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Three new combinations from the Cryptococcus laurentii complex: Cryptococcus aureus, Cryptococcus carnescens and Cryptococcus peneaus.

Fifteen strains of the Cryptococcus laurentii complex were reclassified based on sequence analyses of 18S rDNA, the D1/D2 region of the 26S rDNA and the internal transcribed spacer regions, as well as physiological and biochemical properties. The strains were divided into phylogenetic groups I and II. The type strain of C. laurentii (CBS 139T=ATCC 18803T=JCM 9066T=MUCL 30398T=NRRL Y-2536T) was in phylogenetic group I. Phylogenetic group II, which was phylogenetically distant from phylogenetic group I, clustered with Cryptococcus dimennae and Bullera globispora. In phylogenetic group I, the type strain of Torula aurea (CBS 318T=ATCC 32063T=IFO 0372T=NRRL Y-1582T) appeared to be a separate species from C. laurentii, and the designation Cryptococcus aureus comb. nov. is proposed for Torula aurea. Cryptococcus flavescens (formerly Torula flavescens, type strain CBS 942T=ATCC 10668T=DBVPG 6007T=MUCL 30414T) was treated as a synonym of C. laurentii; however, this is a distinct species. The type strains of Torulopsis carnescens (CBS 973T=ATCC 32064T=MUCL 30641T=NRRL Y-1503T), Rhodotorula peneaus (CBS 2409T=ATCC 13546T=MUCL 30643T=NRRL Y-2005T) and Cryptococcus victoriae belonged to phylogenetic group II. Two new combinations, Cryptococcus carnescens comb. nov. (type strain CBS 973T=ATCC 32064T=MUCL 30641T=NRRL Y-1503T) and Cryptococcus peneaus comb. nov. (type strain CBS 2409T=ATCC 13546T=MUCL 30643T=NRRL Y-2005T), are proposed from this group.

Base Composition↗

Cryptococcus bestiolae and Cryptococcus dejecticola, two new yeast species isolated from frass of the litchi fruit borer Conopomorpha sinensis Bradley.

Two new yeast species, Cryptococcus bestiolae and Cryptococcus dejecticola, were discovered in the frass of the litchi fruit borer Conopomorpha sinensis Bradley. The yeasts utilize inositol, hydrolyze urea, produce starch-like substance, and contain CoQ10. Phylogenetic analyses of D1/D2 26S rDNA and internal transcribed spacer (ITS) sequences indicate that the yeasts are closely related to Bullera dendrophila and an undescribed species of Cryptococcus (strain CBS 8507). The two new species differed from each other by 17 nucleotides in the D1/D2 region and by 68 nucleotides in the ITS region. Cryptococcus bestiolae is a sister species to Cryptococcus sp. CBS 8507, from which it differs by eight nucleotides in the D1/D2 region and 59 nucleotides in the ITS region. Cryptococcus dejecticola and B. dendrophila differed by 13 nucleotides in the D1/D2 and 57 nucleotides in the ITS region. Cryptococcus bestiolae and Cr. dejecticola formed with B. dendrophila a well defined clade consisting of insect associated species. The type strain of Cr. bestiolae is TH3.2.59 (=CBS 10118=NRRL Y-27894), and the type strain of Cr. dejecticola is Litch 17 (=CBS 10117=NRRL Y-27898).

Animals↗

Cryptococcus mujuensis sp. nov. and Cryptococcus cuniculi sp. nov., basidiomycetous yeasts isolated from wild rabbit faeces.

Two previously undescribed anamorphic yeasts, strains T-11(T) and T-26(T), recovered from wild rabbit faecal pellets collected in Muju, Korea, were identified using phenotypic and molecular taxonomic methods. The isolates were characterized by the proliferation of budding cells, positive diazonium blue B and urease reactions, the presence of Q-10 as the major ubiquinone, the presence of xylose in whole-cell hydrolysates and the inability to ferment sugars. Phylogenetic analyses based on 26S rRNA gene partial sequences revealed that strain T-11(T) was located in the Bulleromyces clade and was related to Sirobasidium intermedium, Tremella exigua, Cryptococcus cellulolyticus and Bullera pseudoalba. Strain T-26(T) was located in the Mesenterica clade and was closely related to Cryptococcus sp. F6 and Cryptococcus heveanensis CBS 8976. Sequence divergence values of more than 4 % from other described Cryptococcus species, together with the phenotypic differences, showed that the isolated yeasts represent previously unrecognized members of this genus. Therefore, two novel yeast species are proposed: Cryptococcus mujuensis sp. nov., with strain T-11(T) (=KCTC 17231(T)=CBS 10308(T)) as the type strain, and Cryptococcus cuniculi sp. nov., with strain T-26(T) (=KCTC 17232(T)=CBS 10309(T)) as the type strain.

Animals↗

Pigment production by Cryptococcus neoformans and other Cryptococcus species from aminophenols and diaminobenzenes.

Cryptococcus neoformans and other Cryptococcus species can produce pigment(s) from many aminophenol and diaminobenzene compounds. Pigment production from these compounds is similar to the conversion of diphenols to melanin by C. neoformans. Several pigmentation patterns (resulting in the identification or grouping of Cryptococcus species) have been observed by using diaminobenzene and aminophenol compounds as substrates. The most common pigmentation pattern observed was pigment production by both C. neoformans and C. terreus. In contrast to the diphenols, only two aminophenols (4-hydroxymetanilamide and 3-aminotyrosine) were found to be highly specific as substrates. They allowed only C. neoformans to produce pigment. When 4-aminosalicylic acid was the substrate, a unique pattern was observed because only C. terreus, C. diffluens, and C. albidus produced pigment. Finally, a pattern was observed in which C. neoformans produced large amounts of pigment from aminophenol and diaminobenzene compounds, whereas the other Cryptococcus species produced smaller amounts. A simplified scheme with three substrates resulted in the identification of C. terreus and C. neoformans as well as two groups of other Cryptococcus species, group I (C. albidus and C. diffluens) and group II (C. laurentii and C. luteolus).

Aminobenzoates↗

Feral pigeons as carriers of Cryptococcus laurentii, Cryptococcus uniguttulatus and Debaryomyces hansenii.

We collected fresh droppings and cloaca samples from feral pigeons Columba livia in the southern Swedish city of Malmö, and isolated the following fungi: Debaryomyces hansenii var. hansenii, Cryptococcus laurentii and Cryptococcus uniguttulatus. The first two species are known to be pathogenic to humans. No strains of Cryptococcus neoformans var. neoformans were found. Our results indicate that feral pigeons can be carriers of medically significant fungi other than Cryptococcus neoformans var. neoformans.

Animals↗

Distribution of Cryptococcus gattii and Cryptococcus neoformans in decayed trunk wood of Syzygium cumini trees in north-western India.

The aim of this study is to report the regional distribution of Cryptococcus. gattii and Cryptococcus. neoformans in decayed wood inside trunk hollows of Syzygium cumini trees (Java plum, Indian black berry) investigated in Amritsar (Panjab), Meerut Cantt. and Bulandshahr (Uttar Pradesh) and Delhi, in north-western India. Two hundred and seventeen wood samples collected from 74 S. cumini trees were investigated. This includes 7 known positive S. cumini trees in Delhi subjected to a mycological surveillance for perennial colonization by C. gattii and C. neoformans. Cryptococcus gattii showed the highest prevalence (89%) in S. cumini trees in Delhi, followed by 27%, 12.5% and 9% prevalence in Bulandshahr, Amritsar City and Meerut Cantt., respectively. In contrast, C. neoformans had the highest prevalence (54%) in Amritsar, followed by 44% in Delhi, 9% in Bulandshahr and 0% in Meerut Cantt. Furthermore, 44% of the S. cumini trees in Delhi, 9% in Bulandshahr and 8% in Amritsar were concomitantly colonized by both C. gattii and C. neoformans. A mycological surveillance over 4.8-5.2 years of 7 selected S. cumini trees in Delhi revealed perennial colonization by both the Cryptococcus species. In addition, air samples taken close to the decayed trunk hollows of 4 of the perennially colonized S. cumini trees contained strains of the C. neoformans species complex. Of a random sample of 48 isolates serotyped, 26 (54%) were C. neoformans, serotype A, and 22 (46%) C. gattii, serotype B. Determination of mating type alleles was done in 44 of the isolates, comprising 31 of C. neoformans, serotype A and 13 of C.gattii, serotype B. All of them proved to be mating type alpha (MATalpha). The data on high prevalence, fungal population density, perennial colonization and aerial isolations indicate that decayed wood in trunk hollows of S. cumini trees is to-date the main well documented primary environmental niche of C. gattii and C. neoformans in north-western India. Attention is drawn to the likely health hazard posed by the environmental reservoirs of C. gattii and C. neoformans occurring in tree trunk hollows in proximity to human and animal habitations.

Cryptococcus neoformans↗

Cryptococcus socialis sp. nov. and Cryptococcus consortionis sp. nov., Antarctic basidioblastomycetes.

New yeasts from the Ross Desert (dry valley area) of Antarctica include Cryptococcus socialis sp. nov. and Cryptococcus consortionis sp. nov. Cryptococcus socialis MYSW A801-3aY1 (= ATCC 56685) requires no vitamins, assimilates L-arabinose, cellobiose, D-glucuronate, maltose, melezitose, raffinose, soluble starch, sucrose, and trehalose, and may be distinguished from all other basidioblastomycetes by the combination of amylose production, cellobiose assimilation, and failure to utilize nitrate, D-galactose, myo-inositol, and mannitol. Its guanine-plus-cytosine content is 56 mol%. Cryptococcus consortionis MYSW A801-3aY92 (= ATCC 56686) requires thiamine, assimilates L-arabinose, D-glucuronate, 2-ketogluconate, salicin, succinate, sucrose, trehalose, and D-xylose, and may be distinguished from all other basidioblastomycetes by the combination of amylose production and failure to utilize nitrate, cellobiose, D-galactose, myo-inositol, and mannitol. Its guanine-plus-cytosine content is 56 mol%.

Antarctic Regions↗

Cryptococcus nemorosus sp. nov. and Cryptococcus perniciosus sp. nov., related to Papiliotrema Sampaio et al. (Tremellales).

Three mycocinogenic strains representing the genus Cryptococcus were isolated on glucuronate agar from plants and turf collected in the Prioksko-terrasny biosphere reserve (Russia). These isolates fit the standard description of Cryptococcus laurentii, but differ from its type strain in both their mycocin-sensitivity profiles and the killing patterns of their mycocins. Sequence analyses of the D1/D2 domain of the 26S rDNA and of the internal transcribed spacer region confirmed that these isolates represent two novel species, for which the names Cryptococcus nemorosus sp. nov. (type strain VKM Y-2906(T)) and Cryptococcus perniciosus sp. nov. (type strain VKM Y-2905(T)) are proposed. Morphological, physiological and biochemical characteristics, as well as mycocinotyping and molecular analysis, show a close affinity between these two novel anamorphic species and the teleomorphic species Papiliotrema bandonii (Tremellales).

Basidiomycota↗

Serological relationships of Cryptococcus spp.: distribution of antigenic factors in Cryptococcus and intraspecies diversity.

The antigenic formulas of 34 species in the genus Cryptococcus were determined by using type strains and eight factor sera prepared from adsorption experiments with Cryptococcus neoformans serotypes. These antigenic factors were shared by 19 species. The strains used could be divided into eight serological groups. The patterns of groups 1, 2, 3, 5, and 6 were the same as the patterns of C. neoformans serotypes A, D, A-D, B, and C, respectively. The species belonging to group 4 reacted to factor sera 1, 2, and 3. Group 7 contained one species that reacted only to factor serum 1. The 15 species in group 8 did not react to any of the factor sera used. Compared to the reported molecular phylogenetic tree, the serological and phylogenetic data were correlated in the Filobasidium lineage. All the members of the albidus clade in the Filobasidium lineage had antigens 1, 2, and 3, and all the strains in the magnus clade belonged to serogroup 8. Moreover, intraspecies diversity was examined using strains of C. curvatus, C. humicolus, and C. laurentii. Serological heterogeneity was observed in the species C. humicolus and C. laurentii, as well as in phylogenetic relationships previously published. Using serological features, similarities and differences between Cryptococcus species were demonstrated. Our study contributes to a better description of the genus Cryptococcus and related species phenotypically and phylogenetically.

Agglutination Tests↗

Cryptococcus tephrensis, sp.nov., and Cryptococcus heimaeyensis, sp.nov.; new anamorphic basidiomycetous yeast species from Iceland.

Two new species from Iceland are described on the basis of physiological profiles and sequence data from the D2 region of LSU rDNA: Cryptococcus tephrensis (type ICE99-IToM Y5, ATCC MYA-1765, CBS 8935, GenBank AF317208) and Cryptococcus heimaeyensis (type ICE99-IToM Y8, ATCC MYA-1759, CBS 8933, GenBank AF370717). The two new species are identifiable from sequence data and can be distinguished from their closest relative, Cryptococcus victoriae, by their higher maximum temperatures for growth, failure to utilize nitrate as sole nitrogen source, and utilization of cadaverine and ethylamine as sole nitrogen sources. Cryptococcus tephrensis is distinguishable from C. heimaeyensis by failure to grow on saccharate as sole source of carbon and energy.

Cryptococcus↗

Gene disruption in Cryptococcus neoformans and Cryptococcus gattii by in vitro transposition.

Cryptococcus neoformans and Cryptococcus gattii are basidiomycetous fungi that infect immunocompromised and immunocompetent people. We developed an insertional mutagenesis strategy for these species based on in vitro transposition and we tested the method by disrupting the URA5 gene in a strain of C. neoformans and the CAP10 gene in three strains of C. gattii. We targeted plasmid DNA containing the URA5 gene or plasmid DNA containing the CAP10 gene from genomic libraries from the shotgun sequencing project for the C. gatti strain WM276. In the latter case, the availability of the end sequences of the clones from the assembled genomic sequence allows rapid selection of target genes for disruption. Modified transposons containing the nourseothricin (NAT) or neomycin (Neo) resistance cassettes were randomly inserted into the target DNA by in vitro transposition. The disrupted genes were used for biolistic transformation and homologous integration was subsequently confirmed by PCR and Southern blot analysis. These results demonstrate that the emerging genomic resources, combined with in vitro transposition into plasmid DNAs from shotgun sequencing libraries or cloned PCR products, will facilitate high-throughput genetic analysis in Cryptococcus species.

Biolistics↗

In vitro susceptibilities of Malaysian clinical isolates of Cryptococcus neoformans var. grubii and Cryptococcus gattii to five antifungal drugs.

The in vitro susceptibilities of Malaysian clinical isolates of Cryptococcus neoformans var. grubii and C . gattii to five antifungal drugs (amphotericin B, flucytosine, fluconazole, itraconazole and ketoconazole) were determined using the Etest method. None of the Malaysian isolates was resistant to amphotericin B and ketoconazole. Isolates resistant to flucytosine, fluconazole and itraconazole were observed in this study. Minimum inhibition concentrations (MICs) of > or = 32 microg ml(-1) against flucytosine, > or = 64 microg ml(-1) against fluconazole and > or = 1 microg ml(-1) against itraconazole were noted in four (8.3%), two (4.2%) and one (2.1%) isolates respectively. There was no significant difference in the MICs for both Cryptococcus species (P > 0.05), indicating that C. gattii was as susceptible as var. grubii to all the antifungal drugs tested. No significant difference in the MICs for both Cryptococcus species collected from 1980 to 1990 and 2002 to 2004 were observed (P > 0.05).

Amphotericin B↗

Unique hybrids between the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii.

Cryptococcus neoformans and Cryptococcus gattii are yeasts that cause meningoencephalitis, but that differ in host range and geographical distribution. Cryptococcus neoformans occurs world-wide and mostly infects immunocompromised patients, whereas C. gattii occurs mainly in (sub)tropical regions and infects healthy individuals. Anomalous C. neoformans strains were isolated from patients. These strains were found to be monokaryotic, and diploid or aneuploid. Amplified Fragment Length Polymorphism (AFLP) and sequence analyses indicated that AFLP genotypes 2 (C. neoformans) and 4 (C. gattii) were present. The strains were serologically BD. Mating- and serotype-specific PCR reactions showed that the strains were MATa-serotype D/MATalpha-serotype B. This study is the first to describe naturally occurring hybrids between C. neoformans and C. gattii.

Adult↗

Laccase and melanization in clinically important Cryptococcus species other than Cryptococcus neoformans.

The laccase enzyme and melanin synthesis have been implicated as contributors to virulence in Cryptococcus neoformans. Since isolations of Cryptococcus species other than C. neoformans from clinical specimens have been increasing, we examined the laccase activities of C. albidus, C. laurentii, C. curvatus, and C. humicola. Incubation of cells with epinephrine produced adrenochrome color in C. albidus, C. laurentii, and C. curvatus but not in C. humicola. Activity was always less than in C. neoformans. Laccase was detected in the soluble fractions of disrupted C. albidus, C. laurentii, and C. curvatus cells. Activity staining of partially purified enzyme after nondenaturing polyacrylamide gel electrophoresis revealed that laccases from C. albidus, C. laurentii, and C. curvatus migrated more slowly than that from C. neoformans. One strain of C. curvatus exhibited two melanin bands. Thus, several clinically emerging Cryptococcus species express laccase and can synthesize melanin.

Cryptococcosis↗

Cryptococcus allantoinivorans sp.nov., an anamorphic basidiomycetous yeast (Tremellales) physiologically resembling other species of the Cryptococcus laurentii complex that degrade polysaccharides and C2 compounds.

A novel Cryptococcus species is proposed to accommodate a yeast strain (CBS 9604) able to assimilate allantoin as sole carbon source, a characteristic very uncommon among yeasts. By traditional methods, the strain could not be distinguished from Cryptococcus laurentii, but nucleotide sequences of the D1D2 region of the large subunit (26S) and of the ITS region of ribosomal DNA showed relationship to the Bulleromyces clade of the genus Cryptococcus (order Tremellales) with some Tremella spp. as the closest relatives. A traditional morphological and physiological description of the strain is given. Data on the assimilation of some C2 compounds and polysaccharides are provided and compared with those of other type strains of novel species of the C. laurentii complex.

Allantoin↗

Interspecies differences in the enantioselectivity of epoxide hydrolases in Cryptococcus laurentii (Kufferath) C.E. Skinner and Cryptococcus podzolicus (Bab'jeva & Reshetova) Golubev.

Isolates representing Cryptococcus laurentii and Cryptococcus podzolicus, originating from soil of a heathland indigenous to South Africa, were screened for the presence of enantioselective epoxide hydrolases for 2,2-disubstituted epoxides. Epoxide hydrolase activity for the 2,2-disubstituted epoxide (+/-)-2-methyl-2-pentyl oxirane was found to be abundantly present in all isolates. The stereochemistry of the products formed by the epoxide hydrolase enzymes from isolates belonging to the two species (11 isolates representing C. laurentii and 23 isolates representing C. podzolicus) was investigated. The enantiopreferences of the epoxide hydrolases for 2,2-disubstituted epoxides of these two species were found to be opposite. All strains of C. laurentii preferentially hydrolysed the (S)-epoxides while all C. podzolicus isolates preferentially hydrolysed the (R)-epoxides of (+/-)-2,2-disubstituted epoxides. These findings indicate that the stereochemistry of the products formed from 2,2-disubstituted epoxides by the epoxide hydrolase enzymes of these yeasts should be evaluated as additional taxonomic criterion within the genus Cryptococcus. Also, the selectivity of some epoxide hydrolases originating from isolates of C. podzolicus was high enough to be considered for application in biotransformations for the synthesis of enantiopure epoxides and vicinal diols.

Cryptococcus↗

Recognition of cytoplasmic yeast antigens of Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. gattii by immune human sera.

The humoral immune response of patients infected with Cryptococcus neoformans var. neoformans and C. neoformans var. gattii to cytoplasmic (non-capsular) antigens from the two varieties of Cryptococcus has been investigated. Cytoplasmic antigens from C. neoformans (one clinical isolate and one acapsular mutant of var. neoformans and two clinical isolates from var. gattii) were subject to isoelectric focusing, SDS-PAGE and Western blotting; patients sera was then used in the immunoenzyme development of the Western blots. The humoral response from the 20 patients (all HIV+) infected with var. neoformans against the var. neoformans antigens was predominantly IgG based, with a large number of bands recognised: the most commonly recognised bands were at 26, 52, 74, 100, 115 and 144 kDa. The IgM response was less pronounced and the IgA response was practically non-existent. The humoral response of the sera from the 15 patients (all but one HIV-) infected with var. gattii against var. gattii antigens was also predominantly IgG based with bands at 37, 55, 65, 74, 94 and 115 kDa being most commonly recognised. Periodate treatment of cytoplasmic antigens reduced the intensity of antigen recognition, though it did not absolutely destroy reactivity to any individual antigen. Comparison of immunodevelopment of cytoplasmic antigens from both varieties grown at 25 degrees C and 37 degrees C revealed that culture temperature made no differences in the number of bands recognised although there were differences in the intensity of recognition. This is the first report on the pattern of serological recognition of the non-capsular antigens from the two varieties of Cryptococcus and it identifies a number of major antigenic components.

Antibodies, Fungal↗

Divergence of protein kinase A catalytic subunits in Cryptococcus neoformans and Cryptococcus gattii illustrates evolutionary reconfiguration of a signaling cascade.

Gene duplication and divergence via both the loss and gain of gene activities are powerful evolutionary forces underlying the origin of new biological functions. Here a comparative genetics approach was applied to examine the roles of protein kinase A (PKA) catalytic subunits in three closely related varieties or sibling species of the pathogenic fungus genus Cryptococcus. Previous studies revealed that two PKA catalytic subunits, Pka1 and Pka2, control virulence factor production and mating. However, only one of the two plays the predominant physiological role, and this function has been exchanged between Pka1 and Pka2 in strains of the Cryptococcus neoformans var. grubii serotype A lineage compared to divergent C. neoformans var. neoformans serotype D isolates. To understand the basis for this functional plasticity, here the activities of Pka1 and Pka2 were defined in the two varieties and the related sibling species Cryptococcus gattii by gene disruption and characterization, heterologous complementation, and analysis of serotype AD hybrid mutant strains. The findings provide evidence for a shared ancestral role of PKA in governing mating and virulence factor production and indicate that the exchange of catalytic subunit roles is attributable to loss of function. Our studies illustrate the plasticity of signaling networks enabling rapid rewiring during speciation of a clade of common human fungal pathogens.

Catalytic Domain↗