Cryptococcus neoformans is a haploid environmental organism and the major cause of fungal meningoencephalitis in AIDS patients. Fluconazole (FLC), a triazole, is widely used for the maintenance therapy of cryptococcosis. Heteroresistance to FLC, an adaptive mode of azole resistance, was associated with FLC therapy failure cases but the mechanism underlying the resistance was unknown. We used comparative genome hybridization and quantitative real-time PCR in order to show that C. neoformans adapts to high concentrations of FLC by duplication of multiple chromosomes. Formation of disomic chromosomes in response to FLC stress was observed in both serotype A and D strains. Strains that adapted to FLC concentrations higher than their minimal inhibitory concentration (MIC) contained disomies of chromosome 1 and stepwise exposure to even higher drug concentrations induced additional duplications of several other specific chromosomes. The number of disomic chromosomes in each resistant strain directly correlated with the concentration of FLC tolerated by each strain. Upon removal of the drug pressure, strains that had adapted to high concentrations of FLC returned to their original level of susceptibility by initially losing the extra copy of chromosome 1 followed by loss of the extra copies of the remaining disomic chromosomes. The duplication of chromosome 1 was closely associated with two of its resident genes: ERG11, the target of FLC and AFR1, the major transporter of azoles in C. neoformans. This adaptive mechanism in C. neoformans may play an important role in FLC therapy failure of cryptococcosis leading to relapse during azole maintenance therapy. Cryptococcus neoformans is an environmental fungus that causes life threatening brain disease, primarily in AIDS patients. The disease is estimated to claim 700,000 lives annually world-wide but most heavily in Africa. Fluconazole (FLC), a fungistatic antifungal drug, is commonly used to treat patients for long term maintenance therapy. Recurrence of cryptococcosis in AIDS patients undergoing FLC maintenance therapy has been increasingly reported. Heteroresistance, an adaptive azole resistance, was associated with FLC therapy failure cases but the mechanism underlying the resistance was unknown. We previously described that C. neoformans strains are innately heteroresistant to FLC; each strain producing a fraction of subpopulation that can tolerate a high concentration of the drug. These resistant subpopulations revert to original phenotype during maintenance in drug free media. Various methods including cDNA microarrays, comparative genome hybridization and quantitative PCR have been applied to uncover the mechanism involved in the adaptation of C. neoformans to high concentrations of FLC and subsequent loss of resistance upon the removal of drug pressure. We discovered that C. neoformans adapts to high concentration of FLC by formation of disomy in multiple chromosomes. The removal of drug pressure results in a sequential loss of the extra chromosomal copies. It is likely that this novel mechanism of adaptation contributes to the failure of FLC therapy for cryptococcosis.

Cryptococcosis is a potentially lethal disease of humans/animals caused by Cryptococcus neoformans and Cryptococcus gattii . Distinction between the two species is based on phenotypic and genotypic characteristics. Recently, it was proposed that C. neoformans be divided into two species and C. gattii into five species based on a phylogenetic analysis of 115 isolates. While this proposal adds to the knowledge about the genetic diversity and population structure of cryptococcosis agents, the published genotypes of 2,606 strains have already revealed more genetic diversity than is encompassed by seven species. Naming every clade as a separate species at this juncture will lead to continuing nomenclatural instability. In the absence of biological differences between clades and no consensus about how DNA sequence alone can delineate a species, we recommend using “ Cryptococcus neoformans species complex” and “ C. gattii species complex” as a practical intermediate step, rather than creating more species. This strategy recognizes genetic diversity without creating confusion.

Aspergillus fumigatus , a filamentous fungus producing bluish-green conidia, is an important opportunistic pathogen that primarily affects immunocompromised patients. Conidial pigmentation of A. fumigatus significantly influences its virulence in a murine model. In the present study, six genes, forming a gene cluster spanning 19 kb, were identified as involved in conidial pigment biosynthesis in A. fumigatus . Northern blot analyses showed the six genes to be developmentally regulated and expressed during conidiation. The gene products of alb1 (for “albino 1”), arp1 (for “aspergillus reddish-pink 1”), and arp2 have high similarity to polyketide synthases, scytalone dehydratases, and hydroxynaphthalene reductases, respectively, found in the dihydroxynaphthalene (DHN)-melanin pathway of brown and black fungi. The abr1 gene (for “aspergillus brown 1”) encodes a putative protein possessing two signatures of multicopper oxidases. The abr2 gene product has homology to the laccase encoded by the yA gene of Aspergillus nidulans . The function of ayg1 (for “aspergillus yellowish-green 1”) remains unknown. Involvement of the six genes in conidial pigmentation was confirmed by the altered conidial color phenotypes that resulted from disruption of each gene in A. fumigatus . The presence of a DHN-melanin pathway in A. fumigatus was supported by the accumulation of scytalone and flaviolin in the arp1 deletant, whereas only flaviolin was accumulated in the arp2 deletants. Scytalone and flaviolin are well-known signature metabolites of the DHN-melanin pathway. Based on DNA sequence similarity, gene disruption results, and biochemical analyses, we conclude that the 19-kb DNA fragment contains a six-gene cluster which is required for conidial pigment biosynthesis in A. fumigatus . However, the presence of abr1 , abr2 , and ayg1 in addition to alb1 , arp1 , and arp2 suggests that conidial pigment biosynthesis in A. fumigatus is more complex than the known DHN-melanin pathway.

Aspergillus fumigatus, an important opportunistic pathogen which commonly affects neutropenic patients, produces conidia with a bluish-green color. We identified a gene,alb1, which is required for conidial pigmentation. Thealb1gene encodes a putative polyketide synthase, and disruption ofalb1resulted in an albino conidial phenotype. Expression ofalb1is developmentally regulated, and the 7-kb transcript is detected only during the conidiation stage. Thealb1mutation was found to block 1,3,6,8-tetrahydroxynaphthalene production, indicating thatalb1is involved in dihydroxynaphthalene-melanin biosynthesis. Scanning electron microscopy studies showed that thealb1disruptant exhibited a smooth conidial surface, whereas complementation of thealb1deletion restored the echinulate wild-type surface. Disruption ofalb1resulted in a significant increase in C3 binding on conidial surfaces, and the conidia of thealb1disruptant were ingested by human neutrophils at a higher rate than were those of the wild type. Thealb1-complemented strain producing bluish-green conidia exhibited inefficient C3 binding and neutrophil-mediated phagocytosis quantitatively similar to those of the wild type. Importantly, thealb1disruptant had a statistically significant loss of virulence compared to the wild-type andalb1-complemented strains in a murine model. These results suggest that disruption ofalb1causes pleiotropic effects on conidial morphology and fungal virulence.

Molecular phylogenetic analysis confirmed the phylum Zygomycota to be polyphyletic, and the taxa conventionally classified in Zygomycota are now distributed among the new phylum Glomeromycota and 4 subphyla incertae sedis (uncertain placement). Because the nomenclature of the disease zygomycosis was based on the phylum Zygomycota (Zygomycetes) in which the etiologic agents had been classified, the new classification profoundly affects the name of the disease. Zygomycosis was originally described as a convenient and inclusive name for 2 clinicopathologically different diseases, mucormycosis caused by members of Mucorales and entomophthoramycosis caused by species in the order Entomophthorales of Zygomycota. Without revision of original definition, the name “zygomycosis,” however, has more often been used as a synonym only for mucormycosis. This article reviews the progress and changes in taxonomy and nomenclature of Zygomycota and the disease zygomycosis. The article also reiterates the reasons why the classic names “mucormycosis” and “entomophthoramycosis” are more appropriate than “zygomycosis.”

Cryptococcal meningoencephalitis develops as a result of hematogenous dissemination of inhaled Cryptococcus neoformans from the lung to the brain. The mechanism(s) by which C. neoformans crosses the blood-brain barrier (BBB) is a key unresolved issue in cryptococcosis. We used both an in vivo mouse model and an in vitro model of the human BBB to investigate the cryptococcal association with and traversal of the BBB. Exposure of human brain microvascular endothelial cells (HBMEC) to C. neoformans triggered the formation of microvillus-like membrane protrusions within 15 to 30 min. Yeast cells of C. neoformans adhered to and were internalized by the HBMEC, and they crossed the HBMEC monolayers via a transcellular pathway without affecting the monolayer integrity. The histopathology of mouse brains obtained after intravenous injection of C. neoformans showed that the yeast cells either were associated with endothelial cells or escaped from the brain capillary vessels into the neuropil by 3 h. C. neoformans was found in the brain parenchyma away from the vessels by 22 h. Association of C. neoformans with the choroid plexus, however, was not detected during up to 10 days of observation. Our findings indicate that C. neoformans cells invade the central nervous system by transcellular crossing of the endothelium of the BBB.