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PubMed2005

Antifungal susceptibilities of clinical and environmental isolates of Cryptococcus neoformans in Goiânia city, Goiás, Brazil.

We evaluated the antifungal activities of amphotericin B, fluconazole, itraconazole and voriconazole in 70 Cryptococcus neoformans strains obtained from cerebrospinal fluid from AIDS patients and 40 C. neoformans strains isolated from the environment. Four clinical isolates were identified as C. neoformans var. gattii. The susceptibility test was done using a broth microdilution method according to NCCLS M27-A2. Range minimal inhibitory concentrations (MICs) for C. neoformans clinical isolates were 0.06-1.0 microg/mL for amphotericin B, 0.125-8 microg/mL for fluconazole, 0.03-0.5 microg/mL for itraconazole and 0.03-0.25 microg/mL for voriconazole. C. neoformans environmental isolates showed range MICs 0.015-0.125 microg/mL, 0.25-2.0 microg/mL, 0.007-0.125 microg/mL and 0.03-0.25 microg/mL for amphotericin B, fluconazole, itraconazole and voriconazole respectively. The MICs results obtained from clinical and environmental isolates showed similar pattern of susceptibility and no resistance has been found in our isolates.

Amphotericin B↗
PubMed1999

Hypercalcemia associated with infection by Cryptococcus neoformans and Coccidioides immitis.

BACKGROUND: Of the 13 reported cases of hypercalcemia associated with fungal infection, 1 was caused by Cryptococcus neoformans and probably mediated by increased levels of 1,25-dihydroxyvitamin D [1,25(OH)2D]. Eight others were associated with Coccidioides immitis, of which only 2 had measured 1,25(OH)2D levels; in both, they were diminished. We report a patient with human immunodeficiency virus infection and simultaneous C. immitis and C. neoformans pneumonia and C. immitis fungemia associated with hypercalcemia. METHODS: Consecutive measurements of serum total and ionized calcium, phosphorous, blood urea nitrogen, creatinine, 25(OH)D, 1,25(OH)2D, parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrp) and albumin were performed over a period of 46 months. RESULTS: While the patient was hypercalcemic, intact serum PTH and PTHrp were undetectable, serum 25(OH)D levels were normal, and serum 1,25(OH)2D levels were in the high normal range. Successful treatment of the C. immitis and C. neoformans infections resulted in resolution of the hypercalcemia and increase of PTH and PTHrp to the normal range. CONCLUSION: In some patients with HIV infection, coincident hypercalcemia, and severe fungal infection, the responsible factor may be 1,25(OH)2D. Although total serum levels of this compound may not be frankly elevated, they are inappropriately high for the circumstances. Free 1,25(OH)2D levels should be determined in this situation.

AIDS-Related Opportunistic Infections↗
PubMed1994

Sterol composition of Cryptococcus neoformans in the presence and absence of fluconazole.

Analysis of the sterol compositions of 13 clinical isolates of the pathogenic yeast Cryptococcus neoformans obtained from five patients with recurring cryptococcal meningitis showed that, unlike Candida albicans, the major sterols synthesized by this yeast were obtusifoliol (range, 21.1 to 68.2%) and ergosterol (range, 0.0 to 46.5%). There was considerable variation in the sterol contents among the 13 isolates, with total sterol contents ranging from 0.31 to 5.9% of dry weight. The isolates from the five patients who had relapses had different total sterol contents and compositions in comparison with those of the pretreatment isolates, indicating either that the sterols had been changed by therapy or that the patients were infected with new isolates with different sterol compositions. Growth of the cryptococcal isolates in the presence of subinhibitory concentrations of fluconazole (0.25x the MIC) significantly altered the sterol content and pattern. The total sterol content decreased in nine isolates and increased in four isolates in response to pretreatment with fluconazole. Fluconazole had no consistent effect on ergosterol levels. In contrast, fluconazole caused a decrease in obtusifoliol levels and an increase in 4,14-dimethylzymosterol levels in all isolates. These results indicate extensive diversity in sterol content, sterol composition, and sterol synthesis in response to subinhibitory concentrations of fluconazole in C. neoformans strains. We propose that fluconazole inhibits the sterol synthesis of C. neoformans by interfering with both 14 alpha-demethylase-dependent and -independent pathways. No correlation between the sterol compositions of C. neoformans isolates and their susceptibilities to fluconazole was found.

Cholestadienols↗
PubMed2006

Ecto-glycanases and metabolic stability of the capsule in Cryptococcus neoformans.

We have identified a number of ecto-glycanases (glycosylhydrolases) associated with the capsule and/or the cell wall of Cryptococcus neoformans. The enzyme activities detected included alpha-mannosidase, alpha-, and beta -glucosidase, alpha-, and beta-galactosidase, beta-xylosidase, beta-glucuronidase, and endo-beta-1,3-glucanase. Small portions of the enzymes were also secreted into the growth medium. Cell-wall associated endo-beta-1,3-glucanases exhibited highest activity in the acidic range between pH 2.5 and 5.0. The products of laminarin hydrolysis by the enzymes located on the cell surface were glucose and beta-1,3-linked glucooligosaccharides. The same products were released from isolated cell walls incubated in the buffer. Endo-beta-1,3-glucanase activity extracted from the cell surfaces by mild sonication consisted of six isoforms separable by isoelectric focusing. In spite of the presence of the whole array of glycanase activities on the cell surfaces, capsular polysaccharides released from C. neoformans cells into the growth medium were practically metabolically stable. From the defined polysaccharides tested, only laminarin (beta-1,3-glucan) and to some extent also mixed-linkage beta-1,3/beta-1,4-glucan and/or 4-O-methyl-D-glucurono-D-xylan were able to support the yeast growth. The activities of majority of identified ecto-glycanases were low when the yeast was grown on glucose but were considerably elevated when the cells were grown on glycerol indicating that their synthesis is regulated by catabolite repression.

Cell Wall↗
PubMed2001

Are two Cryptococcus neoformans strains epidemiologically linked?

The aim of this study was to standardize a method to determine whether two strains of Cryptococcus neoformans could be considered epidemiologically linked. We hypothesized that strains isolated from the same patient were epidemiologically linked and that those isolated from different patients were unrelated. We used 17 environmental isolates and 97 clinical isolates from 31 patients diagnosed with cryptococcosis (1 to 14 isolates per patient). Using the plasmid pCnTel-1-labeled probe CENTEL, we were able to differentiate some unrelated strains that yielded the same hybridization profile with the C. neoformans middle-repetitive-element CNRE-1 probe. The genetic distances separating the strains isolated from the same patient and those separating the strains isolated from different patients were estimated, and the results obtained with the two probes were compared. Analysis of the results enabled the calculation of two Dice coefficient limits defining the zones containing the pairs of linked strains and the pairs of unrelated strains, as well as an intermediate uncertainty zone for which it was not possible to establish whether the pairs of strains were linked.

Cryptococcosis↗
PubMed2001

An immunohistochemical method that differentiates Cryptococcus neoformans varieties and serotypes in formalin-fixed paraffin-embedded tissues.

An immunohistochemical method for determining the variety of Cryptococcus neoformans in formalin-fixed paraffin-embedded tissues was developed using mAbs 471, 302 and CRND8. The method was validated primarily using veterinary patients for which both formalin-fixed lesions and a cultured isolate were available. L-Canavanine glycine bromothymol blue (CGB) agar and the 'Crypto-Check' kit were used to determine the variety and serotype, respectively, of cultured isolates. Immunohistochemistry accurately predicted the C. neoformans variety in all tissue specimens. The CGB agar method of determining C. neoformans variety gave the same result as immunohistochemistry for 30/31 specimens. For the single discordant isolate, the serotype, random amplification of polymorphic DNA profile, microscopic and colony morphology all supported the immunohistochemical staining pattern in suggesting C. neoformans var. gattii; however, the CGB agar result was at variance. Of the C. neoformans var. neoformans cases, immunohistochemistry was congruent with variety for 13/13 cases and with serotyping for 10/13 cases. The three discordant cases were classified as having some serotype D reactivity by immunohistochemistry, but were considered to be serotype A using the Crypto-Check kit. This new method should prove a valuable epidemiological tool in studies of cryptococcosis, especially in the veterinary setting where archival tissue specimens may exist but corresponding mycological data is typically absent. The versatility of this method will expand in the future as other monoclonal antibodies with different specificities are developed.

Antibodies, Monoclonal↗
PubMed2002

Anti-CD11 b monoclonal antibody suppresses brain dissemination of Cryptococcus neoformans in mice.

To elucidate the role of the beta2 integrin family of adhesion molecules in the disseminated infection of Cryptococcus neoformans from the lung to the central nervous system, we examined the effects of monoclonal antibodies (mAbs) against CD11a, CD11b, CD11c and CD18 on the number of live microorganisms in both the lung and brain of mice three weeks after intratracheal infection. Administration of anti-CD11b mAb partially, but reproducibly, reduced the fungal loads in the brain in three independent experiments, while the lung loads were not affected. In addition, the same treatment significantly decreased the number of live microorganisms in the blood. In sharp contrast, the brain loads one week after intravenous injection with C. neoformans were not affected by treatment with anti-CD11b mAb. Finally, administration of mAb against other adhesion molecules (CD11a, CD11c or CD18) failed to affect the fungal loads in the brain as well as in the lung three weeks after intratracheal instillation, except for anti-CD18 mAb which rather increased the brain loads. Our results suggested that CD11b might be involved at least in part in the process of fungal dissemination from lung to brain, although the significance of other beta2 integrin family adhesion molecules remains to be substantiated.

Animals↗
PubMed2005

The structure of Cryptococcus neoformans thymidylate synthase suggests strategies for using target dynamics for species-specific inhibition.

The ternary complex crystal structures of Cryptococcus neoformans and Escherichia coli thymidylate synthase (TS) suggest mechanisms of species-specific inhibition of a highly conserved protein. The 2.1 Angstrom structure of C. neoformans TS cocrystallized with substrate and the cofactor analog CB3717 shows that the binding sites for substrate and cofactor are highly conserved with respect to human TS, but that the structure of the cofactor-binding site of C. neoformans TS is less constrained by surrounding residues. This feature might allow C. neoformans TS to form TS-dUMP-inhibitor complexes with a greater range of antifolates than human TS. 3',3''-Dibromophenol-4-chloro-1,8-naphthalein (GA9) selectively inhibits both E. coli TS and C. neoformans TS (K(i) = 4 microM) over human TS (K(i) >> 245 microM). The E. coli TS-dUMP-GA9 complex is in an open conformation, similar to that of the apoenzyme crystal structure. The GA9-binding site overlaps the binding site of the pABA-glutamyl moiety of the cofactor. The fact that human apoTS can adopt an unusual fold in which the GA9-binding site is disordered may explain the poor affinity of GA9 for the human enzyme. These observations highlight the critical need to incorporate multiple target conformations in any computational attempt to facilitate drug discovery.

Algorithms↗
PubMedDate not supplied

Superoxide dismutase of Cryptococcus neoformans: purification and characterization.

We have purified to homogeneity a putative superoxide dismutase of 19.5 kDa from the pathogenic yeast Cryptococcus neoformans by homogenization, isoelectric focusing and gel filtration. The N-terminal amino acid sequence of this protein indicates a significant sequence homology with known manganese-containing superoxide dismutases (Mn-SODs) from various organisms. In addition, the presence of superoxide dismutase activity was confirmed using specific substrate gels which detect this enzyme when nitro-tetrazolium blue reduction is prevented by the photochemical source of superoxide, in the presence of riboflavin when exposed to light. Superoxide dismutase activity was also assayed using cytochrome c. The molecular weight of the native enzyme (on non-denaturing gels) is 80 kDa. The optimum pH for the enzyme is 7.5 and its pi = 6.6. The enzyme was inhibited by sodium dodecyl sulphate, sodium azide, o-phenanthroline, and EDTA, in descending order.

Amino Acid Sequence↗
PubMed2002

Urokinase-type plasminogen activator is required for the generation of a type 1 immune response to pulmonary Cryptococcus neoformans infection.

Urokinase-type plasminogen activator (uPA)(-/-) mice cannot mount protective host defenses during infection with the opportunistic yeast Cryptococcus neoformans (52D). Because effective host defense against C. neoformans requires specific immune responses and the generation of type 1 (T1) cytokines, we determined how the absence of uPA impacts these processes. Wild-type (WT) and uPA(-/-) mice were inoculated with C. neoformans. Macrophage antifungal activity was assessed histologically, T lymphocyte responses in vivo and proliferation in vitro were quantified, and cytokine concentrations were determined by ELISA. uPA(-/-) macrophages have impaired antimicrobial activity. Regional lymph nodes of infected uPA(-/-) mice contained fewer cells than WT, suggesting impaired T cell proliferation in response to the pathogen in vivo. In vitro, uPA(-/-) T lymphocytes had impaired proliferative responses to C. neoformans rechallenge compared with WT. Infected WT mice generated T1 cytokines in the lung, characterized by high levels of IFN-gamma and IL-12. uPA(-/-) mice had decreased levels of IFN-gamma and IL-12, and increased IL-5, a type 2 cytokine. In the absence of uPA, the cytokine profile of regional lymph nodes shifted from a T1 pattern characterized by IFN-gamma and IL-2 to a weak, nonpolarized response. We conclude that in the absence of uPA, lymphocyte proliferative responses are diminished, and mice fail to generate protective T1 cytokines, resulting in impaired antimicrobial activity. This study provides novel evidence that uPA is a critical modulator of immune responses and of immune cell effector functions in vivo.

Adoptive Transfer↗
PubMed2005

Evaluation of Mycograb, amphotericin B, caspofungin, and fluconazole in combination against Cryptococcus neoformans by checkerboard and time-kill methodologies.

This article reported the identification of heat shock protein 90 (hsp90) homologues by immunoblot in Cryptococcus neoformans. Mycograb, a genetically recombinant antibody against hsp90, was evaluated against 8 clinical isolates and the National External Quality Assessment Service for Microbiology strain of C. neoformans alone and in combination with amphotericin B, caspofungin, and fluconazole by checkerboard assay. At the end point of an optically clear well, the minimum inhibitory concentration (MIC) 0's ranged from 256 to 1024 microg/mL for Mycograb, from 0.5 to 1 microg/mL for amphotericin B, and from 16 to 32 microg/mL for caspofungin. The combination of Mycograb and amphotericin B produced a fractional inhibitory concentration index from 0.27 to 0.56, indicating a mainly synergistic effect, whereas for caspofungin, it varied from 0.5 to 2. At an end point of > or =50% inhibition, the MIC-2s varied from 16 to 128 microg/mL for Mycograb and from 0.125 to 16 microg/mL for fluconazole. The fractional inhibitory concentration index classified the combination as indifferent for 5 isolates, additive for 3 more isolates, and synergistic in a single isolate. Time-kill analysis on 2 isolates (F/7844 and F/10156), which had synergistic and additive results with amphotericin B, respectively, on checkerboard was performed with 4-16 microg/mL of Mycograb, 2-8 microg/mL of fluconazole, and 0.0625-2 microg/mL of amphotericin B. This demonstrated an increasingly static effect with augmenting concentrations of fluconazole and an initial static effect with amphotericin B at lower concentrations, which became fungicidal as the level of drug increased. The addition of either 4 or 8 microg/mL of Mycograb to 0.5 microg/mL of amphotericin B with C. neoformans F/7844 changed a static effect to a fungicidal effect at 8 h with an increased killing of 1.2 logs at 48 h. With C. neoformans F/10156, the addition of 16 microg/mL of Mycograb to 0.25 microg/mL of amphotericin B produced a difference in killing from 1 logarithm after 4 h to 1.5 logarithms after 48 h. These data suggest that the combination of amphotericin B and Mycograb would be worth exploring in the treatment of infection due to C. neoformans.

Amphotericin B↗
PubMed2002

Killing of Caenorhabditis elegans by Cryptococcus neoformans as a model of yeast pathogenesis.

We found that the well-studied nematode Caenorhabditis elegans can use various yeasts, including Cryptococcus laurentii and Cryptococcus kuetzingii, as a sole source of food, producing similar brood sizes compared with growth on its usual laboratory food source Escherichia coli OP50. C. elegans grown on these yeasts had a life span similar to (C. laurentii) or longer than (C. kuetzingii) those fed on E. coli. However, the human pathogenic yeast Cryptococcus neoformans killed C. elegans, and the C. neoformans polysaccharide capsule as well as several C. neoformans genes previously shown to be involved in mammalian virulence were also shown to play a role in C. elegans killing. These included genes associated with signal transduction pathways (GPA1, PKA1, PKR1, and RAS1), laccase production (LAC1), and the alpha mating type. C. neoformans adenine auxotrophs, which are less virulent in mammals, were also less virulent in C. elegans. These results support the model that mammalian pathogenesis of C. neoformans may be a consequence of adaptations that have evolved during the interaction of C. neoformans with environmental predators such as free-living nematodes and amoebae and suggest that C. elegans can be used as a simple model host in which C. neoformans pathogenesis can be readily studied.

Animals↗
PubMed1986

The biochemical basis for the distinction between the two Cryptococcus neoformans varieties with CGB medium.

The biochemical basis for the reaction to canavanine-glycine-bromthymol blue (CGB) agar by Cryptococcus neoformans var. gattii and C. neoformans var. neoformans was investigated. All of the var. gattii isolates tested were found to utilize glycine as the sole source of carbon and nitrogen and were resistant to L-canavanine. Only 11% of the serotype D isolates of var. neoformans utilized glycine as the sole source of carbon and nitrogen, but these were all sensitive to canavanine. Nineteen percent of the serotype A isolates of var. neoformans were able to assimilate glycine, and 81% of the glycine users were resistant to canavanine. However, these canavanine-resistant, glycine-assimilating, var. neoformans isolates failed to grow when they were cultured on a medium containing glycine and canavanine. Unlike the var. neoformans isolates, all of the var. gattii isolates tested grew on a medium that contained both of these compounds. Glycine-utilizing isolates exhibited good uptake of the amino acid, and a glycine-cleaving enzyme was discernable in the isolate. The isolates that fail to utilize glycine accumulated the amino acid at a rate which was barely 15% of that seen in the glycine users, and no glycine-cleaving enzyme was apparent within the 48-hr incubation period. When a cell-free extract (which had been derived from a glycine-utilizing isolate), was incubated with 14C-labeled glycine, ammonia, radiolabeled CO2, and serine were produced. The glycine decarboxylase activity of the cell-free extract was found to be enhanced by the addition of dithiothreitol, tetrahydrofolate, pyridoxal phosphate, and nicotinamide adenine dinucleotide (NAD). The ammonia released during glycine cleavage seems to be responsible for the positive reaction on CGB medium.

Biological Transport↗
PubMedDate not supplied

Recovery of Cryptococcus neoformans from the nasopharynx of AIDS patients.

Nasopharyngeal swabbings, obtained from AIDS patients, were plated onto Niger seed agar containing antibiotics Cryptococcus neoformans was isolated from 35 out of 84 patients (41.7%) diagnosed as primary cryptococcal cases before antifungal administration, and 8 out of 86 (9.3%) cryptococcosis patients on antifungal therapy. The fungus could not be isolated from any of 447 samples from 194 AIDS patients not diagnosed with cryptococcosis. These findings are novel in that the presence of C. neoformans in AIDS patients at this site has never been looked at previously.

AIDS-Related Opportunistic Infections↗
PubMed1992

Isolation of Cryptococcus neoformans var. gattii from an Asian patient in France: evidence for dormant infection in healthy subjects.

We report the first case of cryptococcosis due to Cryptococcus neoformans var. gattii in France. This Cambodian, non-AIDS patient had been living in France for 23 years when he developed cryptococcosis due to C. neoformans serotype B in 1985. It is possible that he was infected back in Asia or several months before, in Zaire, where he lived between 1981 and 1984. To our knowledge, this is the second case providing clinical evidence for reactivation of latent infection with C. neoformans. The mechanism is probably similar to that for C. neoformans var. neoformans.

Adult↗
PubMed1989

Murine natural killer cell interactions with a fungal target, Cryptococcus neoformans.

Earlier investigations have shown that murine natural killer (NK) cells bind to and inhibit the growth of the fungal pathogen Cryptococcus neoformans in vitro and in vivo. To define the stages of NK cell-mediated inhibition of C. neoformans growth and the requirements for the completion of these stages, the events which lead to cryptococcal growth inhibition were compared with those previously elucidated for NK cell-mediated tumor cell lysis. Our data indicate that NK cell-cryptococci binding is a distinct event that precedes inhibition; is temperature independent, although it is slowed at 4 degrees C; and is Mg2+ dependent. In contrast to binding, NK cell-mediated cryptococcal growth inhibition is temperature, Mg2+, and Ca2+ dependent. The removal of Ca2+ by EDTA addition within 3 h after maximal NK cell-cryptococci binding significantly reduced cryptococcal growth inhibition, indicating that Ca2+ is required either late in the NK cell trigger stage or early in the inhibitory stage. These stages and requirements are similar to those previously demonstrated for the model of NK cell-mediated tumor cell lysis; however, the NK cell-cryptococci interactions are somewhat slower than the interactions which culminate in the lysis of the YAC-1 tumor cell targets. These results suggest that C. neoformans cells, although structurally distinct from the standard tumor cell targets, are capable of similar cell-to-cell interactions with NK effector cells as the tumor cell targets.

Animals↗