Many globally isolated AD hybrid strains of Cryptococcus neoformans originated in Africa

Interspecific and intervarietal hybridization may contribute to the biological diversity of fungal populations. Cryptococcus neoformans is a pathogenic yeast and the most common fungal cause of meningitis in patients with AIDS. Most patients are infected with either of the two varieties of C. neoformans, designated as serotype A (C. neoformans var. grubii) or serotype D (C. neoformans var. neoformans). In addition, serotype AD strains, which are hybrids of these two varieties, are commonly isolated from clinical and environmental samples. While most isolates of serotype A and serotype D are haploid, AD strains are diploid or aneuploid, and contain two sets of chromosomes and two mating type alleles, MATa and MATalpha, one from each of the serotypes. The global population of serotype A is dominated by isolates with the MATalpha mating type (Aalpha); however, about half of the globally analyzed AD strains possess the extremely rare serotype A MATa allele (Aa). We previously described an unusual population of serotype A in Botswana, in which 25% of the strains contain the rare MATa allele. Here we utilized two methods, phylogenetic analysis of three genes and genotyping by scoring amplified fragment length polymorphisms, and discovered that AD hybrid strains possessing the rare serotype A MATa allele (genotype AaDalpha) cluster with isolates of serotype A from Botswana, whereas AD hybrids that possess the MATalpha serotype A allele (AalphaDa and AalphaDalpha) cluster with cosmopolitan isolates of serotype A. We also determined that AD hybrid strains are more resistant to UV irradiation than haploid serotype A strains from Botswana. These findings support two hypotheses: (i) AaDalpha strains originated in sub-Saharan Africa from a cross between strains of serotypes A and D; and (ii) this fusion produced hybrid strains with increased fitness, enabling the Botswanan serotype A MATa genome, which is otherwise geographically restricted, to survive, emigrate, and propagate throughout the world.     

Cryptococcus neoformans population includes hybrid strains homozygous at mating-type locus

Recent attempts to characterise the hybrid strains of Cryptococcus neoformans have led to the identification of a cryptic population of hybrid strains ('H strains') with double DNA content but only a single mating-type allele. To verify a set of hypotheses concerning their origin, we investigated 14 previously isolated H strains and ten F1-progeny strains arising from H99 and JEC20 mating. The double DNA content was tested by flow cytometry; the presence of only one mating type was tested by amplifying 12 mating-type-specific genes and one gene unlinked with the mating-type locus (URA5). Analysis of the F1 progeny identified two H strains, and electrophoretic karyotyping confirmed the occurrence of genetic recombination. The simultaneous presence of the homozygous and heterozygous loci, and the fact that all of the F1-progeny strains presented a recombinant karyotype, suggest that the H strains originated from the post-meiotic random fusion of two of the four recombinant nuclei. Further studies are required to elucidate the role of the homozygous mating-type loci in the virulence of C. neoformans.     

Specialization of the HOG pathway and its impact on differentiation and virulence of Cryptococcus neoformans

The human pathogenic fungus Cryptococcus neoformans has diverged from a common ancestor into three biologically distinct varieties or sibling species over the past 10-40 million years. During evolution of these divergent forms, serotype A C. neoformans var. grubii has emerged as the most virulent and cosmopolitan pathogenic clade. Therefore, understanding how serotype A C. neoformans is distinguished from less successful pathogenic serotypes will provide insights into the evolution of fungal virulence. Here we report that the structurally conserved Pbs2-Hog1 MAP kinase cascade has been specifically recruited as a global regulator to control morphological differentiation and virulence factors in the highly virulent serotype A H99 clinical isolate, but not in the laboratory-generated and less virulent serotype D strain JEC21. The mechanisms of Hog1 regulation are strikingly different between the two strains, and the phosphorylation kinetics and localization pattern of Hog1 are opposite in H99 compared with JEC21 and other yeasts. The unique Hog1 regulatory pattern observed in the H99 clinical isolate is widespread in serotype A strains and is also present in some clinical serotype D isolates. Serotype A hog1delta and pbs2delta mutants are attenuated in virulence, further underscoring the role of the Pbs2-Hog1 MAPK cascade in the pathogenesis of cryptococcosis.     

新型隐球酵母氯离子通道在阳离子内稳态中的趋异作用

新型隐球酵母Cryptococcus neoformans有两个变种（varieties）,即grubii和neoformans。目前研究最多的两个菌株H99（血清型A）和JEC21（血清型D）分别代表这两个变种,两者的毒性差别显著,为研究新型隐球酵母菌株间毒性的进化提供了良好模型。我们通过比较JEC21的clc1-突变体Tx1与早先鉴定的H99 clc1-菌株Mlac3发现,JEC21 CLC1同样决定铜离子的吸收。Tx1中丧失的漆酶活力可以通过外源Cu2+的加入得以恢复,而漆酶基因LAC1的转录与野生     

Diversity of laccase among Cryptococcus neoformans serotypes

The pathogenic yeast C. neoformans is classified into three varieties with five serotypes; var. grubii (serotype A), var. neoformans (serotype D), var. gattii (serotypes B and C), and serotype AD. Melanin is a virulence factor in the species, and its biosynthesis is catalyzed by laccase, encoded by the LAC1 gene. In order to estimate the natural variability of the LAC1 gene among Cryptococcus serotypes, the laccase protein sequence from 55 strains was determined and the phylogenetic relationships between cryptococcal and related fungal laccases revealed. The deduced laccase proteins consisted of 624 amino acid residues in serotypes A, D and AD, and 613 to 615 residues in serotypes B and C. Intra-serotype amino acid variation was marginal within serotypes A and D, and none was found within serotypes AD and C. Maximum amino acid replacement occurred in two serotype B strains. The similarity in the deduced sequence ranged from 80 to 96% between serotypes. The sequence in the copper-binding regions was strongly conserved in the five serotypes. The laccases of the five serotypes were grouped together in the same clade of the phylogenetic tree reconstructed from different fungal laccases, suggesting a monophyletic clade.     

Chromosomal Rearrangements between Serotype A and D Strains in Cryptococcus neoformans

Cryptococcus neoformans is a major human pathogenic fungus that can cause meningoencephalitis in immunocompromised hosts. It contains two divergent varieties, var. grubii (serotype A) and var. neoformans (serotype D), as well as hybrids (serotype AD) between these two varieties. In this study, we investigated the extent of chromosomal rearrangements between the two varieties, estimated the effects of chromosomal rearrangements on recombination frequencies, and surveyed the potential polymorphisms of the rearrangements among natural strains of the three serotypes. Through the analyses of two sequenced genomes from strains H99 (representing var. grubii) and JEC21 (representing var. neoformans), we revealed a total of 32 unambiguous chromosome rearrangements, including five translocations, nine simple inversions, and 18 complex rearrangements. Our analyses identified that overall, rearranged regions had recombination frequencies about half of those around syntenic regions. Using a direct PCR screening strategy, we examined the potential polymorphisms of 11 rearrangements among 64 natural C. neoformans strains from five countries. We found no polymorphism within var. neoformans and very limited polymorphism within var. grubii. However, strains of serotype AD showed significant polymorphism, consistent with their hybrid origins coupled with differential loss of heterozygosity. We discuss the implications of these results on the genome structure, ecology, and evolution of C. neoformans.     

Strain-related differences in antibody-mediated changes in gene expression are associated with differences in capsule and location of binding

We recently established that antibody (Ab)-binding can induce gene expression changes in a serotype A strain (H99) of the pathogenic yeast, Cryptococcus neoformans. That study showed that monoclonal antibodies (mAbs) differing in epitope specificity and protective efficacy elicited differences in gene expression. Because many mAbs bind to serotypes A and D strains differently, we now investigate the binding of one mAb to two strains representing these serotypes. Cells of the serotype A strain H99 and the serotype D strain 24067 were incubated with near saturating concentrations of the IgG1 capsule-binding mAb 18B7 or MOPC, an irrelevant mAb matched control. Comparative immunofluorescence analysis of mAb 18B7 binding revealed that it bound closer to the cell wall in H99 than 24067, where it was associated with decreased or increased cell diameter, respectively. A comparison of encapsulated cell compressibility showed that strain 24067 was more compressible than that of strain H99. RNA was extracted and used for gene expression analysis using the C. neoformans JEC21 genomic microarray. After 1h incubation with mAb 18B7, there were just 2 gene expression changes observed with strain 24067 or strain JEC21, unlike the 43 seen with strain H99. After 4h incubation with mAb 18B7, there were 14 and 140 gene expression changes observed with strain 24067 and JEC21, respectively. Thus, C. neoformans strains differ both in the response and the time of response to mAb binding and these differences may reflect differences in the location of Ab binding, Ab-mediated changes in cell diameter and compressibility of the capsular polysaccharide.     

Isolation and characterisation of the phospholipase B gene of Cryptococcus neoformans var. gattii

Cryptococcus neoformans var. gattii (serotypes B and C) is a human pathogen, ecologically, biochemically, clinically and genetically different from C. neoformans var. grubii (serotype A) and C. neoformans var. neoformans (serotype D). The phospholipase B (PLB1) gene from serotypes B and C was isolated and characterised. It resembled the serotype A and D genes, with an overall sequence homology of more than 85%. The respective open reading frames were 2236 bp (serotype B) and 2239 bp (serotype C) in length. Each contained six introns and encoded a 68-kDa protein destined for secretion. PLB1 was located on the second smallest chromosome in both serotypes. Gene expression, measured as mRNA, was not regulated by temperature, pH or exogenous nutrients.     

Molecular sequence analyses of the intergenic spacer (IGS) associated with rDNA of the two varieties of the pathogenic yeast, Cryptococcus neoformans

The pathogen Crytococcus neoformans has been traditionally grouped in two varieties, C. neoforrmans var. neoformans (serotypes A, D and AD) and C. neoformans var. gattii (serotypes B and C). A recent taxonomic evaluation of C. neoformans var. neoformans described C. neoformans var. grubii as a new variety represented by serotype A isolates. Despite immunological, biochemical, ecological and molecular differences the three varieties are classified within one species. We examined the genetic variability of one hundred and five clinical and environmental isolates that included all varieties and serotypes. Sequence analysis of the intergenic spacer (IGS) associated with rDNA revealed significant differences in nucleotide composition between and within the varieties. Parsimony analysis showed five different genotypes representing distinct genetic lineages. Although there was a high degree of relatedness between serotype and genotype this relatedness was not exclusive as serotypes were not restricted to one particular genotypic group. Serotyping and sequence analyses indicate that C. neoformans var. grubii (serotype A) should not be recognized as a separate variety. Based on this study we propose to accept two separate species, C. neoformans (serotypes A, D and AD) and C. bacillisporus (serotypes B and C synonymous with C. neoformans var. gattii).     

Heterozygosity in 2n gametes of potato evaluated by RFLP markers

The heterozygosity transmitted through 2n gametes in potato was evaluated by following the segregation of RFLP markers in tetraploid progeny from bilateral sexual polyploidization in a cross between two diploid (2x) interspecific hybrids which produce 2n SDR eggs or 2n FDR pollen. Out of 84 probe/enzyme combinations tested, 23 revealed polymorphism between the parents and were heterozygous in at least one of the parents. These probes characterized 13 loci distributed on five different chromosomes of the potato RFLP map. The heterozygosity transmitted through SDR and FDR gametes was estimated to be 31.8% and 71.4%, respectively. Two different indices (LH and RHI) were used to select plants showing a high level of heterozygosity in the tetraploid progeny. The recombination events and the centromere positions were estimated for chromosomes I, VI and VII, following the segregation ratios of SDR or FDR gametes produced by the parents. A different recombination rate was observed between the two interspecific hybrids.Contribution no. 119 from Research Centre for Vegetable Breeding, C.N.R., Portici, Italy. Research supported by National Research Council of Italy, special Project RAISA, Sub-project No. 2, Paper No. 2090     

Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans

Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans. Fungal Genetics and Biology 29, 38-48. Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle and well-developed genetic and molecular approaches. Two different transformation systems have been developed, and a number of genes have been disrupted by homologous recombination. However, the frequency of homologous recombination achieved by these approaches has differed dramatically between strains of the A and D serotypes. Transformation by electroporation in serotype D strains results in homologous recombination at frequencies of 1/1000 to 1/100,000, whereas transformation by the biolistic method has resulted in gene disruption at frequencies between 2 and 50% in serotype A strains. We find that gene disruption by homologous recombination can be achieved in the congenic serotype D strain series by biolistic transformation with frequencies of approximately 1 to 4%. By this approach, we have readily disrupted the genes encoding a MAPK homolog (CPK1), the calcineurin A catalytic subunit (CNA1), and a G protein alpha subunit (GPA1). By physical and genetic methods, we show that these mutations result from targeted recombination events without ectopic integrations. Because genetic approaches can be applied in the congenic serotype D strains, our observations represent a significant advance in molecular approaches to understand the physiology and virulence of this important human pathogen.     

Characterization of Cryptococcus neoformans by random DNA amplification

Random amplified polymorphic DNA (RAPD) was optimized and used to distinguish between the varieties and serotypes of Cryptococcus neofirmans . The RAPD technique distinguished between serotypes A, D or AD within C. neoformans var. neoformans , and revealed further differentiation within each serotype. Four RAPD profiles were clearly recognizable within C. neofirmans var. gattii , although its two serotypes, B and C, were only differentiated with one primer combination out of seven.     

Identification of Novel Hybrids Between Cryptococcus neoformans var. grubii VNI and Cryptococcus gattii VGII

Cryptococcus neoformans and Cryptococcus gattii are pathogenic yeasts causing meningoencephalitis in immunocompromised and immunocompetent hosts. The fungus is typically haploid, and sexual reproduction occurs normally between individuals with opposite mating types, α and a. C. neoformans var. grubii (serotype A) is comprised of molecular types VNI, VNII, and VNB, and C. neoformans var. neoformans (serotype D) contains the molecular type VNIV. Additionally, diploid or aneuploid AD hybrids (VNIII) have been reported. C. gattii contains the molecular types VGI, VGII, VGIII, and VGIV, which encompass both serotypes B and C. To identify possible hybrid strains, URA5-RFLP analysis was performed on 350 globally obtained clinical, environmental, and veterinary isolates. Four clinical isolates from cerebrospinal fluid showed combination patterns of C. neoformans var. grubii and C. gattii: Brazil (n = 2), Colombia (n = 1), and India (n = 1). These strains were monokaryotic and diploid or aneuploid. M13 PCR fingerprinting showed that they contained fragments of both proposed parental groups. Luminex IGS genotyping identified these isolates as hybrids with two different molecular type combinations: three VNI/VGII and one VNI/VGI. Blue color development on CGB agar was delayed in three isolates and absent in one. C. gattii-specific PCR confirmed the presence of C. gattii in the hybrids. CAP59 allele-specific PCR revealed that all the hybrids contained both serotype A and B alleles. Determination of mating-type allelic patterns by PCR revealed that the isolates were αA aB. This is the first study discovering novel natural hybrids between C. neoformans molecular type VNI and C. gattii molecular type VGII.     

Comparative hybridization reveals extensive genome variation in the AIDS-associated pathogen Cryptococcus neoformans

Background

Genome variability can have a profound influence on the virulence of pathogenic microbes. The availability of genome sequences for two strains of the AIDS-associated fungal pathogen Cryptococcus neoformans presented an opportunity to use comparative genome hybridization (CGH) to examine genome variability between strains of different mating type, molecular subtype, and ploidy.

Results

Initially, CGH was used to compare the approximately 100 kilobase MAT a and MATα mating-type regions in serotype A and D strains to establish the relationship between the Log2 ratios of hybridization signals and sequence identity. Subsequently, we compared the genomes of the environmental isolate NIH433 (MAT a) and the clinical isolate NIH12 (MATα) with a tiling array of the genome of the laboratory strain JEC21 derived from these strains. In this case, CGH identified putative recombination sites and the origins of specific segments of the JEC21 genome. Similarly, CGH analysis revealed marked variability in the genomes of strains representing the VNI, VNII, and VNB molecular subtypes of the A serotype, including disomy for chromosome 13 in two strains. Additionally, CGH identified differences in chromosome content between three strains with the hybrid AD serotype and revealed that chromosome 1 from the serotype A genome is preferentially retained in all three strains.

Conclusion

The genomes of serotypes A, D, and AD strains exhibit extensive variation that spans the range from small differences (such as regions of divergence, deletion, or amplification) to the unexpected disomy for chromosome 13 in haploid strains and preferential retention of specific chromosomes in naturally occurring diploids.     

In vitro susceptibility characteristics of Cryptococcus neoformans varieties from AIDS patients in Goiânia, Brazil

Sixty clinical isolates of Cryptococcus neoformans from AIDS from Goiania, state of Goiás, Brazil, were characterized according to varieties, serotypes and tested for antifungal susceptibility. To differentiate the two varieties was used L-canavanine-glycine-bromothymol blue medium and to separate the serotypes was used slide agglutination test with Crypto Check Iatron. The Minimal Inhibitory Concentration (MIC) of fluconazole, itraconazole, and amphotericin B were determined by the National Committee for Clinical Laboratory Standards macrodilution method. Our results identified 56 isolates as C. neoformans var. neoformans serotype A and 4 isolates as C. neoformans var. gattii serotype B. MIC values for C. neoformans var. gattii were higher than C. neoformans var. neoformans. We verified that none isolate was resistant to itraconazole and to amphotericin B, but one C. neoformans var. neoformans and three C. neoformans var. gattii isolates were resistant to fluconazole. The presence of C. neoformans var. gattii fluconazole resistant indicates the importance of determining not only the variety of C. neoformans infecting the patients but also measuring the MIC of the isolate in order to properly orient treatment.     

Multilocus sequence typing reveals three genetic subpopulations of Cryptococcus neoformans var. grubii (serotype A), including a unique population in Botswana

We applied multilocus sequence typing (MLST) to investigate the population structure and mode of reproduction of Cryptococcus neoformans var. grubii (serotype A). This MLST system utilizes 12 unlinked polymorphic loci, which are dispersed on nine different chromosomes, and allows the unambiguous identification of closely related strains of serotype A. We compared MLST analyses with the conventional genotyping method of detecting amplified fragment length polymorphisms (AFLPs), and there was excellent correlation between the MLST and AFLP results. However, MLST differentiated a larger number of strains. We analyzed a global collection of isolates of serotype A using both methods, and the results identified at least three genetically distinct subpopulations, designated groups VNI, VNII, and VNB. Groups VNI and VNII are widespread, dominated by isolates with the MATalpha mating type, and predominantly clonal. Conversely, isolates of group VNB are unique to Botswana, include a significant proportion of fertile strains with the MATa mating type, and manifest compelling evidence of recombination. We have AFLP genotyped >1000 strains of serotype A from different parts of the world, including isolates from several African countries, and, to date, haploid serotype A isolates of group VNB have been found only in Botswana.     

Mitochondria are inherited from the MATa parent in crosses of the basidiomycete fungus Cryptococcus neoformans

Previous studies demonstrated that mitochondrial DNA (mtDNA) was uniparentally transmitted in laboratory crosses of the pathogenic yeast Cryptococcus neoformans. To begin understanding the mechanisms, this study examined the potential role of the mating-type locus on mtDNA inheritance in C. neoformans. Using existing isogenic strains (JEC20 and JEC21) that differed only at the mating-type locus and a clinical strain (CDC46) that possessed a mitochondrial genotype different from JEC20 and JEC21, we constructed strains that differed only in mating type and mitochondrial genotype. These strains were then crossed to produce hyphae and sexual spores. Among the 206 single spores analyzed from six crosses, all but one inherited mtDNA from the MATa parents. Analyses of mating-type alleles and mtDNA genotypes of natural hybrids from clinical and natural samples were consistent with the hypothesis that mtDNA is inherited from the MATa parent in C. neoformans. To distinguish two potential mechanisms, we obtained a pair of isogenic strains with different mating-type alleles, mtDNA types, and auxotrophic markers. Diploid cells from mating between these two strains were selected and 29 independent colonies were genotyped. These cells did not go through the hyphal stage or the meiotic process. All 29 colonies contained mtDNA from the MATa parent. Because no filamentation, meiosis, or spore formation was involved in generating these diploid cells, our results suggest a selective elimination of mtDNA from the MATalpha parent soon after mating. To our knowledge, this is the first demonstration that mating type controls mtDNA inheritance in fungi.     

Diversity among strains of Cryptococcus neoformans var. gattii as revealed by a sequence analysis of multiple genes and a chemotype analysis of capsular polysaccharide.

We demonstrated the diversity of Cryptococcus neoformans var. gattii strains by a sequence analysis of multiple genes: (i) the intergenic spacer (IGS) 1 and 2 regions of the rRNA gene; (ii) the internal transcribed spacer (ITS) region, including 5.8S of the rRNA gene; (iii) TOP1 (topoisomerase); and (iv) CAP59. In these studies, we compared C. neoformans var. gattii with varieties grubii, and neoformans of C. neoformans. Phylogenetic analysis indicated that both C. neoformans var. grubii and C neoformans var. neoformans are monophyletic, but C. neoformans var. gattii showed polyphyletic. C. neoformans var. gattii can be divided into three phylogenetic groups, I, II, and III, with high bootstrap support. Phylogenetic group I contains serotype B and C strains, and groups II and III include serotype B strains. Because the serotype B strains of C. neoformans var. gattii exhibited more genetic divergence, the serological characteristics and chemotypes of their capsular polysaccharide were further investigated. No remarkable difference among the serotype B strains was found in the reactivities to factor serum 5, which is specific for serotype B. The NMR spectra of the capsular polysaccharide from serotype B strains could be divided into three characteristic patterns, but the chemical shifts were very similar. These results suggested that the serotype B strain of C. neoformans var. gattii has more genetic diversity than the serotype C strain of C. neoformans var. gattii or the varieties grubii and neoformans of C. neoformans, but there was no correlation between genotype and chemotype.     

The internal transcribed spacers and 5.8S rRNA gene show extensive diversity among isolates of the Cryptococcus neoformans species complex

Sequences of the internal transcribed spacer (ITS) region including the 5.8S rRNA gene delineated seven genotypes within the three varieties of Cryptococcus neoformans via specific combinations of eight nucleotide differences located at positions 10, 11, 15, 19, 108 (ITS1), 221 (5.8S), 298 and 346 (ITS2). The ITS types correlated to polymerase chain reaction fingerprint/random amplification of polymorphic DNA (RAPD) molecular types: with ITS type 1 (ATACTAGC)=C. neoformans var. grubii, molecular types VNI+VNII and the serotype A allele of the AD hybrid, VNIIIA; ITS type 2 (ATATAGGC)=the serotype D allele of the AD hybrid, VNIIIB, and C. neoformans var. neoformans, VNIV; and ITS type 3 (GCGCTGGC) and ITS type 7 (ACGCTGGC)=VGI=RAPD type III, ITS type 4 (ACACTGAC)=VGII=RAPD type II, ITS type 5: (ACACTGGG)=VGIII=RAPD type I, ITS type 6 (ACACTGGC)=VGIV=RAPD type IV, all corresponding to C. neoformans var. gattii. Cloned sequences from serotype AD revealed that the hybrid serotype is diploid at the ITS1-5.8S-ITS2 locus carrying the ITS type 1 (ATACTAGC) and the ITS type 2 (ATATAGGC) alleles. ITS sequencing is a useful technique for genotyping the three C. neoformans varieties and for subtyping within C. neoformans var. gattii.     

Mating types and serotypes of Cryptococcus neoformans isolated in Japan

Thirty-two isolates of Cryptococcus neoformans from patients and one from the wild obtained in Japan were characterized for their serotype, self-fertility, and mating behaviour by crossing them with two mating types of Filobasidiella neoformans var. neoformans and F. neoformans var. bacillispora. Of the 32 isolates from patients, 31 were of serotype A and the remaining one was of serotype D. Although these 32 isolates were all self-sterile, 23 serotype A and one serotype D isolates produced a complete sexual state when mixed with the alpha mating type of F. neoformans var. neoformans. The one natural isolate was of serotype A-D and self-fertile. The Japanese clinical isolates of C. neoformans appear to be predominantly serotype A and alpha mating type of F. neoformans var. neoformans as is the case in the U.S.A.