Cryptococcus neoformans and Cryptococcus gattii are the two etiologic agents of cryptococcosis. They belong to the phylum Basidiomycota and can be readily distinguished from other pathogenic yeasts such as Candida by the presence of a polysaccharide capsule, formation of melanin, and urease activity, which all function as virulence determinants. Infection proceeds via inhalation and subsequent dissemination to the central nervous system to cause meningoencephalitis. The most common risk for cryptococcosis caused by C. neoformans is AIDS, whereas infections caused by C. gattii are more often reported in immunocompetent patients with undefined risk than in the immunocompromised. There have been many chapters, reviews, and books written on C. neoformans. The topics we focus on in this article include species description, pathogenesis, life cycle, capsule, and stress response, which serve to highlight the specializations in virulence that have occurred in this unique encapsulated melanin-forming yeast that causes global deaths estimated at more than 600,000 annually.

Evaluation of Cryptococcus neoformans virulence in a number of nonmammalian hosts suggests that C. neoformans is a nonspecific pathogen. We used the killing of Galleria mellonella (the greater wax moth) caterpillar by C. neoformans to develop an invertebrate host model system that can be used to study cryptococcal virulence, host immune responses to infection, and the effects of antifungal compounds. All varieties of C. neoformans killed G. mellonella . After injection into the insect hemocoel, C. neoformans proliferated and, despite successful phagocytosis by host hemocytes, killed caterpillars both at 37°C and 30°C. The rate and extent of killing depended on the cryptococcal strain and the number of fungal cells injected. The sequenced C. neoformans clinical strain H99 was the most virulent of the strains tested and killed caterpillars with inocula as low as 20 CFU/caterpillar. Several C. neoformans genes previously shown to be involved in mammalian virulence ( CAP59 , GPA1 , RAS1 , and PKA1 ) also played a role in G. mellonella killing. Combination antifungal therapy (amphotericin B plus flucytosine) administered before or after inoculation was more effective than monotherapy in prolonging survival and in decreasing the tissue burden of cryptococci in the hemocoel. The G. mellonella - C. neoformans pathogenicity model may be a substitute for mammalian models of infection with C. neoformans and may facilitate the in vivo study of fungal virulence and efficacy of antifungal therapies.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence. Cryptococcus neoformans var. grubii is a major human pathogen responsible for deadly meningoencephalitis in immunocompromised patients. Here, we report the sequencing and annotation of its genome. Evidence for extensive intron splicing, antisense transcription, non-coding RNAs, and alternative polyadenylation indicates the potential for highly intricate regulation of gene expression in this opportunistic pathogen. In addition, detailed molecular, genetic, and genomic studies were performed to characterize structural features of the genome, including centromeres and origins of replication. Finally, the phenotypic and genome re-sequencing analysis of a collection of isolates of the reference H99 strain resulting from laboratory passage revealed that microevolutionary processes during in vitro culturing of pathogenic fungi can impact virulence.

Rhizopus oryzae is the primary cause of mucormycosis, an emerging, life-threatening infection characterized by rapid angioinvasive growth with an overall mortality rate that exceeds 50%. As a representative of the paraphyletic basal group of the fungal kingdom called “zygomycetes,” R. oryzae is also used as a model to study fungal evolution. Here we report the genome sequence of R. oryzae strain 99–880, isolated from a fatal case of mucormycosis. The highly repetitive 45.3 Mb genome assembly contains abundant transposable elements (TEs), comprising approximately 20% of the genome. We predicted 13,895 protein-coding genes not overlapping TEs, many of which are paralogous gene pairs. The order and genomic arrangement of the duplicated gene pairs and their common phylogenetic origin provide evidence for an ancestral whole-genome duplication (WGD) event. The WGD resulted in the duplication of nearly all subunits of the protein complexes associated with respiratory electron transport chains, the V-ATPase, and the ubiquitin–proteasome systems. The WGD, together with recent gene duplications, resulted in the expansion of multiple gene families related to cell growth and signal transduction, as well as secreted aspartic protease and subtilase protein families, which are known fungal virulence factors. The duplication of the ergosterol biosynthetic pathway, especially the major azole target, lanosterol 14α-demethylase (ERG11), could contribute to the variable responses of R. oryzae to different azole drugs, including voriconazole and posaconazole. Expanded families of cell-wall synthesis enzymes, essential for fungal cell integrity but absent in mammalian hosts, reveal potential targets for novel and R. oryzae-specific diagnostic and therapeutic treatments. Rhizopus oryzae is a widely dispersed fungus that can cause fatal infections in people with suppressed immune systems, especially diabetics or organ transplant recipients. Antibiotic therapy alone is rarely curative, particularly in patients with disseminated infection. We sequenced the genome of a pathogenic R. oryzae strain and found evidence that the entire genome had been duplicated at some point in its evolution and retained two copies of three extremely sophisticated systems involved in energy generation and utilization. The ancient whole-genome duplication, together with recent gene duplications, has led to the expansion of gene families related to pathogen virulence, fungal-specific cell wall synthesis, and signal transduction, which may contribute to the aggressive and frequently life-threatening growth of this organism. We also identified cell wall synthesis enzymes, essential for fungal cell integrity but absent in mammals, which may present potential targets for developing novel diagnostic and therapeutic treatments. R. oryzae represents the first sequenced fungus from the early lineages of the fungal phylogenetic tree, and thus the genome sequence sheds light on the evolution of the entire fungal kingdom.