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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↗
PubMed1995

Electrophoretic karyotype and in vitro antifungal susceptibility of Cryptococcus neoformans isolates from AIDS patients.

Electrophoretic karyotype (EK) was used to type 13 clinical isolates of Cryptococcus neoformans from eight AIDS patients. All of the isolates were also tested for their in vitro susceptibilities to fluconazole, itraconazole, D0870, flucytosine, and amphotericin B by a broth macrodilution technique performed according to the National Committee for Clinical Laboratory Standards recommendations. Although all strains were isolated from a limited geographic area, DNA typing showed a wide genetic variation in this group of patients, yielding seven different patterns. Two patients in whom C. neoformans was isolated in the same time period shared similar EK profiles, suggesting the possibility of cross-infection. In three patients, sequential isolates were evaluated: in two of them, EK analysis showed the persistence of the same genotype throughout the infection, whereas from the third, two isolates of C. neoformans with two different DNA profiles were obtained. Despite the small number of strains considered in this study, our susceptibility data indicate that C. neoformans isolates are very susceptible to the new triazoles.

AIDS-Related Opportunistic Infections↗
PubMed2006

Multifocal choroiditis in disseminated Cryptococcus neoformans infection.

PURPOSE: To report an uncommon case of multifocal choroiditis as the result of disseminated Cryptococcus neoformans infection in a patient who is HIV-positive. DESIGN: Interventional case report. METHODS: A 27-year-old HIV-positive woman with fever, headache, and vomiting was examined. Lumbar puncture was performed and revealed C neoformans infection. Her condition evolved with sudden bilateral blindness and deafness. Ophthalmologic examination revealed multiple yellowish choroidal lesions in the posterior pole of both eyes. RESULTS: Postmortem examination showed disseminated C neoformans infection. Histologic examination of the eyes confirmed the presence of C neoformans in the choroiditis. CONCLUSION: Multifocal choroiditis in C neoformans infection is a rare ophthalmic manifestation. The recognition of this condition by ophthalmologists can help physicians to diagnose a disseminated and fatal disease.

AIDS-Related Opportunistic Infections↗
PubMed2004

Cloning and characterization of the lanosterol 14alpha-demethylase (ERG11) gene in Cryptococcus neoformans.

The ergosterol pathway in fungal pathogens is an attractive antimicrobial target because it is unique from the major sterol (cholesterol) producing pathway in humans. Lanosterol 14alpha-demethylase is the target for a major class of antifungals, the azoles. In this study we have isolated the gene for this enzyme from Cryptococcus neoformans. The gene, ERG11, was recovered using degenerate PCR with primers designed with a novel algorithm called CODEHOP. Sequence analysis of Erg11p identified a highly conserved region typical of the cytochrome P450 class of mono-oxygenases. The gene was present in single copy in the genome and mapped to one end of the largest chromosome. Comparison of the protein sequence to a number of major human fungal pathogen Erg11p homologs revealed that the C. neoformans protein was highly conserved, and most closely related to the Erg11p homologs from other basidiomycetes. Functional studies demonstrated that the gene could complement a Saccharomyces cerevisiae erg11 mutant, which confirmed the identity of the C. neoformans gene.

Algorithms↗
PubMed2005

Genomic organization and expression of 23 new genes from MATalpha locus of Cryptococcus neoformans var. gattii.

The pathogenic yeast Cryptococcus neoformans (Cn) causes cryptococcosis, a life-threatening disease of the brain. Molecular studies of Cn variety gattii have lagged behind other two varieties (var. grubii and var. neoformans) although they have distinct biology and disease patterns. We focused on gene discovery in MATalpha locus because it predominates in clinical strains. A var. gattii cosmid library was screened with DNA probes from other two varieties. Two positive clones were sequenced to identify ORFs based on similarities to known proteins, and to ESTs using bioinformatics, and manually by a curator. Approximately 76kb sequenced DNA revealed 23 genes and ORFs. The existence of predicted genes was verified by RT-PCR analyses designed to amplify spliced sequences. The results confirmed that the transcripts were expressed both at 30 and 37 degrees C. The var. gattii MATalpha locus genes showed rearrangements in order and orientation vis-a-vis other two varieties. Mating-specific genes showed higher nonsynonymous mutation rates, and gene trees showed var. gattii strains in a distinct clade. The identification of the largest number, thus far, of var. gattii structural genes should set the stage for future molecular pathogenesis studies.

Chromosome Mapping↗
PubMed2005

Carbonic anhydrase and CO2 sensing during Cryptococcus neoformans growth, differentiation, and virulence.

The gas carbon dioxide (CO2) plays a critical role in microbial and mammalian respiration, photosynthesis in algae and plants, chemoreception in insects, and even global warming . However, how CO2 is transported, sensed, and metabolized by microorganisms is largely not understood. For instance, CO2 is known to induce production of polysaccharide capsule virulence determinants in pathogenic bacteria and fungi via unknown mechanisms . Therefore, we studied CO2 actions in growth, differentiation, and virulence of the basidiomycetous human fungal pathogen Cryptococcus neoformans. The CAN2 gene encoding beta-carbonic anhydrase in C. neoformans was found to be essential for growth in environmental ambient conditions but dispensable for in vivo proliferation and virulence at the high CO2 levels in the host. The can2Delta mutant in vitro growth defect is largely attributable to defective fatty acid synthesis. CO2 was found to inhibit cell-cell fusion but not filamentation during sexual reproduction. The can2 mutation restored early mating events in high CO2 but not later steps (fruiting body formation, sporulation), indicating a major role for carbonic anhydrase and CO2/HCO3- in this developmental cascade leading to the production of infectious spores. Our studies illustrate diverse roles of an ancient enzyme class in enabling environmental survival of a ubiquitous human pathogen.

Base Sequence↗
PubMed2006

Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages.

Cryptococcus neoformans (Cn) is an encapsulated yeast that is a facultative intracellular pathogen and a frequent cause of human disease. The interaction of Cn with alveolar macrophages is critical for containing the infection , but Cn can also replicate intracellularly and lyse macrophages . Cn has a unique intracellular pathogenic strategy that involves cytoplasmic accumulation of polysaccharide-containing vesicles and intracellular replication leading to the formation of spacious phagosomes in which multiple cryptococcal cells are present . The Cn intracellular pathogenic strategy in macrophages and amoebas is similar, leading to the proposal that it originated as a mechanism for survival against phagocytic predators in the environment . Here, we report that under certain conditions, including phagosomal maturation, possible actin depolymerization, and homotypic phagosome fusion, Cn can exit the macrophage host through an extrusion of the phagosome, while both the released pathogen and host remain alive and able to propagate. The phenomenon of "phagosomal extrusion" indicates the existence of a previously unrecognized mechanism whereby a fungal pathogen can escape the intracellular confines of mammalian macrophages to continue propagation and, possibly, dissemination.

Actins↗
PubMed2004

In vitro activities of voriconazole, posaconazole, and fluconazole against 4,169 clinical isolates of Candida spp. and Cryptococcus neoformans collected during 2001 and 2002 in the ARTEMIS global antifungal surveillance program.

We examined the in vitro activities of voriconazole, posaconazole, and fluconazole against 3,932 isolates of Candida spp. and 237 isolates of Cryptococcus neoformans obtained from over 100 medical centers worldwide during 2001 and 2002. The MICs of the antifungal drugs were determined by broth microdilution tests performed according to the National Committee for Clinical Laboratory Standards (NCCLS) methods using RPMI 1640 as the test medium. Voriconazole and posaconazole were very active against Candida spp. (97-98% susceptible at MICs < or =1 microg/ml) and C. neoformans (98-100% susceptible at MICs < or =1 microg/mL). C. albicans (MIC90, 0.015-0.03 microg/ml) was the most susceptible species of Candida to both agents and C. glabrata (MIC90, 1-2 microg/mL) was the least susceptible. Both voriconazole and posaconazole were more active than fluconazole against all Candida spp. and C. neoformans. These results provide further evidence for the increased spectrum and potency of the new triazoles against a large and geographically diverse collection of opportunistic fungal pathogens.

Candida↗
PubMed2004

Clinical evaluation of a dried commercially prepared microdilution panel for antifungal susceptibility testing of five antifungal agents against Candida spp. and Cryptococcus neoformans.

A commercially prepared dried-broth microdilution panel (Sensititre, TREK Diagnostic Systems, Cleveland, OH) was compared with a reference frozen-broth microdilution panel for antifungal susceptibility testing of 728 clinical isolates of Candida spp. and 98 clinical isolates of Cryptococcus neoformans. The antifungal agents tested were amphotericin B, fluconazole, 5-fluorocytosine (5FC), itraconazole, and voriconazole. Microdilution testing was performed according to NCCLS recommendations. Minimum inhibitory concentration (MIC) endpoints were read visually after 48 hours of incubation (72 hours for C. neoformans) and were assessed independently for each microdilution panel. Discrepancies among MIC endpoints of no more than 2 log(2) dilutions were used to calculate the percentage of agreement. Overall levels of agreement between the study and reference panels were 98% for Candida spp. and 93% for C. neoformans. The agreement for each antifungal agent ranged from 96.6% for voriconazole to 99.4% for amphotericin B. The TREK dried microdilution panel appears to be a viable alternative to frozen-broth microdilution panels for testing of Candida spp. and C. neoformans.

Antifungal Agents↗
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↗
PubMed2006

Trypanosoma lewisi-induced immunosuppression: the effects on alveolar macrophage activities against Cryptococcus neoformans.

The immunosuppressive effect of Trypanosoma lewisi infection on alveolar macrophage (AM) activities against Cryptococcus neoformans was studied in an animal model. Two groups of rats were treated with T. lewisi and killed after 4 (4d-rats) and 7 days (7d-rats), respectively. A third group not given T. lewisi, served as control. AM were challenged in vitro with C. neoformans. Phagocytosis was assessed with a fluorescence method. Superoxide anion production was evaluated with the nitroblue tetrazolium (NBT) test. The survival of cryptococci was estimated by counting colony-forming units. The numbers of detached AM from culture plates were determined using a Bürker chamber. The NBT response, adhesion to plate surface and killing activity, but not the phagocytosis of AM from 4d-rats were significantly impaired compared to control or 7d-rats. Thus, T. lewisi causes transitory immunosuppressive effects on AM activities. This rapid T. lewisi immunosuppression model may be useful to study new approaches to anticryptococcal therapy.

Animals↗
PubMed2004

The human immunodeficiency virus (HIV) protease inhibitor indinavir directly affects the opportunistic fungal pathogen Cryptococcus neoformans.

Highly active antiretroviral therapy (HAART), that includes human immunodeficiency virus (HIV) protease inhibitors (PIs), has been remarkably efficacious including against some opportunistic infections. In this report we investigated the effect(s) of the PI indinavir on protease activity by Cryptococcus neoformans, an opportunistic fungal pathogen responsible for recurrent meningoencephalitis in AIDS patients. Indinavir was also tested for potential effects on other parameters, such as fungal viability, growth ability and susceptibility to immune effector cells. It was found that indinavir impaired cryptococcal protease activity in a time- and dose-dependent fashion. The phenomenon was similarly detectable in ATCC/laboratory strains and clinical isolates. C. neoformans growth rate was also significantly reduced upon exposure to indinavir, while fungal viability was not affected and mitochondrial toxicity not detected. Furthermore, as assessed by an in vitro infection model, indinavir significantly and consistently augmented C. neoformans susceptibility to microglial cell-mediated phagocytosis and killing. Overall, by providing the first evidence that indinavir directly affects C. neoformans, these data add new in vitro insights on the wide-spectrum efficacy of PIs, further arguing for the clinical relevance of HAART against opportunistic infections in AIDS.

Antifungal Agents↗
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↗
PubMed2004

Synthesis and immunogenicity of polysaccharide-protein conjugate composed of galactoglucoxylomannan of Cryptococcus laurentii.

Galactoglucoxylomannan (GalGXMan) antigen of Cryptococcus laurentii was conjugated to protein carrier by a simple one step reaction. The conjugate was immunogenic in rabbits and reinjection elicited booster response with significant increase of serum IgG (H+L) level. Induction of this Ig-isotype was confirmed in experiments with Protein A. Effectiveness of immune serum to inhibit the growth of C. laurentii was demonstrated. The results indicate that the prepared conjugate could be considered as effective immunogen with potential for incorporation in the vaccine.

Animals↗
PubMed2004

Isolation of a CDC28 homologue from Cryptococcus neoformans that is able to complement cdc28 temperature-sensitive mutants of Saccharomyces cerevisiae.

A partial cDNA fragment of the Cryptococcus neoformans homologue of the main cell cycle control gene CDC28/cdc2 was isolated using degenerate primer RT-PCR. A subsequent search in the C. neoformans genome database identified several sequences similar to CDC28/cdc2. A part of the sequence which showed the highest similarity to CDC28/cdc2 turned out to be identical to the partial cyclin-dependent kinase (Cdk) cDNA fragment isolated by degenerate RT-PCR. The full-length coding region of this Cdk homologue was amplified by RT-PCR using primers designed to target regions around start and stop codons, and the gene was named CnCdk1. To determine its function, an analysis of deduced amino acid sequence of the CnCdk1 was performed and its ability to rescue Saccharomyces cerevisiae cdc28-temperature sensitive mutants was tested. S. cerevisiae cdc28-4 and cdc28-1N strains transformed with the pYES2- CnCdk1 construct exhibited growth at 36.5 degrees C in galactose-raffinose medium, but not in glucose medium. Results of the sequence analysis and the fact that CnCdk1 is able to complement the S. cerevisiae cdc28-ts mutation support its assumed role as the CDC28/cdc2 homologue in C. neoformans.

Amino Acid Sequence↗
PubMed2004

Cryptococcus neoformans capsule biosynthesis and regulation.

The capsule is certainly the most prominent virulence factor in Cryptococcus neoformans: acapsular strains are avirulent, and capsular polysaccharides have a deleterious effect on the immune system. Until very recently, very few genes involved in capsule biosynthesis had been identified - and this despite the existence of a detailed body of work concerning the capsule's composition, structure and their regulation by environmental factors. The tremendous development of experimental tools and techniques suited to the study of C. neoformans biology together with the sequencing of three complete genomes have, over the last three years, enabled the identification of a number of proteins which participate directly in biosynthesis of the capsule or which regulate its size. Even though this knowledge is still preliminary, it gives us a clearer picture of the various events needed for biosynthesis of this fascinating structure.

Cryptococcus neoformans↗
PubMed2004

Role of laccase in the biology and virulence of Cryptococcus neoformans.

Laccase is an important virulence factor for the human pathogen, Cryptococcus neoformans. In this review, we examine the structural, biological and genetic features of the enzyme and its role in the pathogenesis of cryptococcosis. Laccase is expressed in C. neoformans as a cell wall enzyme that possesses a broad spectrum of activity oxidizing both polyphenolic compounds and iron. Two paralogs, CNLAC1 and CNLAC2, are present in the fungus, of which the first one expresses the dominant enzyme activity under glucose starvation conditions. Regulation of the enzyme is in response to various environmental signals including nutrient starvation, the presence of multivalent cations and temperature stress, and is mediated through multiple signal transduction pathways. Study of the function and regulation of this important virulence factor has led to further understanding of mechanisms of fungal pathogenesis and the regulation of stress response in the host cell environment.

Amino Acid Sequence↗
PubMed2005

Characterization of an ecto-ATPase activity in Cryptococcus neoformans.

Cryptococcus neoformans is the causative agent of pulmonary cryptococcosis and cryptococcal meningoencephalitis, which are major clinical manifestations in immunosuppressed patients. In the present study, a surface ATPase (ecto-ATPase) was identified in C. neoformans yeast cells. Intact yeasts hydrolyzed adenosine-5'-triphosphate (ATP) at a rate of 29.36+/-3.36nmol Pi/hx10(8) cells. In the presence of 5 mM MgCl(2), this activity was enhanced around 70 times, and an apparent K(m) for Mg-ATP corresponding to 0.61mM was determined. Inhibitors of phosphatases, mitochondrial Mg(2+)-ATPases, V-ATPases, Na(+)-ATPases or P-ATPases had no effect on the cryptococcal ATPase, but extracellular impermeant compounds reduced enzyme activity in living cells. ATP was the best substrate for the cryptococcal ecto-enzyme, but it also efficiently hydrolyzed inosine 5'-triphosphate (ITP), cytidine 5'-triphosphate (CTP), guanosine 5'-triphosphate (GTP) and uridine-5'-triphosphate (UTP). In the presence of ATP, C. neoformans became less susceptible to the antifungal action of fluconazole. Our results are indicative of the occurrence of a C. neoformans ecto-ATPase that may have a role in fungal physiology.

Adenosine Triphosphatases↗