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PubMed2001

A nuclear-encoded intein in the fungal pathogen Cryptococcus neoformans.

We have used comparative sequence analysis to identify an intein-like sequence (protein splicing element) present in Cryptococcus neoformans, a fungal pathogen of humans. The sequence encoding this element is present in the C. neoformans PRP8 gene, as an in-frame insertion relative to the PRP8 genes of other organisms. It contains sequences similar to those of the protein-splicing domains of two previously described yeast inteins (in Saccharomyces cerevisiae and Candida tropicalis), although it lacks any recognizable internal endonuclease domain. The Cryptococcus neoformans intein (Cne PRP8) is only the second to be found in a eukaryote nuclear genome; the previously described yeast inteins occur at the same site in the VMA gene homologues of S. cerevisiae and C. tropicalis. The host gene of the Cryptococcus intein, PRP8, encodes a highly conserved mRNA splicing protein found as part of the spliceosome. The Cne PRP8 intein may be a useful drug target in addressing the cryptococcal infections so prevalent in AIDS patients.

Amino Acid Sequence↗

PubMed2004

Efficacy of immune sera from human immunoglobulin transgenic mice immunized with a peptide mimotope of Cryptococcus neoformans glucuronoxylomannan.

The efficacy of antibody mediated immunity against Cryptococcus neoformans has not been established experimentally for human antibodies. Our group has previously shown that immunization with a conjugate consisting of a peptide mimotope of the C. neoformans capsular polysaccharide glucuronoxylomannan (GXM), P13, and diphtheria toxoid (P13-DT) prolonged survival of transgenic mice with human immunoglobulin loci, XenoMouse mice, which were challenged with a lethal dose of C. neoformans. In the study reported herein, we determined the efficacy of human antibodies in the sera of immunized XenoMouse mice against C. neoformans in passive transfer experiments in naïve BALB/c mice. Survival studies were performed with sera from XenoMouse mice expressing human IgG2/kappa (G2/k mice) or IgG4/kappa (G4/k mice) that had been immunized with P13-tetanus toxoid (TT)/Alhydrogel with or without CpG, and G2/k mice that had been immunized with P13-DT/Alhydrogel/CpG or Alhydrogel/CpG, obtained on day 7 (early sera) and days 30 or 35-59 (late sera) after primary immunization. Compared to mice receiving sera from G2/k-PBS-treated mice, the survival of naïve mice was prolonged by both early and late sera from G2/k-P13-DT/Alhydrogel/CpG-immunized mice, but only late sera from G2/k-P13-TT/Alhydrogel/CpG-immunized mice. Late, but not early sera from G2/k-Alhydrogel/CpG-immunized mice also prolonged survival. For all sera, prolongation of survival was associated with GXM-specific serum IgM. Sera from G2/k mice that received P13-TT without CpG, and all groups of G4/k mice had low to undetectable levels of antibody to GXM and were not protective. Our findings suggest that GXM-specific human IgM may be a functional mediator of protection against C. neoformans.

Adjuvants, Immunologic↗

PubMed1996

In vitro susceptibilities of clinical and environmental isolates of Cryptococcus neoformans to five antifungal drugs.

A total of 53 Cryptococcus neoformans strains, including clinical and environmental Brazilian isolates, were tested for their susceptibilities to amphotericin B, 5-flucytosine, ketoconazole, fluconazole, and itraconazole. The tests were performed according to the National Committee of Clinical Laboratory Standards recommendations (document M27-P). In general, there was a remarkable homogeneity of results for all strains, and comparable MICs were found for environmental and clinical isolates. This paper represents the first contribution in which susceptibility data for Brazilian C. neoformans isolates are provided.

AIDS-Related Opportunistic Infections↗

PubMed1981

Pseudohyphal forms of Cryptococcus neoformans: decreased survival in vivo.

Three pseudohyphal isolates of Cryptococcus neoformans were inoculated intracranially into mice. Four weeks post-inoculation the animals showed no symptoms of disease and the number of viable cells per brain decreased to zero. Possible roles of pseudohyphal forms of C. neoformans in the immunology and pathogenesis of cryptococcosis are discussed.

Animals↗

PubMedDate not supplied

Differential killer toxin sensitivity patterns of varieties of Cryptococcus neoformans.

Ten different killer sensitivity types are distinguished within Cryptococcus neoformans, namely four in var. neoformans and six in var. gattii. All strains of the var. gattii investigated were inhibited by killer toxins of C. laurentii CBS 139, whereas those of the var. neoformans were not. Killer sensitivity patterns are an easy-to-use method to differentiate between the two varieties of the clinically important yeast C. neoformans, and may be of help in epidemiological surveys.

AIDS-Related Opportunistic Infections↗

PubMed1994

Susceptibility of melanized and nonmelanized Cryptococcus neoformans to nitrogen- and oxygen-derived oxidants.

Melanized Cryptococcus neoformans cells were less susceptible than nonmelanized cells to the fungicidal effects of nitrogen- and oxygen-derived oxidants. The results support the hypothesis that the phenoloxidase enzyme system contributes to virulence by protecting C. neoformans against nitrogen- and oxygen-derived oxidative antimicrobial molecules produced by immune effector cells.

Antifungal Agents↗

PubMed1983

Mating types and serotypes of Cryptococcus neoformans isolated in Japan.

Thirty-two isolates of Cryptococcus neoformans from patients and one from the wild obtained in Japan were characterized for their serotype, self-fertility, and mating behaviour by crossing them with two mating types of Filobasidiella neoformans var. neoformans and F. neoformans var. bacillispora. Of the 32 isolates from patients, 31 were of serotype A and the remaining one was of serotype D. Although these 32 isolates were all self-sterile, 23 serotype A and one serotype D isolates produced a complete sexual state when mixed with the alpha mating type of F. neoformans var. neoformans. The one natural isolate was of serotype A-D and self-fertile. The Japanese clinical isolates of C. neoformans appear to be predominantly serotype A and alpha mating type of F. neoformans var. neoformans as is the case in the U.S.A.

Conjugation, Genetic↗

PubMed1994

Serotypes and mating types of clinical strains of Cryptococcus neoformans isolated in Taiwan.

Twenty-one strains of Cryptococcus neoformans isolated from patients in Taiwan were characterized for serotypes and mating types. Slide agglutination test was performed with 8 factor-specific sera (Iatron Company, Japan) to determine the serotypes. Wheat bran agar (WBA) and malt extract agar (MEA, Wickerham) media were used for the mating tests. Twenty of the isolates were of serotype A, and one was serotype B. Except for 2 strains of serotype A, all of the serotype A strains mated with Filobasidiella neoformans var. neoformans, mating type a. The only serotype B strain mated with F. neoformans var. bacillispora mating type a in MEA medium. These data revealed the low prevalence (1/21; 4.8%) of C. neoformans var. gattii in Taiwan, a subtropically located isoland.

Cryptococcosis↗

PubMed1992

[Detection of anti-Cryptococcus neoformans antibodies in 3 groups of human sera].

The tube agglutination (TA) technique was normalized in order to determine anti-Cryptococcus neoformans antibodies, according to Palmer et al., by employing positive and negative control sera and an antigenic suspension prepared from an autochtonous strain of Cryptococcus neoformans varneofornnans. Three groups of human sera were studied and the role of TA in detecting antibodies in the group of sera from patients with active cryptococcosis (100%) was shown. In sera from banks positiveness was only 6%, while in the group of fowl breeders, considered to be "exposed", positiveness was found to be 16%, although with low titres. A discussion is carried out about the value of this technique as a complement in the diagnosis and prognosis of cryptococcosis.

Agglutination↗

PubMed1998

Comparative evaluation of National Committee for Clinical Laboratory Standards broth macrodilution and agar dilution screening methods for testing fluconazole susceptibility of Cryptococcus neoformans.

A simple screening method for fluconazole susceptibility of Cryptococcus neoformans using 2% dextrose Sabouraud dextrose agar (SabDex) with fluconazole was compared to the National Committee for Clinical Laboratory Standards (NCCLS) broth macrodilution method. By this method, fluconazole-susceptible C. neoformans isolates are significantly smaller on medium with fluconazole than on fluconazole-free medium. Isolates with decreased susceptibility have normal-size colonies on medium containing fluconazole. The 48-h NCCLS broth macrodilution MICs (NCCLS MICs) for isolates with normal-size colonies on 8- or 16-microg/ml fluconazole plates were predicted to be > or =8 or > or =16 microg/ml, respectively. On medium with 16 microg of fluconazole per ml, all strains (84 of 84) for which the NCCLS MICs were <16 microg/ml were correctly predicted, as were all isolates (7 of 7) for which the MICs were > or =16 microg/ml. Agar dilution appears to be an effective screening method for fluconazole resistance in C. neoformans.

Antifungal Agents↗

PubMed1995

Detection of Cryptococcus neoformans in bronchial lavage cytology: report of four cases.

Four cases of pulmonary cryptococcosis were diagnosed by cytological detection of Cryptococcus neoformans in bronchial lavage. Three patients had underlying diseases, but not HIV infection. The chest X-rays showed 2 patients with nodular lesions and 2 with cavitary lesions. The cryptococcal antigen in the serum was positive in all four patients. In the cytology of bronchial lavage, Cryptococcus neoformans was detected after period-acid-Schiff (PAS) staining and was cultured in Sabouraud-dextrose agar. The cytology of bronchial lavage is useful for the rapid diagnosis of pulmonary cryptococcosis.

Adult↗

PubMed2005

Cryptococcus neoformans: a sugar-coated killer with designer genes.

Cryptococcus neoformans has become a common central nervous system pathogen as the immunocompromised populations enlarge world-wide. This encapsulated yeast has significant advantages for the study of fungal pathogenesis and these include: (1) a clinically important human pathogen; (2) a tractable genetic system; (3) advanced molecular biology foundation; (4) understanding of several virulence phenotypes; (5) well-studied pathophysiology; and (6) robust animal models. With the use of a sequenced genome and site-directed mutagenesis to produce specific null mutants, the virulence composite of C. neoformans has begun to be identified one gene at a time. Studies into capsule production, melanin synthesis, high temperature growth, metabolic pathways and a variety of signaling pathways have led to understandings of what makes this yeast a pathogen at the molecular level. Multiple principles of molecular pathogenesis have been demonstrated in virulence studies with C. neoformans. These include evolutionary differences between the varieties of C. neoformans in their genes for virulence, quantitative impact of genes on the virulence composite, species and site-specific importance of a virulence gene, gene expression correlation with its functional importance or phenotype and the impact of a pathogenesis gene on the host immune response. C. neoformans has now become a primary model to study molecular fungal pathogenesis with the goal of identifying drug targets or vaccine strategies.

Animals↗

PubMedDate not supplied

The ecology of Cryptococcus neoformans and the epidemiology of cryptococcosis.

The ecology of Cryptococcus neoformans and the epidemiology of cryptococcosis are reviewed. Two varieties of C. neoformans have been recognized. C. neoformans variety neoformans has been found in nature worldwide, primarily in association with bird droppings, although nonavian sources have also been found. Most cases of human cryptococcosis are caused by this variety. C. neoformans var. gattii has recently been isolated in nature in association with Eucalyptus trees. Infections caused by this variety occur mainly in tropical and subtropical regions. Because exposure to C. neoformans is probably common and clinically apparent cases of cryptococcosis in healthy hosts are rare, it is presumed that most people can mount adequate host defenses upon exposure to the organism. At least 5%-10% of patients with AIDS become infected with Cryptococcus; the epidemiology of this infection is different in many respects from that seen in patients without AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed1964

CHARACTERIZATION OF THE PYROGENICITY OF CANDIDA ALBICANS, SACCHAROMYCES CEREVISIAE, AND CRYPTOCOCCUS NEOFORMANS.

Kobayashi, George S. (Tulane University, New Orleans, La.), and Lorraine Friedman. Characterization of the pyrogenicity of Candida albicans, Saccharomyces cerevisiae, and Cryptococcus neoformans. J. Bacteriol. 88:660-666. 1964.-The intravenous injection into rabbits of 10(9) yeast cells of Candida albicans, Saccharomyces cerevisiae, or Cryptococcus neoformans (both slightly and heavily encapsulated forms) induced a febrile response indistinguishable from that elicited by gram-negative bacterial endotoxin. There was a brisk rise in body temperature which began as early as 30 min after injection, peaked once or twice, and then returned to normal after about 10 hr. With viable C. albicans, the febrile response did not return to normal but remained elevated for several days and terminated at death of the animal. Of three extraction procedures employed in attempts to isolate the endotoxin-like pyrogenically active substances from C. albicans, only one, the phenol extraction method, was successful. Pyrogenic substances were more easily extractable from S. cerevisiae, but extracted cells of both species were still highly pyrogenic. It was concluded that the particulate nature of the yeast cell did not contribute to the induction of fever, for latex particles of a similar size were nonpyrogenic. Viable or heat-killed C. albicans, phenol extract of C. albicans, zymosan, and polystyrene latex particles all failed to induce in rabbits increased dermal reactivity to epinephrine.

Animals↗

PubMed1997

The in vitro interaction of Cryptococcus neoformans with human lung epithelial cells.

The interaction of Cryptococcus neoformans with a human lung epithelial cell line (A549) is described. Encapsulated and acapsular strains adhered to epithelial cells in a time-dependent manner, with the acapsular strain being the most adherent under all conditions tested. Internalized cryptococci were additionally observed. The expression of the adhesins responsible for adherence to the epithelial cells was induced by growth at 37 degrees C. Adhesin expression was repressed in all strains by growth with sucrose as the sole carbon source. A strain-specific repression of adhesin expression was observed after growth with galactose and xylose. A variety of carbohydrates included in the assay suspensions blocked adherence, implicating certain carbohydrate moieties that might serve as ligands for the yeast adhesin. Finally, a monoclonal antibody is described that inhibited cryptococcal adherence to the epithelial cells. Collectively, the results demonstrate a specific interaction between C. neoformans and lung epithelial cells mediated by yeast adhesins whose expression is regulated by environmental factors.

Adhesiveness↗

PubMed1995

Effects of the two varieties of Cryptococcus neoformans cells and culture filtrate antigens on neutrophil locomotion.

Cryptococcus neoformans var. gattii (serotype B and C) isolates have a relative predilection for immunocompetent hosts, and C. neoformans var. neoformans (serotype A and D) isolates have a relative predilection for immunocompromised hosts, suggesting that normal host resistance to the former may be relatively inefficient compared with that to the latter variety. In order to assess the possibility that normal cellular host defense is inadequate in protecting against C. neoformans var. gattii, we compared the two varieties of C. neoformans cells and their culture filtrate antigens (CneF) with respect to effects on neutrophil (polymorphonuclear leukocyte [PMN]) locomotion. In a 48-well modified Boyden chamber, the cells and CneF of C. neoformans var. neoformans (serotype A and D) isolates stimulated chemotaxis and chemokinesis of human PMN and activated a complement component(s) in pooled human serum to become a chemoattractant(s) for human PMN. In contrast, the cells and CneF of C. neoformans var. gattii (serotype B and C) isolates did not stimulate chemotaxis or chemokinesis in human PMN but rather inhibited chemokinesis and chemotactic responses of PMN to pooled human serum and formylmethionyl leucyl phenylalanine. Neither of the CneF from the C. neoformans var. gattii isolates was cytotoxic to PMN. Furthermore, with the mouse model, we found that CneF from C. neoformans var. neoformans caused migration of PMN into gelatin sponges implanted in naive and immunized mice, whereas CneF from C. neoformans var. gattii inhibited PMN migration into sponges. Our results, combined with findings of others showing reduced PMN infiltration in lungs of mice infected with C. neoformans var. gattii compared with PMN infiltration in lungs of mice infected with C. neoformans var. neoformans, indicate that the relative inadequacy of normal host resistance mechanisms to prevent infection with C. neoformans var. gattii results, in part, from inhibition of PMN migration to the site of the organism.

Animals↗

PubMed2006

Superoxide dismutase in Cryptococcus neoformans varieties gattii, grubi, and neoformans.

Some clear dissimilarities occur among the varieties of Cryptococcus neoformans but there are few studies about the differences among individual yeast antioxidant enzymes. The total superoxide dismutase (SOD) activities and the copper, zinc-depend SOD (Cu,ZnSOD) and manganese-dependent SOD (MnSOD) isoenzymes of five reference C. neoformans strains belonged to A, B, C, AD and D serotypes (Table I) and other nine C. neoformans isolates (Table II) were determined. There were significant differences (p < 0.01 and p < 0.05) in total SOD activity among the varietie gattii (serotype C) and the other varieties. Cu,ZnSOD showed difference (p < 0.05) between A and D serotypes. These results point out a variety and serotype-independent SOD activity in C. neoformans reference strains and the other isolates that were evaluated.

Cryptococcus neoformans↗

PubMedDate not supplied

Enhanced activity of antifungal drugs by lysozyme against Cryptococcus neoformans.

The in vitro susceptibility of 16 isolates of Cryptococcus neoformans to three antifungal drugs and lysozyme in combination was determined using an urea broth microdilution method. The antifungal activities of each drug alone against 16 isolates of Cr. neoformans were determined as mean minimal inhibitory concentrations (MICs). MICs of fluconazole, itraconazole and terbinafine were 2.0 micrograms ml-1, 0.004 microgram ml-1 and 0.25 microgram ml-1, respectively. Lysozyme alone inhibited the growth of Cr. neoformans in a dose-dependent manner, although the lysozyme was unable to kill the cells of Cr. neoformans at the highest concentration of 20 micrograms ml-1. The mean MICs of fluconazole, itraconazole and terbinafine in combination with lysozyme were 0.13 microgram ml-1, 0.004 microgram ml-1 and 0.03 microgram ml-1 respectively. The antifungal activity of fluconazole and terbinafine in combination with lysozyme against Cr. neoformans was greatly enhanced compared with that of each drug alone. Itraconazole was unable to enhance the antifungal activity, as it demonstrated higher activity against Cr. neoformans when alone rather than in combination. Lysozyme was confirmed to enhance the antifungal activity of fluconazole and terbinafine in vitro.

Antifungal Agents↗