, CAP60, required for virulence in Cryptococcus neoformans.

A polysaccharide capsule is one of the most important virulence factors for the pathogenic fungus Cryptococcus neoformans. We previously characterized two capsule-associated genes, CAP59 and CAP64. To further dissect the molecular mechanism of capsule synthesis, 16 acapsular mutants induced by 4-nitroquinoline-1-oxide were obtained. The acapsular phenotype of one of these mutants was complemented. The cloned gene was designated CAP60, and deletion of this newly described capsule-associated gene resulted in an acapsular phenotype. The proposed 67-kDa Cap60p contains 592 amino acids and appears to have a putative transmembrane domain close to the N terminus. DNA sequence analysis revealed that CAP60 has similarity to CAP59 at the center portion of its coding regions. Contour-clamped homogeneous electric field blot analysis suggested that these two genes are on the same chromosome. CAP60 and CAP59, however, could not be functionally substituted for each other by direct complementation or by domain swap experiments. In addition, CAP60 is closely linked to a gene which is similar to a cellulose growth-specific gene of Agaricus bisporus, CEL1. Immunogold electron microscopy studies of the epitope-tagged CAP60 gene revealed that Cap60p was primarily localized to the nuclear membrane. Animal model studies indicated that CAP60 is essential for virulence. Thus, CAP60 is required for both capsule formation and virulence.

Amino Acid Sequence↗
PubMed2004

Glucuronoxylomannan, the major capsular polysaccharide of Cryptococcus neoformans, inhibits the progression of group B streptococcal arthritis.

Glucuronoxylomannan (GXM), the principal constituent of the Cryptococcus neoformans capsule, modulates the inflammatory response of human monocytes in vitro. Here we examine the efficacy of GXM as a novel anti-inflammatory compound for use against experimental septic arthritis. Arthritis was induced in mice by the intravenous injection of 8 x 10(6) CFU of type IV group B streptococcus (GBS). GXM was administered intravenously in different doses (50, 100, or 200 microg/mouse) 1 day before and 1 day after bacterial inoculation. GXM treatment markedly decreased the incidence and severity of articular lesions. Histological findings showed limited periarticular inflammation in the joints of GXM-treated mice, confirming the clinical observations. The amelioration of arthritis was associated with a significant reduction in the local production of interleukin-6 (IL-6), IL-1beta, macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-2 and an increase in systemic IL-10 levels. Moreover, peritoneal macrophages derived from GXM-treated mice and stimulated in vitro with heat-inactivated GBS showed a similar pattern of cytokine production. The present study provides evidence for the modulation of the inflammatory response by GXM in vivo and suggests a potential therapeutic use for this compound in pathologies involving inflammatory processes.

Animals↗
PubMed2003

A CLC-type chloride channel gene is required for laccase activity and virulence in Cryptococcus neoformans.

Laccase is a major virulence factor required for infection caused by the human pathogenic yeast Cryptococcus neoformans. However, cellular processes involved in the regulation and expression of laccase remain largely unknown in C. neoformans. Here we report the identification of a chloride channel gene CLC-A which is essential for laccase activity in C. neoformans. CLC-A shares homology to CLC-type voltage-gated chloride channels from other organisms; for example, 63% homology to GEF1, a chloride channel gene from Saccharomyces cerevisiae. A clc-a mutant, Mlac3, generated by insertional mutagenesis as well as a targeted Deltaclc-a mutant produced undetectable laccase in a liquid assay and produced no melanin on asparagine agar containing norepinephrine. Mlac3 was complemented with wild-type CLC-A which restored laccase activity and melanin biosynthesis. The clc-a mutants also showed reduced synthesis of another important virulence factor, capsule, and showed reduced growth at elevated pH. In addition, the clc-a mutation resulted in attenuated virulence in a mouse cryptococcosis model that was restored by complementation with wild-type CLC-A, indicating that the chloride channel plays an important role in the virulence of the organism. Further analysis revealed that the basis for absent laccase expression in the clc-a mutant was a laccase transcriptional defect that could be restored by adding exogenous copper. In conclusion, our findings show that CLC-A plays a role in the expression of two important virulence factors, capsule and laccase expression, which are required for virulence of the fungal pathogen.

Animals↗
PubMed2000

Th1-Th2 cytokine kinetics in the bronchoalveolar lavage fluid of mice infected with Cryptococcus neoformans of different virulences.

Th1 immune response plays an important role in protection against infection with Cryptococcus neoformans in mice. We investigated the effect of virulence of C. neoformans on cytokine production in the lung of a mouse model of pulmonary cryptococcosis. BALB/c mice were inoculated intratracheally with a high or low virulence strain of C. neoformans, followed by serial measurements of Th1 and Th2 cytokine concentrations in the bronchoalveolar lavage (BAL) fluid using appropriate enzyme-linked immunosorbent assay kits. The number of colony-forming units (CFU) increased with time, and all mice infected with the highly virulent strain were dead at 28 days after inoculation. In contrast, the number of microorganisms diminished with time in the mice infected with the low virulence strain during the 4-week study. The numbers of neutrophils and lymphocytes in the BAL fluid paralleled those of CFU. High neutrophil counts were observed in the BAL fluid of mice infected with the highly virulent strain, while lymphocyte counts were increased only in the later part of the study in mice infected with the high and low virulence strains. The concentrations of Th2 cytokine, interleukin (IL)-4 were significantly higher in mice infected with the highly virulent strain at days 14 and 21 of infection, whereas the level of Th1 cytokine, interferon-gamma, was significantly higher in the latter strain at days 7 and 14. Our results suggest that strain-specific difference in the organism's ability to induce (or evade) the host immune system contributes to the outcome of infection.

Animals↗
PubMed2006

Systemic and cerebrospinal fluid T-helper cytokine responses in organ transplant recipients with Cryptococcus neoformans infection.

BACKGROUND: The role of Th1 and Th2 mediated cytokine responses in the pathogenesis of Cryptococcus neoformans infection in organ transplant recipients has not been defined. METHODS: We assessed cytokine levels in the sera and CSF collected prospectively at the time of diagnosis of infection in 25 transplant recipients with cryptococcosis. Serum levels were compared with those in healthy individuals and transplant recipients without cryptococcosis. IFN-gamma or IL-12 (Th1)/IL-10 (Th2) ratio < 1.0 was considered a dominant Th2 response. RESULTS: Cases had lower ratios of IFN-gamma/IL-10 (p = 0.03) and IL-12/IL-10 (p = 0.03) compared to healthy individuals. Cytokine responses, however, did not differ significantly for cases vs. transplant controls. Cases with fungemia compared to those without fungemia tended to have higher serum IL-10 levels (p = 0.07) and lower IL-12/IL-10 ratios (p = 0.06). CSF ratios of IFN-gamma/IL-10 (p = 0.04) and IL-12/IL-10 (p = 0.04) were lower in cases with cryptococcal meningitis compared to those without meningitis; 80% (8/10) of the cases with cryptococcal meningitis vs. 0% (4/4) of those without meningitis had CSF IFN-gamma/IL-10 ratio of < 1.0 (p = 0.015). The levels of IL-10 (p = 0.04) and IFN-gamma (p = 0.04) in the CSF in cases with cryptococcal meningitis were significantly higher than those in their serum, respectively. CONCLUSIONS: High expression of Th2 phenotype in cryptococcal meningitis and in fungemia suggests that Th dysregulation may contribute to the pathogenesis of cryptococcosis in organ transplant recipients.

Cryptococcus neoformans↗
PubMed1998

Ferrous iron uptake in Cryptococcus neoformans.

Previous studies have implicated ferric reduction in the iron uptake pathway of the opportunistic pathogen Cryptococcus neoformans. Here we studied iron uptake directly, using 55Fe in the presence of reductants. Uptake was linear with respect to time and number of yeast cells. The plot of uptake versus concentration exhibited a steep rise up to about 1 microM, a plateau between 1 and 25 microM, and a second steep rise above 25 microM, consistent with high- and low-affinity uptake systems. A Km for high-affinity uptake was estimated to be 0.6 microM Fe(II); 1 microM was used for standardized uptake assays. At this concentration, the uptake rate was 110 +/- 3 pmol/10(6) cells/h. Iron repletion (15 microM) and copper starvation drastically decreased high-affinity iron uptake. Incubation at 0 degreesC or in the presence of 2 mM KCN abolished high-affinity iron uptake, suggesting that uptake requires metabolic energy. When exogenous reducing agents were not supplied and the culture was washed free of secreted reductants, uptake was reduced by 46%; the remaining uptake activity presumably was dependent upon the cell membrane ferric reductase. Further decreases in free Fe(II) levels achieved by trapping with bathophenanthroline disulfonate or reoxidizing with potassium nitrosodisulfonate reduced iron uptake very drastically, suggesting that it is the Fe(II) species which is transported by the high-affinity transporter. The uptake of Fe was stimulated two- to threefold by deferoxamine, but this increment could be abolished by copper starvation or inhibition of the ferric reductase by Pt, indicating that Fe solubilized by this molecule also entered the reductive iron uptake pathway.

Biological Transport↗
PubMed2024

Virulence-associated variants in Cryptococcus neoformans sequence type 93 are less likely to be associated with population structure compared to independent rare mutations.

Cryptococcus neoformans is a pathogenic yeast that is the causative agent of cryptococcal meningitis. While it is well known that the genotype of C. neoformans impacts patient outcomes, the reason for this association has not been well elucidated. In this study, we examined the relationship between two subpopulations in the sequence type 93 clade of C. neoformans: ST93A and ST93B. We found extensive linkage disequilibrium (LD) among the single nucleotide polymorphisms (SNPs) that differentiate ST93A from ST93B. We also found differences in the extent of linkage among SNPs within each subpopulation; LD was more extensive within ST93B than ST93A. SNPs associated with virulence were in long-range linkage disequilibrium with less frequency than recurrent SNPs not associated with virulence. We investigated the karyotype of ST93A and ST93B using contour-clamped gel electrophoresis and long-read sequencing and found that the extensive long-range linkage was not due to chromosomal rearrangements. Overall, we found that the two subpopulations in ST93 are driven by SNPs in LD. We additionally found that recurrent SNPs associated with virulence were less frequently evolutionarily linked and were two times more likely to be independent, congruent mutations rather than tied to phylogeny.IMPORTANCECryptococcus neoformans is an important pathogen that is widely distributed and ubiquitous in the environment. The majority of the human population has a latent, controlled infection suggesting that C. neoformans is uniquely adapted to cause infection. In spite of this, the reason C. neoformans is a pathogen remains unknown; interestingly, most environmental isolates are avirulent but are genetically very similar to disease-causing virulent isolates. Recent evidence from genome-wide association studies shows that small mutations in key virulence-associated genes are associated with the virulence of specific isolates. The data presented here provide an evolutionary framework for those small mutations. The mutations that impact disease are not being collected over long-term evolution. The mutations may instead occur independently during infection. Identifying these genes that are more likely to be mutated during infection will be fundamental for understanding C. neoformans virulence.

Cryptococcus neoformans↗