Clonality and recombination in genetically differentiated subgroups of Cryptococcus gattii

Cryptococcus gattii is a pathogenic yeast that together with Cryptococcus neoformans causes cryptococcosis in humans and animals. High numbers of viable C. gattii propagules can be obtained from certain species of Australian Eucalyptus camaldulensis trees, and an epidemiological link between Eucalyptus colonization and human exposure has been proposed. However, the highest prevalence of C. gattii cryptococcosis occurs in Papua New Guinea and in regions of Australia where the eucalypt species implicated to date are not endemic. This study investigated the population structure of three geographically distinct clinical and veterinary populations of C. gattii from Australia and Papua New Guinea. All populations that consisted of a genotype found frequently in Australia (VGI) were strongly clonal and were highly differentiated from one another. Two populations of the less common VGII genotype from Sydney and the Northern Territory had population structures inferring recombination. In addition, there was some evidence of reduced genetic differentiation between these geographically remote regions. In a companion study presented in this issue, VGII isolates were overwhelmingly more fertile than those of the VGI genotype, giving biological support to the indirect assessment of sexual exchange. It appears that the VGI genotype propagates clonally on eucalypts in Australia and on an unknown substrate in Papua New Guinea, with infection initiated by an unidentified infectious propagule. VGII isolates are completing their life cycles and may be dispersed via sexually produced basidiospores, which are also likely to initiate respiratory infection.     

Genetic differentiation, recombination and clonal expansion in environmental populations of Cryptococcus gattii in India

Cryptococcus gattii is a ubiquitous eukaryotic pathogen capable of causing life-threatening infections in a wide variety of hosts, including both immunocompromised and immunocompetent humans. Since infections by C. gattii are predominantly obtained from environmental exposures, understanding environmental populations of this pathogen is critical, especially in countries like India with a large population and with environmental conditions conducive for the growth of C. gattii. In this study, we analysed 109 isolates of C. gattii obtained from hollows of nine tree species from eight geographic locations in India. Multilocus sequence typing was conducted for all isolates using nine gene fragments. All 109 isolates belonged to the VGI group and were mating type α. Population genetic analyses revealed limited evidence of recombination but unambiguous evidence for clonal reproduction and expansion. However, the observed clonal expansion has not obscured the significant genetic differentiation among populations from either different geographic areas or different host tree species. A positive correlation was observed between genetic distance and geographic distance. The results obtained here for environmental populations of C. gattii showed both similarities and differences with those of the closely related Cryptococcus neoformans var. grubii from similar locations and host tree species in India.     

格特隐球菌核基因、交配型位点和线粒体基因的多位点序列分型及重组分析

研究格特隐球菌VGI和VGII基因型菌株间线粒体基因重组与核基因、交配型位点重组间的关系。采用分子鉴定区分受试格特隐球菌的血清型、基因型和交配型;选取包含核基因和线粒体基因10个位点的多位点序列分型（ MLST）进行基因型和系统发育分析;采用同质性检验评价基因系谱间的一致性。多位点的序列和系统发育分析结合同质性检验表明,受试位点中,仅ATP6位点发现VGI和VGII菌株间基因的杂交和重组,该基因系谱与其余位点的基因系谱呈现不一致性。结果显示,受试的部分VGI菌株中的ATP6位点含有VGII菌株的基因序列,表明VGI和VGII菌株间线粒体基因的重组;没有发现核基因及交配型位点中两者间的重组,提示VGI和VGII菌株间线粒体基因的重组现象与交配行为无关。     

Promiscuous mitochondria in Cryptococcus gattii

Cryptococcus gattii is a primary pathogenic basidiomycetous yeast comprising four genotypic groups. Here we present data on two mitochondrial loci (MtLrRNA and ATP6 ). Two of the genotypic groups, namely amplified fragment length polymorphism (AFLP)5/VGIII and AFLP6/VGII, formed monophyletic lineages. The AFLP4/VGI genotypic group, however, possessed five different mitochondrial genotypes that did not form a monophyletic lineage. The majority of these isolates contained mitochondrial genomes that are partially identical to those found in isolates belonging to AFLP6/VGII, which is causing the ongoing and expanding Vancouver Island outbreak. Two out of four AFLP7/VGIV isolates contained an AFLP4/VGI allele of MtLrRNA. These observations are best explained by assuming a process of mitochondrial recombination. If this is true, mitochondrial recombination seems possible between cells belonging to different genotypic groups of C. gattii , especially between AFLP6/VGII or AFLP7/VGIV and AFLP4/VGI. We also have to assume that mitochondria, most likely, were transferred from cells belonging to AFLP6/VGII to AFLP4/VGI. As such a process of mitochondrial recombination is only possible after cell–cell conjugation, this may also allow the further exchange of genetic material, for example nuclear or plasmid in nature, between different genotypes of C. gattii . This may be relevant as it may provide a possible mechanism contributing to the modulation of virulence attributes of isolates, such as has been observed in the ongoing Vancouver Island outbreak of C. gattii .     

Cryptococcus neoformans/Cryptococcus gattii Species Complex in Southern Italy: An Overview on the Environmental Diffusion of Serotypes, Genotypes and Mating-Types

Given the lack of comprehensive molecular epidemiology studies in Reggio Calabria and Messina, Italy, we decided to perform an extensive environmental sampling to describe the current molecular epidemiology of C. neoformans/C. gattii species complex in southern Italy. In this study, we report the occurrence of serotypes, genotypes and mating-types of isolates of the C. neoformans/C. gattii species complex recovered from environmental sources. In addition, a number of environmental C. neoformans var. grubii strains, isolated in 1997 by our laboratory, were also retrospectively examined in order to compare their genotypes with those recently found and to infer the possible epidemiological changes in our country. One hundred and twenty-two isolates were identified as being C. neoformans, whereas only one was found to belong to C. gattii serotype B, genotype VGI and mating-type alpha. Our data revealed that all environmental isolates of C. neoformans recovered here as well as those previously isolated in 1997 belong to serotype A and genotype VNI and posses a mating-type alpha allele.     

Comparative gene genealogies indicate that two clonal lineages of Cryptococcus gattii in British Columbia resemble strains from other geographical areas

Cryptococcus gattii has recently emerged as a pathogen of humans and animals in the temperate climate of Vancouver Island, British Columbia (B.C.). The majority (approximately 95%) of the isolates from the island belong to the VGII molecular type, and the remainder belong to the VGI molecular type. The goals of this study were to compare patterns of molecular variation among C. gattii isolates from B.C. with those from different areas of the world and to investigate the population structure using a comparative gene genealogy approach. Our results indicate that the C. gattii population in B.C. comprises at least two divergent lineages, corresponding to previously identified VGI and VGII molecular types. The genealogical analysis of strains suggested a predominantly clonal population structure among B.C. isolates, while there was evidence for sexual recombination between different molecular types on a global scale. We found no geographic pattern of strain relationships, and nucleotide sequence comparisons revealed that genotypes among isolates from B.C. were also present among isolates from other areas of the world, indicating extensive strain dispersal. The nucleotide sequence diversity among isolates from B.C. was similar to that among isolates from other areas of the world.     

Clinical and environmental isolates of Cryptococcus gattii from Australia that retain sexual fecundity

Cryptococcus gattii is a primary pathogenic yeast that causes disease in both animals and humans. It is closely related to Cryptococcus neoformans and diverged from a common ancestor approximately 40 million years ago. While C. gattii has a characterized sexual cycle dependent upon a dimorphic region of the genome known as the MAT locus, mating has rarely been observed in this species. In this study, we identify for the first time clinical (both human and veterinary) and environmental isolates from Australia that retain sexual fecundity. A collection of 120 isolates from a variety of geographic locations was analyzed for molecular type, mating type, and the ability to develop mating structures when cocultured with fertile tester strains. Nine isolates produced dikaryotic filaments with paired nuclei, fused clamp connections, and basidiospores. DNA sequence analysis of three genes (URA5, the MATalpha-specific SXI1alpha gene, and the MATa-specific SXI2a gene) revealed little or no variability in URA5 and SXI2a, respectively. However across the 108 MATalpha strains sequenced, the SXI1alpha gene was found to exist as 11 different alleles. Phylogenetic analysis found most variation to occur in the more fertile genotypes. Although some lineages of Australian C. gattii have retained the ability to mate, the majority of isolates were sterile, suggesting that asexuality is the dominant mode of propagation in these populations.     

Genotype and mating type analysis of Cryptococcus neoformans and Cryptococcus gattii isolates from China that mainly originated from non-HIV-infected patients

Cryptococcosis has been reported to be mostly associated with non-HIV-related patients in China. However, little is known about the molecular characteristics of clinical isolates from the Cryptococcus neoformans species complex in this country. In this study, 115 clinical isolates were included. Molecular type VNI was the most representative ( n =103), followed by VGI ( n =8), VNIII ( n =2), VNIV ( n =1), and VGII ( n =1). With the exception of a serotype D mating type a isolate, all possessed the MAT α locus. Multilocus sequence typing (MLST) revealed that most Cryptococcus gattii isolates from China shared identical MLST profiles with the most common MLST genotype reported in the VGI group, and the only one VGII isolate resembled the Vancouver Island outbreak minor genotype. The C. gattii strains involved in this study were successfully grouped according to their molecular type and mating types by PCR-restriction fragment length polymorphism (RFLP) analysis of the GEF1 gene. Our results suggest that (1) in China, cryptococcosis is mostly caused by C. neoformans var. grubii (molecular type VNI), and mating type α; (2) The most common causative agents of C. gattii infection in China are closely related to a widely distributed MLST genotype; and (3) The PCR-RFLP analysis of the GEF1 gene has the potential to identify the molecular and mating types of C. gattii simultaneously.     

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.     

Cryptococcus neoformans var. gattii isolates from two Peruvian patients.

The ecology of Cryptococcus neoformans var. gatti is restricted to tropical and subtropical areas whereas C. neoformans var. neoformans is cosmopolitan. Perú is a country with three different well defined geographic areas, some of them have the conditions for the presence of the variety gattii. In order to determine the presence of the two varieties of C. neoformans in Perú, we made the C. neoformans differentiation from the clinical isolates. C. neoformans var. gattii was identified by their ability to assimilate D-proline. We tested 68 strains; only two of them were recognized as the variety gattii and were recovered from two patients without any predisposing factor. We described the clinical spectrum of these two patients, who were diagnosed with disseminated cryptococcosis and cryptococcal meningitis. Neither the presence of Eucalyptus camaldulensis nor Eucalyptus tereticornis has been reported in Perú. So there should exist other ecologic niches for the presence of C. neoformans var. gattii in our country, different from those mentioned.     

Divergence, hybridization, and recombination in the mitochondrial genome of the human pathogenic yeast Cryptococcus gattii

The inheritance of mitochondrial genes and genomes are uniparental in most sexual eukaryotes. This pattern of inheritance makes mitochondrial genomes in natural populations effectively clonal. Here, we examined the mitochondrial population genetics of the emerging human pathogenic fungus Cryptococcus gattii . The DNA sequences for five mitochondrial DNA fragments were obtained from each of 50 isolates belonging to two evolutionary divergent lineages, VGI and VGII. Our analyses revealed a greater sequence diversity within VGI than that within VGII, consistent with observations of the nuclear genes. The combined analyses of all five gene fragments indicated significant divergence between VGI and VGII. However, the five individual genealogies showed different relationships among the isolates, consistent with recent hybridization and mitochondrial gene transfer between the two lineages. Population genetic analyses of the multilocus data identified evidence for predominantly clonal mitochondrial population structures within both lineages. Interestingly, there were clear signatures of recombination among mitochondrial genes within the VGII lineage. Our analyses suggest historical mitochondrial genome divergence within C. gattii , but there is evidence for recent hybridization and recombination in the mitochondrial genome of this important human yeast pathogen.     

First environmental isolation of Cryptococcus neoformans var. neoformans and var. gatti from the Gharbia Governorate, Egypt

Flowers from two Eucalyptus camaldulensis trees in the Qutur area and one tree from the Tanta area yielded three isolates of Cryptococcus neoformans var. gattii. Pigeon and sparrow droppings were also investigated for the occurrence of C. neoformans within the study area. Ninety five isolates of the neoformans variety of C. neoformans were recovered from 550 samples of avian droppings.     

Isolation and characterization of Cryptococcus neoformans var. gattii from samples of Eucalyptus camaldulensis in Mexico city.

The habitat of Cryptococcus neoformans var. gattii is not fully known in Mexico. We investigated the relationship of the yeast with Eucalyptus camaldulensis soil in three main Avenues of the city. A total of 135 trees of the species E. camaldulensis, were selected. Samples were taken in duplicate from the ground containing vegetable debris, tree cortex, leaves and flowers. Isolation of the yeast was made on Guizotia abyssinica media, using Staib technique. The identification was accomplished by biochemical and morphologic tests, and caraterization of the variety was made by bromotimol canavanine-glycine-blue (CGB) and D-proline tests. Isolation of 87 strains of Cryptococcus spp. was acomplished and eight of them were identified as C. neoformans var. gattii. These findings confirmed the close relationship of C. neoformans var. gattii and E. camaldulensis. To our knowledge this is the first report concerning the isolation of this variety from E. camaldulensis trees in Mexico.     

<Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> Isolated from Passerine and Psittacine Bird Excreta in the State of Paraná, Brazil

Cryptococcus neoformans is an opportunistic basidiomycete yeast that causes life-threatening infections as meningoencephalitis primarily in immunocompromised hosts, generally associated with AIDS. The source of this organism is mainly pigeon excreta; however, other avian species' excreta are implicated as a source of this yeast. The occurrence of C. neoformans and Cryptococcus gattii in bird excreta in the state of Paraná in Brazil was determined in this study. A total of 141 samples of Passerine and Psittacine excreta from captive birds were collected. Additionally, 25 clinical samples from Hospital de Clínicas, in the state of Paraná were also analyzed. The determination of molecular and mating type of the isolates was performed by PCR fingerprinting, multiplex PCR, and mating type PCR. Cryptococcus neoformans var. grubii (VNI) was isolated from 36 (25.53%) of Passerine and Psittacine excreta samples. Almost all clinical samples, except one (C. gattii VGI), were classified as C. neoformans var. grubii (VNI). All environmental and clinical isolates were mating type alpha. These findings reinforce that, besides pigeon excreta, the excreta of these birds can also be a reservoir of C. neoformans in domestic and public environments and is of zoonotic importance to immunocompromised patients.     

Whole genome sequence analysis of Cryptococcus gattii from the Pacific Northwest reveals unexpected diversity

A recent emergence of Cryptococcus gattii in the Pacific Northwest involves strains that fall into three primarily clonal molecular subtypes: VGIIa, VGIIb and VGIIc. Multilocus sequence typing (MLST) and variable number tandem repeat analysis appear to identify little diversity within these molecular subtypes. Given the apparent expansion of these subtypes into new geographic areas and their ability to cause disease in immunocompetent individuals, differentiation of isolates belonging to these subtypes could be very important from a public health perspective. We used whole genome sequence typing (WGST) to perform fine-scale phylogenetic analysis on 20 C. gattii isolates, 18 of which are from the VGII molecular type largely responsible for the Pacific Northwest emergence. Analysis both including and excluding (289,586 SNPs and 56,845 SNPs, respectively) molecular types VGI and VGIII isolates resulted in phylogenetic reconstructions consistent, for the most part, with MLST analysis but with far greater resolution among isolates. The WGST analysis presented here resulted in identification of over 100 SNPs among eight VGIIc isolates as well as unique genotypes for each of the VGIIa, VGIIb and VGIIc isolates. Similar levels of genetic diversity were found within each of the molecular subtype isolates, despite the fact that the VGIIb clade is thought to have emerged much earlier. The analysis presented here is the first multi-genome WGST study to focus on the C. gattii molecular subtypes involved in the Pacific Northwest emergence and describes the tools that will further our understanding of this emerging pathogen.     

Phenotype characterization of environmental Cryptococcus neoformans isolates

Cryptococcosis is caused by the three varieties of C. neoformans with physiological and virulence differences, some of which have been studied to determine biological aspects of this microorganism. The phenotypical aspects of environmental isolates from varieties grubii and gattii were evaluated to establish differences associated with their life cycle and virulence. To this end, 28 and 31 strains of C. neoformans serotypes A (var. grubii) and C (var. gattii) were studied. The microscopic and macroscopic morphology on Sabouraud agar and soils, growth rate at 37 degrees C, production of 22 extracellular enzymes, haploid fructification, mating type, killer toxin sensitivity patterns and virulence in BALB/c mice were evaluated. No differences were observed between the two varieties regarding microscopic and macroscopic morphology or growth at 37 degrees C (p > 0.05). However, a decrease in the cellular and capsular sizes of yeast in soil, as compared to Sabouraud, was observed (p < 0.05). Additionally, higher enzimatic activity of proteases, phospholipases, phenoloxidase and beta-glucosidase was observed in var. grubii isolates as compared to var. gattii (p < 0.05). In both varieties, structures related with haploid fruitification were observed and all isolates were mating type alpha. Killer toxin sensitivity patterns of the isolates of var. grubii were I and II; in contrast, in var. gattii, seven different patterns were found: I, V, IX-XIII. In the animal model we found that 12 of 22 (54.5%) isolates of var. grubii caused the death of the mice during the observation period, while none of the 14 var. gattii isolates caused it. The decrease in capsular and cellular sizes of the yeast in soil and the frequency of mating type alpha with structures related to haploid fructification suggest an important mechanism of production of infectious particles in nature. Additionally, greater enzimatic activity of var. grubii can be associated with the virulence in the animal model.     

A Decade of Experience: Cryptococcus gattii in British Columbia

It has been over a decade since Cryptococcus gattii was first recognized as the causative organism of an outbreak of cryptococcosis on Vancouver Island, British Columbia. A number of novel observations have been associated with the study of this emergent pathogen. A novel genotype of C. gattii, VGIIa was described as the major genotype associated with clinical disease. Minor genotypes, VGIIb and VGI, are also responsible for disease in British Columbians, in both human and animal populations. The clinical major genotype VGIIa and minor genotype VGIIb are identical to C. gattii isolated from the environment of Vancouver Island. There is more heterogeneity in VGI, and a clear association with the environment is not apparent. Between 1999 and 2010, there have been 281 cases of C. gattii cryptococcosis. Risk factors for infection are reported to be age greater than 50 years, history of smoking, corticosteroid use, HIV infection, and history of cancer or chronic lung disease. The major C. gattii genotype VGIIa is as virulent in mice as the model Cryptococcus, H99 C. neoformans, although the outbreak strain produces a less protective inflammatory response in C57BL/6 mice. The minor genotype VGIIb is significantly less virulent in mouse models. Cryptococcus gattii is found associated with native trees and soil on Vancouver Island. Transiently positive isolations have been made from air and water. An ecological niche for this organism is associated within a limited biogeoclimatic zone characterized by daily average winter temperatures above freezing.     

First Reported Case of Cryptococcus gattii in the Southeastern USA: Implications for Travel-Associated Acquisition of an Emerging Pathogen

In 2007, the first confirmed case of Cryptococcus gattii was reported in the state of North Carolina, USA. An otherwise healthy HIV negative male patient presented with a large upper thigh cryptococcoma in February, which was surgically removed and the patient was started on long-term high-dose fluconazole treatment. In May of 2007, the patient presented to the Duke University hospital emergency room with seizures. Magnetic resonance imaging revealed two large CNS lesions found to be cryptococcomas based on brain biopsy. Prior chest CT imaging had revealed small lung nodules indicating that C. gattii spores or desiccated yeast were likely inhaled into the lungs and dissemination occurred to both the leg and CNS. The patient''s travel history included a visit throughout the San Francisco, CA region in September through October of 2006, consistent with acquisition during this time period. Cultures from both the leg and brain biopsies were subjected to analysis. Based on phenotypic and molecular methods, both isolates were C. gattii, VGI molecular type, and distinct from the Vancouver Island outbreak isolates. Based on multilocus sequence typing of coding and noncoding regions and virulence in a heterologous host model, the leg and brain isolates are identical, but the two differed in mating fertility. Two clinical isolates, one from a transplant recipient in San Francisco and the other from Australia, were identical to the North Carolina clinical isolate at all markers tested. Closely related isolates that differ at only one or a few noncoding markers are present in the Australian environment. Taken together, these findings support a model in which C. gattii VGI was transferred from Australia to California, possibly though an association with its common host plant E. camaldulensis, and the patient was exposed in San Francisco and returned to present with disease in North Carolina.     

Cryptococcus gattii Infection in an Immunocompetent Patient from Southern Italy

Cryptococcus gattii has becoming more prevalent in temperate climate zones, during the past decades. We describe a C. gattii serotype B infection in an immunocompetent Italian patient with sclerosing cholangitis. The patient traveled once to Eastern Canada and otherwise no other countries than Italy were visited. Molecular analysis revealed that the C. gattii isolate belong to genotype AFLP4/VGI and has mating-type α which is the most common genotype in the Mediterranean environment. The C. gattii strain was found to be closely related, but not identical, to other C. gattii strains from the Mediterranean area.     

Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil

The molecular types of 443 Brazilian isolates of Cryptococcus neoformans and Cryptococcus gattii were analyzed to determine their geographic distribution within Brazil and their underlying host conditions. The following data, imported from previous epidemiological studies as well as two culture collections, were analyzed for: place of isolation, source (clinical or environmental), host risk factors, species, serotype, mating type, and molecular type. Molecular typing by PCR-fingerprinting using primers for the minisatellite-specific core sequence of the wild-type phage M13 or microsatellites [(GACA)4, (GTG)5], restriction fragment length polymorphism of URA5 gene analysis, and/or amplified fragment length polymorphism (AFLP) identified eight major genotypes: VNI/AFLP1, VNII/AFLP1A, VNIII/AFLP2, and VNIV/AFLP3 for C. neoformans, and VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5, and VGIV/AFLP7 for C. gattii. The most common molecular type found in Brazil was VNI (64%), followed by VGII (21%), VNII (5%), VGIII (4%), VGI and VNIV (3% each), and VNIII (< 1%). Primary cryptococcosis caused by the molecular type VGII (serotype B, MAT) prevails in immunocompetent hosts in the North and Northeast regions, disclosing an endemic regional pattern for this specific molecular type in the Northern Brazil.