The Evolution of Sexual Reproduction and the Mating-Type Locus: Links to Pathogenesis of Cryptococcus Human Pathogenic Fungi.

Cryptococcus species utilize a variety of sexual reproduction mechanisms, which generate genetic diversity, purge deleterious mutations, and contribute to their ability to occupy myriad environmental niches and exhibit a range of pathogenic potential. The bisexual and unisexual cycles of pathogenic Cryptococcus species are stimulated by properties associated with their environmental niches and proceed through well-characterized signaling pathways and corresponding morphological changes. Genes governing mating are encoded by the mating-type (MAT) loci and influence pathogenesis, population dynamics, and lineage divergence in Cryptococcus. MAT has undergone significant evolutionary changes within the Cryptococcus genus, including transition from the ancestral tetrapolar state in nonpathogenic species to a bipolar mating system in pathogenic species, as well as several internal reconfigurations. Owing to the variety of established sexual reproduction mechanisms and the robust characterization of the evolution of mating and MAT in this genus, Cryptococcus species provide key insights into the evolution of sexual reproduction.

Kosakonia radicincitans and Cryptococcus laurentii controlled Penicillium expansum rot and decreased patulin production at 4 and 25 °C.

In the present work, we evaluated the effects of a mixture of biocontrol agents against two toxigenic strains of Penicillium expansum isolated in Argentine Patagonia from pome fruits. The two strains, INTA-5 and INTA-10, were previusly selected among ten strains coming from the Alto Valle (Rio Negro-Argentina) for their high production of patulin. For the biocontrol, Kosakonia radicincitans, Cryptococcus laurentii, and Rhodosporidium fluviale were tested in vitro experiments on Potato Dextrose Agar (PDA) dishes against the INTA-5 and INTA-10 strains. The bacterium K. radicincitans and the yeast C. laurentii were selected to be used in a mixture due to their capacity to control the fungus and reduce the mycotoxin severely. In vitro assays with the mixture showed a high antagonism against P. expansum INTA-5 and INTA-10, at 21 d of incubation at 25 °C and a patulin reduction of 98%. The mixture of microorganisms was also effective in apples stored at 25 °C for 10 d and 4 °C for 30 d. At cold storage, the mixture controlled moderately the development of rot and decreased patulin concentration. At 25 °C, the pathogen's optimal growth temperature, the mixture of Biological Control Agent (BCAs) assured both the control of rot and decrease of patulin concentration. The combination of two microorganisms, with different requirements and abilities, resulted in a mix with a strong antagonism against P. expansum with the capability to decrease the patulin concentration. Treatment with the selected mixture could be a good option for controlling strains with different behaviours and in different environmental conditions.

Diverse species in the genus Cryptococcus: Pathogens and their non-pathogenic ancestors.

The genus Cryptococcus comprises of more than 30 species. It consists of clinically significant pathogenic Cryptococcus neoformans/Cryptococcus gattii species complex comprising of a minimum of seven species. These pathogens cost more than 200,000 lives annually by causing cryptococcal meningoencephalitis. The evolution of the pathogenic species from closely related non-pathogenic species of the Cryptococcus amylolentus complex is of particular importance and several advances have been made to understand their phylogenetic and genomic relationships. The current review briefly describes the sexual reproduction process followed by an individual description of the members focusing on their key attributes and virulence mechanisms of the pathogenic species. A special section on phylogenetic studies is aimed at understanding the evolutionary divergence of pathogens from non-pathogens. Recent findings from our group pertaining to parameters affecting codon usage bias in six pathogenic and three non-pathogenic ancestral species and their corroboration with existing phylogenetic reports are also included in the current review.

Fungal genome and mating system transitions facilitated by chromosomal translocations involving intercentromeric recombination.

Species within the human pathogenic Cryptococcus species complex are major threats to public health, causing approximately 1 million annual infections globally. Cryptococcus amylolentus is the most closely known related species of the pathogenic Cryptococcus species complex, and it is non-pathogenic. Additionally, while pathogenic Cryptococcus species have bipolar mating systems with a single large mating type (MAT) locus that represents a derived state in Basidiomycetes, C. amylolentus has a tetrapolar mating system with 2 MAT loci (P/R and HD) located on different chromosomes. Thus, studying C. amylolentus will shed light on the transition from tetrapolar to bipolar mating systems in the pathogenic Cryptococcus species, as well as its possible link with the origin and evolution of pathogenesis. In this study, we sequenced, assembled, and annotated the genomes of 2 C. amylolentus isolates, CBS6039 and CBS6273, which are sexual and interfertile. Genome comparison between the 2 C. amylolentus isolates identified the boundaries and the complete gene contents of the P/R and HD MAT loci. Bioinformatic and chromatin immunoprecipitation sequencing (ChIP-seq) analyses revealed that, similar to those of the pathogenic Cryptococcus species, C. amylolentus has regional centromeres (CENs) that are enriched with species-specific transposable and repetitive DNA elements. Additionally, we found that while neither the P/R nor the HD locus is physically closely linked to its centromere in C. amylolentus, and the regions between the MAT loci and their respective centromeres show overall synteny between the 2 genomes, both MAT loci exhibit genetic linkage to their respective centromere during meiosis, suggesting the presence of recombinational suppressors and/or epistatic gene interactions in the MAT-CEN intervening regions. Furthermore, genomic comparisons between C. amylolentus and related pathogenic Cryptococcus species provide evidence that multiple chromosomal rearrangements mediated by intercentromeric recombination have occurred during descent of the 2 lineages from their common ancestor. Taken together, our findings support a model in which the evolution of the bipolar mating system was initiated by an ectopic recombination event mediated by similar repetitive centromeric DNA elements shared between chromosomes. This translocation brought the P/R and HD loci onto the same chromosome, and further chromosomal rearrangements then resulted in the 2 MAT loci becoming physically linked and eventually fusing to form the single contiguous MAT locus that is now extant in the pathogenic Cryptococcus species.

Yeast Volatomes Differentially Affect Larval Feeding in an Insect Herbivore.

Yeasts form mutualistic interactions with insects. Hallmarks of this interaction include provision of essential nutrients, while insects facilitate yeast dispersal and growth on plant substrates. A phylogenetically ancient chemical dialogue coordinates this interaction, where the vocabulary, the volatile chemicals that mediate the insect response, remains largely unknown. Here, we used gas chromatography-mass spectrometry, followed by hierarchical cluster and orthogonal partial least-squares discriminant analyses, to profile the volatomes of six Metschnikowia spp., Cryptococcus nemorosus, and brewer's yeast (Saccharomyces cerevisiae). The yeasts, which are all found in association with insects feeding on foliage or fruit, emit characteristic, species-specific volatile blends that reflect the phylogenetic context. Species specificity of these volatome profiles aligned with differential feeding of cotton leafworm (Spodoptera littoralis) larvae on these yeasts. Bioactivity correlates with yeast ecology; phylloplane species elicited a stronger response than fruit yeasts, and larval discrimination may provide a mechanism for establishment of insect-yeast associations. The yeast volatomes contained a suite of insect attractants known from plant and especially floral headspace, including (Z)-hexenyl acetate, ethyl (2E,4Z)-deca-2,4-dienoate (pear ester), (3E)-4,8-dimethylnona-1,3,7-triene (DMNT), linalool, α-terpineol, ß-myrcene, or (E,E)-α-farnesene. A wide overlap of yeast and plant volatiles, notably floral scents, further emphasizes the prominent role of yeasts in plant-microbe-insect relationships, including pollination. The knowledge of insect-yeast interactions can be readily brought to practical application, as live yeasts or yeast metabolites mediating insect attraction provide an ample toolbox for the development of sustainable insect management.IMPORTANCE Yeasts interface insect herbivores with their food plants. Communication depends on volatile metabolites, and decoding this chemical dialogue is key to understanding the ecology of insect-yeast interactions. This study explores the volatomes of eight yeast species which have been isolated from foliage, from flowers or fruit, and from plant-feeding insects. These yeasts each release a rich bouquet of volatile metabolites, including a suite of known insect attractants from plant and floral scent. This overlap underlines the phylogenetic dimension of insect-yeast associations, which according to the fossil record long predate the appearance of flowering plants. Volatome composition is characteristic for each species, aligns with yeast taxonomy, and is further reflected by a differential behavioral response of cotton leafworm larvae, which naturally feed on foliage of a wide spectrum of broad-leaved plants. Larval discrimination may establish and maintain associations with yeasts and is also a substrate for designing sustainable insect management techniques.

Pulmonary cryptococcosis: A review of pathobiology and clinical aspects.

Pulmonary cryptococcosis is an important opportunistic invasive mycosis in immunocompromised patients, but it is also increasingly seen in immunocompetent patients. The main human pathogens are Cryptococcus neoformans and C. gattii, which have a worldwide distribution. In contrast to cryptococcal meningitis, pulmonary cryptococcosis is still underdiagnosed because of limitations in diagnostic tools. It can mimic lung cancer, pulmonary tuberculosis, bacterial pneumonia, and other pulmonary mycoses both clinically and radiologically. Pulmonary nodules are the most common radiological feature, but these are not specific to pulmonary cryptococcosis. The sensitivity of culture of respiratory samples for Cryptococcus is poor and a positive result may also reflect colonisation. Cryptococcal antigen (CrAg) with lateral flow device is a fast and sensitive test and widely used on serum and cerebrospinal fluid, but sera from patients with pulmonary cryptococcosis are rarely positive in the absence of disseminated disease. Detection of CrAg from respiratory specimens might assist the diagnosis of pulmonary cryptococcosis but there are very few data. Molecular detection techniques such as multiplex reverse transcription polymerase chain reaction (RT-PCR) could also provide better sensitivity but these still require validation for respiratory specimens. The first line of treatment for pulmonary cryptococcosis is fluconazole, or amphotericin B and flucytosine for those with central nervous system involvement. Pulmonary cryptococcosis worsens the prognosis of cryptococcal meningitis. In this review, we summarize the biological aspects of Cryptococcus and provide an update on the diagnosis and management of pulmonary cryptococcosis.

Broad-spectrum antifungal activity of spirooxindolo-pyrrolidine tethered indole/imidazole hybrid heterocycles against fungal pathogens.

Invasive fungal infections are one of the leading causes of nosocomial bloodstream infections with a limited treatment option. A series of derivatized spirooxindolo-pyrrolidine tethered indole and imidazole heterocyclic hybrids have been synthesized, and their antifungal activity against fungal strains were determined. Here we characterize the antifungal activity of a specific spirooxindolo-pyrrolidine hybrid, dubbed compound 9c, a spirooxindolo-pyrrolidine tethered imidazole synthesized with a 2-chloro and trifluoromethoxy substituent. The compound 9c exhibited no cytotoxicity against mammalian cell line at concentrations that inhibited fungal strains. Compound 9c also significantly inhibited the fungal hyphae and biofilm formation. Our results indicate that spirooxindolo-pyrrolidine heterocyclic hybrids potentially represent a broad class of chemical agents with promising antifungal potential.

New Insights Into Cryptococcus Spp. Biology and Cryptococcal Meningitis.

PURPOSE OF REVIEW: Defective cell-mediated immunity is a major risk factor for cryptococcosis, a fatal disease if untreated. Cryptococcal meningitis (CM), the main presentation of disseminated disease, occurs through hematogenous spread to the brain from primary pulmonary foci, facilitated by yeast virulence factors. We revisit remarkable recent improvements in the prevention, diagnosis and management of CM. RECENT FINDINGS: Cryptococcal antigen (CrAg), main capsular polysaccharide of Cryptococcus spp. is detectable in blood and cerebrospinal fluid of infected patients with point of care lateral flow assays. Recent World Health Organization guidelines recommend 7-day amphotericin B plus flucytosine, then 7-day high dose (1200 mg/day) fluconazole for induction treatment of HIV-associated CM. Management of raised intracranial pressure, a consequence of CM, should rely mainly on daily therapeutic lumbar punctures until normalisation. In HIV-associated CM, following introduction of antifungal therapy, (re)initiation of antiretroviral therapy should be delayed by 4-6 weeks to prevent immune reconstitution inflammatory syndrome, common in CM. CM is a fatal disease whose diagnosis has recently been simplified. Treatment should always include antifungal combination therapy and management of raised intracranial pressure. Screening for immune deficiency should be mandatory in all patients with cryptococcosis.

Analysis of sphingolipids, sterols, and phospholipids in human pathogenic Cryptococcus strains.

Cryptococcus species cause invasive infections in humans. Lipids play an important role in the progression of these infections. Independent studies done by our group and others provide some detail about the functions of these lipids in Cryptococcus infections. However, the pathways of biosynthesis and the metabolism of these lipids are not completely understood. To thoroughly understand the physiological role of these Cryptococcus lipids, a proper structure and composition analysis of Cryptococcus lipids is demanded. In this study, a detailed spectroscopic analysis of lipid extracts from Cryptococcus gattii and Cryptococcus grubii strains is presented. Sphingolipid profiling by LC-ESI-MS/MS was used to analyze sphingosine, dihydrosphingosine, sphingosine-1-phosphate, dihydrosphingosine-1-phosphate, ceramide, dihydroceramide, glucosylceramide, phytosphingosine, phytosphingosine-1-phosphate, phytoceramide, α-hydroxy phytoceramide, and inositolphosphorylceramide species. A total of 13 sterol species were identified using GC-MS, where ergosterol is the most abundant species. The 31P-NMR-based phospholipid analysis identified phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidyl-N,N-dimethylethanolamine, phosphatidyl-N-monomethylethanolamine, phosphatidylglycerol, phosphatidic acid, and lysophosphatidylethanolamine. A comparison of lipid profiles among different Cryptococcus strains illustrates a marked change in the metabolic flux of these organisms, especially sphingolipid metabolism. These data improve our understanding of the structure, biosynthesis, and metabolism of common lipid groups of Cryptococcus and should be useful while studying their functional significance and designing therapeutic interventions.

It's not all about us: evolution and maintenance of Cryptococcus virulence requires selection outside the human host.

Cryptococcus is predominantly an AIDS-related pathogen that causes significant morbidity and mortality in immunocompromised patients. Research studies have historically focused on understanding how the organism causes human disease through the use of in vivo and in vitro model systems to identify virulence factors. Cryptococcus is not an obligate pathogen, however, as human-human transmission is either absent or rare. Selection in the environment must thus be invoked to shape the evolution of this taxa, and directly influences genotypic and trait diversity. Importantly, the evolution and maintenance of pathogenicity must also stem directly from environmental selection. To that end, here we examine abiotic and biotic stresses in the environment, and discuss how they could shape the factors that are commonly identified as important virulence traits. We identify a number of important unanswered questions about Cryptococcus diversity and evolution that are critical for understanding this deadly pathogen, and discuss how implementation of modern sampling and genomic tools could be utilized to answer these questions. Copyright © 2016 John Wiley & Sons, Ltd.

Cryptococcal Maxillary Osteomyelitis and Osteonecrosis in a 18-Month-Old Dog.

An 18-month-old neutered male labradoodle was treated with surgical debridement for maxillary osteomyelitis and sequestrum formation. Histopathologic findings of the necrotic bone were consistent with Cryptococcus subspecies, confirmed with latex agglutination serum titer testing. The patient responded to a combination of fluconazole and surgical debridement and was titer negative after 8 months of medical therapy. The patient never exhibited signs of systemic illness which is commonly reported with cryptococcosis. Cryptococcus subspecies infection in dogs in the Pacific Northwest is part of an ongoing outbreak in the region, first reported in 2001, and is associated with specific risk factors. This is the first published case of oral cryptococcosis from primary inoculation.

Growth of high-elevation Cryptococcus sp. during extreme freeze-thaw cycles.

Soils above 6000 m.a.s.l. are among the most extreme environments on Earth, especially on high, dry volcanoes where soil temperatures cycle between -10 and 30 °C on a typical summer day. Previous studies have shown that such sites are dominated by yeast in the cryophilic Cryptococcus group, but it is unclear if they can actually grow (or are just surviving) under extreme freeze-thaw conditions. We carried out a series of experiments to determine if Cryptococcus could grow during freeze-thaw cycles similar to those measured under field conditions. We found that Cryptococcus phylotypes increased in relative abundance in soils subjected to 48 days of freeze-thaw cycles, becoming the dominant organisms in the soil. In addition, pure cultures of Cryptococcus isolated from these same soils were able to grow in liquid cultures subjected to daily freeze-thaw cycles, despite the fact that the culture medium froze solid every night. Furthermore, we showed that this organism is metabolically versatile and phylogenetically almost identical to strains from Antarctic Dry Valley soils. Taken together these results indicate that this organism has unique metabolic and temperature adaptations that make it able to thrive in one of the harshest and climatically volatile places on Earth.

Yeast microbiota of natural cavities of manatees (Trichechus inunguis and Trichechus manatus) in Brazil and its relevance for animal health and management in captivity.

The aim of this study was to characterize the yeast microbiota of natural cavities of manatees kept in captivity in Brazil. Sterile swabs from the oral cavity, nostrils, genital opening, and rectum of 50 Trichechus inunguis and 26 Trichechus manatus were collected. The samples were plated on Sabouraud agar with chloramphenicol and incubated at 25 °C for 5 days. The yeasts isolated were phenotypically identified by biochemical and micromorphological tests. Overall, 141 strains were isolated, of which 112 were from T. inunguis (Candida albicans, Candida parapsilosis sensu stricto, Candida orthopsilosis, Candida metapsilosis, Candida guilliermondii, Candida pelliculosa, Candida tropicalis, Candida glabrata, Candida famata, Candida krusei, Candida norvegensis, Candida ciferri, Trichosporon sp., Rhodotorula sp., Cryptococcus laurentii) and 29 were from T. manatus (C. albicans, C. tropicalis, C. famata, C. guilliermondii, C. krusei, Rhodotorula sp., Rhodotorula mucilaginosa, Rhodotorula minuta, Trichosporon sp.). This was the first systematic study to investigate the importance of yeasts as components of the microbiota of sirenians, demonstrating the presence of potentially pathogenic species, which highlights the importance of maintaining adequate artificial conditions for the health of captive manatees.

Discovery of a modified tetrapolar sexual cycle in Cryptococcus amylolentus and the evolution of MAT in the Cryptococcus species complex.

Sexual reproduction in fungi is governed by a specialized genomic region called the mating-type locus (MAT). The human fungal pathogenic and basidiomycetous yeast Cryptococcus neoformans has evolved a bipolar mating system (a, α) in which the MAT locus is unusually large (>100 kb) and encodes >20 genes including homeodomain (HD) and pheromone/receptor (P/R) genes. To understand how this unique bipolar mating system evolved, we investigated MAT in the closely related species Tsuchiyaea wingfieldii and Cryptococcus amylolentus and discovered two physically unlinked loci encoding the HD and P/R genes. Interestingly, the HD (B) locus sex-specific region is restricted (∼2 kb) and encodes two linked and divergently oriented homeodomain genes in contrast to the solo HD genes (SXI1α, SXI2a) of C. neoformans and Cryptococcus gattii. The P/R (A) locus contains the pheromone and pheromone receptor genes but has expanded considerably compared to other outgroup species (Cryptococcus heveanensis) and is linked to many of the genes also found in the MAT locus of the pathogenic Cryptococcus species. Our discovery of a heterothallic sexual cycle for C. amylolentus allowed us to establish the biological roles of the sex-determining regions. Matings between two strains of opposite mating-types (A1B1×A2B2) produced dikaryotic hyphae with fused clamp connections, basidia, and basidiospores. Genotyping progeny using markers linked and unlinked to MAT revealed that meiosis and uniparental mitochondrial inheritance occur during the sexual cycle of C. amylolentus. The sexual cycle is tetrapolar and produces fertile progeny of four mating-types (A1B1, A1B2, A2B1, and A2B2), but a high proportion of progeny are infertile, and fertility is biased towards one parental mating-type (A1B1). Our studies reveal insights into the plasticity and transitions in both mechanisms of sex determination (bipolar versus tetrapolar) and sexual reproduction (outcrossing versus inbreeding) with implications for similar evolutionary transitions and processes in fungi, plants, and animals.

Cryptococcosis in patients living with hepatitis C and B viruses.

Cryptococcosis, a systemic fungal infection, has become a significant, global public health problem. Patients with liver disease have an increased predisposition to infections, such as Cryptococcosis. To report the underlying disease, the variety of etiologic agents involved and the outcomes of the Cryptococcosis in patients living with HBV and/or HCV, we reviewed 34 medical records of patients who were diagnosed with Cryptococcosis by the Mycology Laboratory of Santa Casa Hospital, Porto Alegre, Brazil. Males corresponded to 79% of the patients, and the average patient age was 46.9 years. The cultures of 26/34 patients were positive: 25 patients were infected with Cryptococcus neoformans and one with C. gattii. A total of 14 deaths (41%) occurred. As a criterion of our study, all patients had viral hepatitis infection: 27 (80%) were infected with HCV, five (15%) were infected with HBV, and two patients were infected with both viruses. Because HBV and/or HCV are transmitted among drug users through infected blood, and the end-stage cirrhotic liver must be transplanted, these two population types were well represented in this study and were analyzed in detail. Cryptococcosis patients living with HCV and/or HBV appear to have the same symptoms, mean age and gender distribution as the general Cryptococcosis population. Once Cryptococcosis affects the brain, a high mortality rate ensues; therefore, physicians must be aware of the possible occurrence of this disease in patients living with HCV and HBV.

First case of disseminated cryptococcosis in a Gorilla gorilla.

In humans, Cryptococcus mainly infects individuals with HIV infection or other types of immunosuppression. Here, we report the first case of disseminated cryptococcosis in a simian immunodeficiency virus-negative 27-year-old female Gorilla gorilla presenting with lethargy, progressive weight loss and productive cough. The diagnosis was confirmed by positive lung biopsy culture, serum cryptococcal antigen, and cerebral histopathology demonstrating encapsulated yeasts. Molecular characterisation of lung culture isolate yielded Cryptococcus neoformans var. grubii. An immune-deficiency could not be demonstrated.

A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric microbial viability assay.

UNLABELLED: A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric microbial viability assay based on the reduction in a tetrazolium salt 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) with 2-methyl-1,4-napthoquinone as the electron mediator was developed. The proposed method was useful to measure the proliferation of 18 kinds of moulds and seven kinds of yeasts, including representative pathogens such as Aspergillus spp., Candida spp. and Cryptococcus spp. Linear relationships between the absorbance and viable fungal cell density were obtained for all fungi, suggesting that the absorbance change reflected the fungal proliferation. In addition, the minimum inhibitory concentrations (MICs) against a variety of different pathogenic moulds and yeasts for amphotericin B, itraconazole and 5-flucytosine were determined by susceptibility testing using the proposed method and compared with those obtained using the conventional broth microdilution method. There was an excellent agreement between the results obtained using the WST-8 colorimetric method and those obtained using the conventional Clinical and Laboratory Standard Institute method. The WST-8 colorimetric assay is a useful method for rapid determination of accurate MICs for a variety of different fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric microbial viability assay based on reduction in a tetrazolium salt (WST-8) was developed. The WST-8 colorimetric method was useful to measure the proliferation of a variety of different fungi. In the antifungal susceptibility testing, there was a good agreement between the MICs determined after 24 h using the WST-8 colorimetric method and those obtained after 48-96 h using the broth microdilution method. The proposed method was superior to conventional methods in terms of its rapidity towards a variety of different fungi.

Host immune response against fungal biofilms.

Fungal biofilms are a multilayered community of cells attached to mucosal or abiotic surfaces enclosed in a coating of self-produced extracellular polymeric matrix. The sheer density of cells protected by a polymeric shield not only makes the biofilm impermeable to antimicrobials or immune cells but also hidden from host recognition. Biofilms also serve as a reservoir of drug-resistant persister cells and dispersal cells armored with virulence factors adept at evading the immune system. Here, we summarize the latest knowledge on the immunomodulatory properties of biofilms formed by Candida species and by other biofilm-forming fungal pathogens such as Aspergillus and Cryptococcus. Finally, we deliberate on promising strategies to help activate the immune system for combating fungal biofilms.

The mating type locus (MAT) and sexual reproduction of Cryptococcus heveanensis: insights into the evolution of sex and sex-determining chromosomal regions in fungi.

Mating in basidiomycetous fungi is often controlled by two unlinked, multiallelic loci encoding homeodomain transcription factors or pheromones/pheromone receptors. In contrast to this tetrapolar organization, Cryptococcus neoformans/Cryptococcus gattii have a bipolar mating system, and a single biallelic locus governs sexual reproduction. The C. neoformans MAT locus is unusually large (>100 kb), contains >20 genes, and enhances virulence. Previous comparative genomic studies provided insights into how this unusual MAT locus might have evolved involving gene acquisitions into two unlinked loci and fusion into one contiguous locus, converting an ancestral tetrapolar system to a bipolar one. Here we tested this model by studying Cryptococcus heveanensis, a sister species to the pathogenic Cryptococcus species complex. An extant sexual cycle was discovered; co-incubating fertile isolates results in the teleomorph (Kwoniella heveanensis) with dikaryotic hyphae, clamp connections, septate basidia, and basidiospores. To characterize the C. heveanensis MAT locus, a fosmid library was screened with C. neoformans/C. gattii MAT genes. Positive fosmids were sequenced and assembled to generate two large probably unlinked MAT gene clusters: one corresponding to the homeodomain locus and the other to the pheromone/receptor locus. Strikingly, two divergent homeodomain genes (SXI1, SXI2) are present, similar to the bE/bW Ustilago maydis paradigm, suggesting one or the other homeodomain gene was recently lost in C. neoformans/C. gattii. Sequencing MAT genes from other C. heveanensis isolates revealed a multiallelic homeodomain locus and at least a biallelic pheromone/receptor locus, similar to known tetrapolar species. Taken together, these studies reveal an extant C. heveanensis sexual cycle, define the structure of its MAT locus consistent with tetrapolar mating, and support the proposed evolutionary model for the bipolar Cryptococcus MAT locus revealing transitions in sexuality concomitant with emergence of a pathogenic clade. These studies provide insight into convergent processes that independently punctuated evolution of sex-determining loci and sex chromosomes in fungi, plants, and animals.

Cryptococcus cyanovorans sp. nov., a basidiomycetous yeast isolated from cyanide-contaminated soil.

Eighteen yeast strains were isolated and identified from cyanide-contaminated soil in South Africa. According to sequence-based analyses using the D1/D2 region of the large ribosomal subunit and ITS region, three of these strains were found to be identical and represent a novel species. Phylogenetic analysis based on the combined dataset of the D1/D2 and ITS regions revealed a grouping with Cryptococcus curvatus, representing a defined clade (Curvatus) in the order Trichosporonales. The three strains were demarcated from Cryptococcus curvatus by standard physiological tests such as assimilation of lactose, xylitol, 5-keto-D-gluconate, succinate and citrate as well as growth on media containing 10 % (w/v) NaCl and 5 % (w/v) glucose. In addition, it was established that these strains could utilize up to 10 mM NaCN as sole carbon source on solid media and as sole nitrogen source in liquid media. On the basis of these findings, it is suggested that the three strains represent a novel species for which the name Cryptococcus cyanovorans sp. nov. is given (type strain CBS 11948(T) = NRRL Y-48730(T)).