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PubMed2005

Capsule structure changes associated with Cryptococcus neoformans crossing of the blood-brain barrier.

Cryptococcus neoformans is a yeast responsible for disseminated meningoencephalitis in patients with cellular immune defects. The major virulence factor is the polysaccharide capsule. We took advantage of a relevant murine model of disseminated meningoencephalitis to study the early events associated with blood-brain barrier (BBB) crossing. Mice were sacrificed at 1, 6, 24, and 48 hours post-intravenous inoculation, and classical histology, electron microscopy, and double immunofluorescence were used to study tissues and yeasts. Crossing of the BBB occurred early after inoculation, did not involve the choroid plexus but instead occurred at the level of the cortical capillaries, and caused early and severe damage to the structure of the microvessels. Seeding of the leptomeninges was not the primary event but occurred secondary to leakage of cortical pseudocysts. Organ invasion was associated with changes in cryptococcal capsule structure and cell size, which differed in terms of magnitude and kinetics, depending on both the organs involved, and potentially, on the bed structure of the local capillary. The rapid changes in capsule structure could contribute to inability of the host immune response to control cryptococcal infection in extrapulmonary spaces.

Animals↗
PubMed1994

Cloning, expression and characterization of thymidylate synthase from Cryptococcus neoformans.

The thymidylate synthase (TS)-encoding gene from Cryptococcus neoformans (Cn) has been isolated from cDNA and genomic libraries. The 1127-bp gene contains three introns and a 951-bp open reading frame encoding a 35,844-Da protein. The cDNA clones lack 324 bp of the 5' coding region of the gene. The complete coding sequence was assembled as an expression cassette in pUC19 using parts of the coding sequence from the cDNA and genomic DNA and completing the sequence using synthetic DNA. Production of active TS from Cn (CnTS) was first demonstrated by complementation of a thymine(Thy)-requiring Escherichia coli strain. The expression cassette was subsequently subcloned into the T7 polymerase vector pET15-b. In this construct, CnTS is produced as approximately 10% of the total soluble protein in E. coli. Homogeneous enzyme was obtained at a 36% yield after consecutive chromatography on DEAE-cellulose, Q-Sepharose, phenyl-Sepharose and Affi-Gel Blue. Steady-state kinetic analysis showed that the Km values for dUMP and CH2H4.folate were 2.7 +/- 0.5 microM and 38.2 +/- 2.5 microM, respectively, and the kcat was 5.1 s-1. The enzyme was stable upon storage at -80 degrees C in Tris.HCl pH 7.4 and thiol.

Amino Acid Sequence↗
PubMedDate not supplied

Purification and characterization of fatty acid synthetase from Cryptococcus neoformans.

Fatty acid synthetase has been purified from Cryptococcus neoformans 450 fold to a specific activity of 3.6 units per mg protein with an overall yield of 23%. The purified enzyme contained two non-identical subunits, Mr approximately 2.1 x 10(5) and 1.8 x 10(5). Under optimum conditions, 100 mM KCl and pH 7.5, apparent K(m) values for the substrates were: Acetyl CoA, 19 microM; Malonyl CoA, 5 microM; and NADPH, 6 microM. Product inhibition patterns were determined to be: CoA, competitive versus acetyl CoA and malonyl CoA, uncompetitive versus NADPH; NADP, competitive versus NADPH, uncompetitive versus acetyl CoA and malonyl CoA; Palmitoyl CoA, competitive versus malonyl CoA, noncompetitive versus acetyl CoA and NADPH; Bicarbonate, uncompetitive versus malonyl CoA. These product inhibition patterns are consistent with the multisite ping-pong mechanism previously proposed for the avian fatty acid synthetase complex. The cryptococcal fatty acid synthetase was inhibited by the polyanionic polymers, heparin and dextran sulfate, an effect never before demonstrated for a fatty acid synthetase. This inhibition exhibited a marked dependence on the length of the polymer chain, with dextran sulfate fractions with Mr of 6 x 10(5) and above having Ki values below 100 nanomolar. A model is presented that involves initial binding of the anionic polymer to the enzyme complex at a region of high positive charge density, followed by interaction of the end of the tethered polymer with the catalytic site. This study represents the first purification of fatty acid synthetase from a basidiomycete.

Anions↗
PubMed1985

Virulence of Cryptococcus neoformans. Regulation of capsule synthesis by carbon dioxide.

Cryptococcus neoformans is variably encapsulated in vitro, whereas in tissues it develops a large capsule. We observed that cells of a strain with thin capsules, when growing in a standard fungal culture medium, became heavily encapsulated when incubated in serum-free cell culture medium (Dulbecco's modified Eagle's medium [DME]). Capsule size was quantitated physically by measuring cell volume, and chemically by determining the content of a capsular monosaccharide, glucuronate. The CO2/HCO-3 couple stimulated capsule development, resulting in visible enlargement by 3 h after exposure to high CO2/HCO-3. The amount of capsule per cell was directly proportional to the total millimolar CO2/HCO-3 concentration between 24 and 2.4 mM at pH 7.35, but at constant PCO2 (40 torr) and varying [HCO-3], the cells were heavily encapsulated down to pH 6.8. Concentration of CO2/HCO-3 in the physiologic range increased elaboration of polysaccharide into the medium and slowed the cell generation time from 2 to 6 h. Four other first-passage clinical isolates were all heavily encapsulated in DME with CO2/HCO-3, but variably encapsulated in DME without CO2/HCO-3. Exposure of yeast to increased CO2/HCO-3 caused a marked reduction in complement-mediated phagocytosis by mouse macrophages. A stable clone was isolated which contained capsular polysaccharide, but lacked the CO2-inducible phenotype. This clone was avirulent for steroid-treated rabbits. Thus, the prevailing CO2 concentration in mammalian tissues may be one stimulus for capsular polysaccharide synthesis. This could serve as an adaptive mechanism favoring parasite survival in the host.

Animals↗
PubMed2004

CNLAC1 is required for extrapulmonary dissemination of Cryptococcus neoformans but not pulmonary persistence.

The pathogenic yeast Cryptococcus neoformans produces a laccase enzyme (CNLAC1), which catalyzes the synthesis of melanin in the presence of phenolic compounds. A number of genes have been implicated in the regulation of laccase and melanization, including IPC1, GPA1, MET3, and STE12. Albino mutants derived from random mutagenesis techniques may contain mutations in genes that regulate multiple virulence factors, including CNLAC1. The goal of our study is to investigate the role of CNLAC1 in virulence and evasion of pulmonary host defenses after infection via the respiratory tract. Using a set of congenic laccase-positive (2E-TUC-4) and laccase-deficient (2E-TU-4) strains, we found that both strains are avirulent at a lower dose (10(4) CFU/mouse) in mice. After the infectious dose was increased to 10(6) CFU/mouse, 70% mortality was observed in mice infected with 2E-TUC-4 compared to no mortality in mice infected with 2E-TU-4 at day 30 postinfection. This observation confirms the requirement for CNLAC1 in virulence. Interestingly, we observed no differences between the two strains in pulmonary growth or in elicitation of cellular immune responses in the lung. The only measurable defect of 2E-TU-4 was in dissemination to extrapulmonary sites. To examine the role of CNLAC1 in dissemination, mice were infected intravenously. By week 3 postinfection, equal numbers of strains 2E-TUC-4 and 2E-TU-4 were recovered from the brain and spleen. This observation indicates that CNLAC1 facilitates escape from the lung, but not growth in the lungs or brain, and suggests a novel role for CNLAC1 in virulence during an infection aquired via the respiratory tract.

Animals↗
PubMed2002

High affinity mimotope of the polysaccharide capsule of Cryptococcus neoformans identified from an evolutionary phage peptide library.

Cryptococcus neoformans causes a life-threatening meningoencephalitis in a significant percentage of AIDS patients. Mice immunized with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid (TT) produce Abs that, based on the epitope recognized, can be either protective or nonprotective. Since nonprotective Abs block the efficacy of protective Abs, we are interested in developing a vaccine that would focus the immune response specifically to protective epitopes. Previously, we screened a phage display library with 2H1, a protective anti-GXM mAb, and isolated PA1, a representative peptide that had a K(d) of 295 nM for 2H1. Mice immunized with PA1 conjugated to keyhole limpet hemocyanin developed high anti-peptide (1/13,000), but low anti-GXM (maximum, 1/200) titers. We now report our efforts to improve this vaccine by screening a sublibrary with six random amino acids added to either end of the PA1 motif to identify higher affinity peptides. P206.1, a peptide isolated from this sublibrary, had 80-fold higher affinity for 2H1 (K(d) = 3.7 nM) than PA1. P206.1 bound protective, but not nonprotective, anti-GXM Abs. Mice immunized with P206.1 conjugated to various carriers did not mount an Ab response to GXM despite developing high anti-peptide titers. However, mice primed with GXM-TT and boosted with P206.1-TT developed significant anti-GXM titers (maximum, 1/180,000). This latter immunization scheme focused the immune response on protective epitopes, since only 2-5% of these titers were directed against nonprotective de-O-acetylated GXM epitopes compared with 20-60% in animals primed and boosted with GXM-TT.

Amino Acid Sequence↗
PubMed1995

Cryptococcus neoformans survive and replicate in human microglia.

BACKGROUND: Cryptococcus neoformans (CN) is an opportunistic pathogen that can cause fatal meningoencephalitis in patients with immune deficiency. Among the central nervous system (CNS) cells that are infected by CN are perivascular microglia and macrophages. Little is known about the interaction of CN and CNS phagocytes at the cellular level. EXPERIMENTAL DESIGN: To better understand the fate of CN in microglia, we followed Ab-opsonized CN in human fetal microglial culture by phase-contrast microscopy, combined lighted microscopy and transmission electron microscopy of plastic-embedded monolayers, and immunocytochemistry for localization of capsular Ag. RESULTS: Phase-contrast microscopy revealed that microglia initially internalized and contained Ab-opsonized yeast cells within phagolysosomes. However, CN escaped from microglia and resumed extracellular growth 16 to 24 hours after being phagocytosed. Transmission electron microscopy/1-mu epoxy sections revealed that intracellular CN were localized in two types of phagosomes in microglia: spacious phagosomes (SP) and close-fitting phagosomes (CP). Three lines of evidence indicate that SP are the primary sites for intracellular CN survival and replication: (a) SP contained multiple, budding yeast cells, whereas CP contained only single yeast cells within a tightly bound phagosomal membrane; (b) the number of SP and the number of CN within SP increased considerably at 24 hours compared with at 2 hours; and (c) microglial cultures challenged with heat- or amphotericin B-treated CN had significantly fewer SP than those challenged with live CN. Both SP and CP phagosomes fused with lysosomes, suggesting that CN survival in SP was not due to failure of phagolysosomal fusion. In SP, there was attenuation and diffusion of capsular polysaccharide within the phagosome, whereas in CP the fungal capsules remained compact and homogeneous. Immunocytochemistry with an mAb directed to capsular glucoronoxylomannan supported continued synthesis of polysaccharide within SP. CONCLUSION: The results suggest that, in human microglia, CN survive and replicate within SP. Modification of CN capsular polysaccharide within SP may be a contributing factor to this aberrant microglial-CN interaction.

Animals↗
PubMed2001

Flucytosine primary resistance in Candida species and Cryptococcus neoformans.

The in vitro activity of flucytosine (5FC) against 1,140 clinical isolates of Candida spp. and Cryptococcus neoformans was evaluated and compared with the activity of amphotericin B, fluconazole and itraconazole. Overall, 87.72% (1,000/1,140) of yeasts were susceptible to 5FC. This agent showed less potent in vitro activity against Candida glabrata, Candida krusei, Candida guilliermondii and Cryptococcus neoformans (MIC90s, 8-16 microg/ml) and intermediate activity or resistance to 6.5% of Candida albicans, 5.1% of Candida tropicalis and 0.8% of Candida parapsilosis strains. Amphotericin B showed potent activity against isolates with an MIC of 5FC > or = 8 microg/ml. A total of 112 of 140 strains that were SFC-intermediate or -resistant showed decreased susceptibility to azoles (P < 0.01).

Antifungal Agents↗
PubMed1986

Cryptococcus neoformans: comparisons of in vitro antifungal susceptibilities of serotypes AD and BC.

Thirty-nine isolates of Cryptococcus neoformans, nineteen serotype AD and twenty serotype BC, were assayed for susceptibility to eight antifungal agents using an in vitro agar dilution assay. Media employed were Kimmig agar and yeast nitrogen base supplemented with 10% glucose. The antifungal agents used were ketoconazole, amphotericin B, 5-fluorocytosine, nystatin, miconazole, BAY N 7133, ICI 153,066, and itraconazole. No clinically significant differences in vitro minimum inhibitory concentrations were detected between serotypes AD and BC against any of the compounds tested. An adverse medium effect was observed in two of the assays, but the outcome of the AD/BC comparison was not affected. This is the first report in which the in vitro antifungal susceptibilities of Cryptococcus neoformans serotypes are analyzed.

Amphotericin B↗
PubMed1992

Aerosolized liposomal amphotericin B for treatment of pulmonary and systemic Cryptococcus neoformans infections in mice.

Cryptococcus infections of the lung and central nervous system have become major problems in immuno-compromised patients, leading to the need for additional treatment protocols. We have utilized a Cryptococcus-mouse model that mimics human cryptococcal disease to evaluate the efficacy of amphotericin B-liposomes (AmpB-Lip) when delivered by small-particle aerosol (SPA). In the model, initial intranasal inoculation leads to a pulmonary infection that spreads after 2 to 3 weeks to distant organs, including the brain. Aerosols of AmpB-Lip that were generated by a Collison nebulizer had mass median aerodynamic diameters of 1.8 microns and contained 10.3 micrograms of AmpB per liter. When AmpB-Lip SPA was begun at 24 h postinoculation, a single 2-h treatment (0.3 mg of AmpB per kg of body weight) was effective in reducing pulmonary Cryptococcus infection. This regimen was more effective than intravenous administration of AmpB-Lip given for 3 continuous days. This single 2-h exposure to AmpB-Lip also was effective in reducing pulmonary Cryptococcus infection when treatment was delayed for 7 or 14 days. At day 21, when organisms had spread to the brain in all animals, the single 2-h aerosol treatment reduced the number of cryptococci in the brain as well as in the lungs. AmpB-Lip SPA administered once for 2 h on days 7, 14, and 21 also was effective in increasing the duration of survival of infected animals. These results demonstrate that aerosolized AmpB-Lip can be effective in treating both local, pulmonary Cryptococcus disease and systemic disease.

Aerosols↗
PubMed2004

Detection of Cryptococcus neoformans var. grubii in honeybee (Apis mellifera) colonies.

The plant flora has an important role in the ecology of Cryptococcus neoformans. It is estimated that the environmental spreading and contamination of human beings with this yeast occurs via contaminated particles of plants. Cultivation of canopy parts of plants in selective media is the most widely used isolation method of this yeast. Cryptococcus neoformans var. grubii was isolated from honeybee colonies in Eucalyptus forests but was not isolated from the places where this flora did not exist. Our results indicate that the occurrence of C. neoformans in honeybee colonies during the flowering season of Eucalyptus spp. trees can be an important bioindicator for environmental yeast presence. The screening of honeybee colonies is a practical and a rapid method for the monitoring of the C. neoformans presence in flowering plants.

Animals↗
PubMed2001

Glyphosate inhibits melanization of Cryptococcus neoformans and prolongs survival of mice after systemic infection.

Cryptococcus neoformans is a major fungal pathogen and is a relatively common cause of life-threatening meningoencephalitis. Glyphosate is a widely used herbicide that inhibits the synthesis of aromatic amino acids via the shikimate acid pathway. This study investigated the effects of glyphosate on C. neoformans growth, melanization, and murine infection. C. neoformans was relatively resistant to glyphosate, requiring concentrations >250 microg/mL for inhibition. Melanization of C. neoformans in the presence of L-dopa was inhibited by subinhibitory concentrations of glyphosate. Glyphosate inhibited autopolymerization of L-dopa and oxidation of L-epinephrine by cryptococcal cells, which is mediated by a laccase. Administration of glyphosate to mice infected with C. neoformans delayed melanization of yeast cells in vivo and prolonged average mouse survival. The results suggest that inhibition of melanization in vivo may facilitate control of C. neoformans infection.

Animals↗
PubMed1981

Particle size of airborn Cryptococcus neoformans in a tower.

Nearly 10(6) cells of Cryptococcus neoformans were cultured per g of pigeon droppings in a vacant tower. The air in the tower contained an average of 45 viable cells of C. neoformans per 100 liters: 60% of the cells were less than 4.7 micron in diameter. It is estimated that a human exposed to this atmosphere for 1 h would have 41 cells of c. neoformans deposited in the lungs. Sweeping resulted in the aerosolization of large numbers of cells of C. neoformans from 4.7 to 11 micron in diameter, the number of cells less than 4.7 micron remained relatively constant. One minute after sweeping, 4.4% of viable airborne cells of C. neoformans were less than 1.1 micron in diameter. We believe that this is the first report of isolating such small cells of C. neoformans from a natural site.

Aerosols↗
PubMed1990

Comparison of Guizotia abyssinica seed extract (birdseed) agar with conventional media for selective identification of Cryptococcus neoformans in patients with acquired immunodeficiency syndrome.

Growth of Cryptococcus neoformans from the sputum of patients with acquired immunodeficiency syndrome may be obscured by oral contamination with Candida albicans on conventional media. We prospectively compared direct plating of sputum and urine onto birdseed agar and compared birdseed agar plating with plating onto Mycosel and Sabouraud dextrose agar cultures. Thirty-two sputum and three urine specimens were compared. C. neoformans was isolated from five specimens. In two specimens, one of sputum and one of urine, C. neoformans was detected only on the birdseed agar plate because of overgrowth on the conventional media by C. albicans. C. neoformans produced dark colonies on birdseed agar, unlike C. albicans, which produces white colonies. The use of birdseed agar as the primary culture medium for sputum and urine specimens from patients with acquired immunodeficiency syndrome increases sensitivity for C. neoformans.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Isotype switching from IgG3 to IgG1 converts a nonprotective murine antibody to Cryptococcus neoformans into a protective antibody.

Passively administered mAbs to Cryptococcus neoformans capsular polysaccharide can alter the course of infection in mouse models. In preliminary studies of passive Ab efficacy, most IgM, IgA, and IgG1 mAbs were protective, but the few IgG3 mAbs tested did not confer significant protection. Because IgG3 is effective in pneumococcal infections, this phenomenon was examined more rigorously by generating an IgG1 switch variant from the non-protective IgG3 mAb 3E5 and comparing its protective efficacy in a murine model of i.v. infection by using strains of both the A and D serotypes. The 3E5 IgG3 mAb did not prolong survival or reduce organ fungal burden. Rather, the IgG3 decreased survival relative to controls. In contrast, the IgG1 switch variant of 3E5 significantly prolonged survival, reduced organ colony-forming units, and reduced serum polysaccharide Ag level in infected mice. The results establish that isotype is important for Ab efficacy against C. neoformans.

Animals↗
PubMed1993

Identification of the perfect state of Cryptococcus neoformans from 195 clinical isolates including 84 from AIDS patients.

Filobasidiella neoformans is the teleomorphic state of Cryptococcus neoformans and it is a heterothalic. The purpose of this study was to establish the proportions of each mating types (a, alpha) from among 195 strains of C. neoformans isolated from clinical material. The culture medium used was sunflower agar. Cultures were incubated at 20-22 degrees C for 15 days and observed periodically for one month. Non-reactive strains were mated several times with different reactive strains. Under these conditions 96.8% of the strains were found to be reactors. Among both varieties of C. neoformans, mating type alpha was found to have the highest frequency of 95% in the variety neoformans and 84% in the variety gattii. These results showed a higher reactivity in comparison with other investigators. This difference could be due to the medium used or to repeated mating with different reactive tested strains.

AIDS-Related Opportunistic Infections↗