Project description:A singleplex PCR assay using a single primer pair targeting the putative sugar transporter gene was developed here to distinguish Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii according to the distinct size of the amplicon. The interspecies and intravarietal hybrids were also characterized on the basis of distinct combined profiles of amplicons. This PCR assay is a rapid, simple, and reliable approach suitable for laboratory diagnoses and large-scale epidemiologic studies.
Project description:Rapid identification of Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii is imperative for facilitation of prompt treatment of cryptococcosis and for understanding the epidemiology of the disease. Our purpose was to evaluate a test algorithm incorporating commercial rapid biochemical tests, differential media, and DNA sequence analysis that will allow us to differentiate these taxa rapidly and accurately. We assessed 147 type, reference, and clinical isolates, including 6 other Cryptococcus spp. (10 isolates) and 14 other yeast species (24 isolates), using a 4-hour urea broth test (Remel), a 24-hour urea broth test (Becton Dickinson), a 4-hour caffeic acid disk test (Hardy Diagnostics and Remel), 40- to 44-hour growth assessment on l-canavanine glycine bromothymol blue (CGB) agar, and intergenic spacer (IGS) sequence analysis. All 123 Cryptococcus isolates hydrolyzed urea, along with 7 isolates of Rhodotorula and Trichosporon. Eighty-five of 86 C. neoformans (99%) and 26 of 27 C. gattii (96%) isolates had positive caffeic acid results, unlike the other cryptococci (0/10) and yeast species (0/24). Together, these two tests positively identified virtually all C. neoformans/C. gattii isolates (98%) within 4 h. CGB agar or IGS sequencing further differentiated these isolates within 48 h. On CGB, 25 of 27 (93%) C. gattii strains induced a blue color change, in contrast to 0 of 86 C. neoformans isolates. Neighbor-joining cluster analysis of IGS sequences differentiated C. neoformans var. grubii, C. neoformans var. neoformans, and C. gattii. Based on these results, we describe a rapid identification algorithm for use in a microbiology laboratory to distinguish clinically relevant Cryptococcus spp.
Project description:The vast burden of cryptococcal meningitis occurs in immunosuppressed patients, driven by HIV, and is caused by Cryptococcus neoformans var. grubii. We previously reported cryptococcal meningitis in Vietnam arising atypically in HIV uninfected, apparently immunocompetent patients, caused by a single amplified fragment length polymorphism (AFLP) cluster of C. neoformans var. grubii (VNI?). This variant was less common in HIV infected individuals; it remains unclear why this lineage is associated with apparently immunocompetent patients. To study this host tropism we aimed to further our understanding of clinical phenotype and genomic variation within Vietnamese C. neoformans var. grubii. After performing MLST on C. neoformans clinical isolates we identified 14 sequence types (STs); ST5 correlated with the VNI? cluster. We next compared clinical phenotype by lineage and found HIV infected patients with cryptococcal meningitis caused by ST5 organisms were significantly more likely to have lymphadenopathy (11% vs. 4%, p = 0.05 Fisher's exact test) and higher blood lymphocyte count (median 0.76 versus 0.55 X109 cells/L, p = 0.001, Kruskal-Wallis test). Furthermore, survivors of ST5 infections had evidence of worse disability outcomes at 70 days (72.7% (40/55) in ST5 infections versus 57.1% (52/91) non-ST5 infections (OR 2.11, 95%CI 1.01 to 4.41), p = 0.046). To further investigate the relationship between strain and disease phenotype we performed genome sequencing on eight Vietnamese C. neoformans var. grubii. Eight genome assemblies exhibited >99% nucleotide sequence identity and we identified 165 kbp of lineage specific to Vietnamese isolates. ST5 genomes harbored several strain specific regions, incorporating 19 annotated coding sequences and eight hypothetical proteins. These regions included a phenolic acid decarboxylase, a DEAD-box ATP-dependent RNA helicase 26, oxoprolinases, a taurine catabolism dioxygenase, a zinc finger protein, membrane transport proteins and various drug transporters. Our work outlines the complexity of genomic pathogenicity in cryptococcal infections and identifies a number of gene candidates that may aid the disaggregation of the pathways associated with the pathogenesis of Cryptococcus neoformans var. grubii.
Project description:UNLABELLED:The accumulation of genomic structural variation between closely related populations over time can lead to reproductive isolation and speciation. The fungal pathogen Cryptococcus is thought to have recently diversified, forming a species complex containing members with distinct morphologies, distributions, and pathologies of infection. We have investigated structural changes in genomic architecture such as inversions and translocations that distinguish the most pathogenic variety, Cryptococcus neoformans var. grubii, from the less clinically prevalent Cryptococcus neoformans var. neoformans and Cryptococcus gattii. Synteny analysis between the genomes of the three Cryptococcus species/varieties (strains H99, JEC21, and R265) reveals that C. neoformans var. grubii possesses surprisingly few unique genomic rearrangements. All but one are relatively small and are shared by all molecular subtypes of C. neoformans var. grubii. In contrast, the large translocation peculiar to the C. neoformans var. grubii type strain is found in all tested subcultures from multiple laboratories, suggesting that it has possessed this rearrangement since its isolation from a human clinical sample. Furthermore, we find that the translocation directly disrupts two genes. The first of these encodes a novel protein involved in metabolism of glucose at human body temperature and affects intracellular levels of trehalose. The second encodes a homeodomain-containing transcription factor that modulates melanin production. Both mutations would be predicted to increase pathogenicity; however, when recreated in an alternate genetic background, these mutations do not affect virulence in animal models. The type strain of C. neoformans var. grubii in which the majority of molecular studies have been performed is therefore atypical for carbon metabolism and key virulence attributes. IMPORTANCE:The fungal pathogen Cryptococcus is a major cause of mortality among the immunocompromised population, primarily in AIDS patients of sub-Saharan Africa. Most research into the particular variety of Cryptococcus responsible for the vast majority of infections, Cryptococcus neoformans var. grubii, is performed using the type strain isolated in 1978 from a Hodgkin's disease patient from North Carolina. We have determined that this particular isolate contains a chromosomal translocation that directly interrupts two genes, which all descendants of this strain from various research laboratories appear to possess. Disruption of these two genes affects multiple virulence factors of Cryptococcus, particularly the ability to grow at human body temperature, which could have wide-ranging implications for molecular genetic studies and virulence assays using this important strain.
Project description:Most of the species of fungi that cause disease in mammals, including Cryptococcus neoformans var. grubii (serotype A), are exogenous and non-contagious. Cryptococcus neoformans var. grubii is associated worldwide with avian and arboreal habitats. This airborne, opportunistic pathogen is profoundly neurotropic and the leading cause of fungal meningitis. Patients with HIV/AIDS have been ravaged by cryptococcosis--an estimated one million new cases occur each year, and mortality approaches 50%. Using phylogenetic and population genetic analyses, we present evidence that C. neoformans var. grubii may have evolved from a diverse population in southern Africa. Our ecological studies support the hypothesis that a few of these strains acquired a new environmental reservoir, the excreta of feral pigeons (Columba livia), and were globally dispersed by the migration of birds and humans. This investigation also discovered a novel arboreal reservoir for highly diverse strains of C. neoformans var. grubii that are restricted to southern Africa, the mopane tree (Colophospermum mopane). This finding may have significant public health implications because these primal strains have optimal potential for evolution and because mopane trees contribute to the local economy as a source of timber, folkloric remedies and the edible mopane worm.
Project description:Although cryptococcosis is usually associated with respiratory and neurologic signs in domestic species (such as sneeze, cough, nasal discharge, seizures, ataxia), clinical manifestations of the disease may be more subtle and nonspecific. A 3-year-old male castrated Boxer dog presented with a history of chronic vomiting, diarrhea, weight loss, and lethargy. At no time had respiratory or neurologic signs been noted by the owners or the primary care veterinarian. Palpation of an abdominal mass revealed an atypical lesion location: a large (16 × 9 × 7 cm) mass at the root of the mesentery. Diagnosis was achieved through cytology of this mass and a positive serologic Cryptococcus capsular antigen titer; polymerase chain reaction was utilized for speciation of the abdominal isolate as Cryptococcus neoformans variety grubii. The animal was euthanized due to poor prognosis. After necropsy and histopathologic analysis, the mesenteric mass and associated lymph nodes were identified as large fungal granulomas. This is a rare manifestation of cryptococcosis, involving several visceral organs, with no remaining evidence of the route of entry of the organism. As prompt diagnosis of mycotic illness is paramount to successful management, this case indicates that cryptococcal infection should be considered as a differential diagnosis in dogs with gastrointestinal signs and lymphadenopathy. The protean nature of cryptococcosis is discussed within the context of a brief review of emerging and unresolved issues in pathogenesis.
Project description:Cryptococcus neoformans is a human-pathogenic fungus that has evolved into three distinct varieties that infect most prominently the central nervous system. A sexual cycle involving haploid cells of a and alpha mating types has been reported for two varieties (C. neoformans var. neoformans, serotype D, and C. neoformans var. gattii, serotypes B and C), yet the vast majority of infections involve a distinct variety (C. neoformans var. grubii, serotype A) that has been thought to be clonal and restricted to the alpha mating type. We recently identified the first serotype A isolate of the a mating type which had been thought to be extinct (strain 125.91). Here we report that this unusual strain can mate with a subset of pathogenic serotype A strains to produce a filamentous dikaryon with fused clamp connections, basidia, and viable recombinant basidiospores. One meiotic segregant mated poorly with the serotype A reference strain H99 but robustly with a crg1 mutant that lacks a regulator of G protein signaling and is hyperresponsive to mating pheromone. This meiotic segregant was used to create congenic a and alpha mating type serotype A strains. Virulence tests with rabbit and murine models of cryptococcal meningitis showed that the serotype A congenic a and alpha mating type strains had equivalent virulence in animal models, in contrast to previous studies linking the alpha mating type to increased virulence in congenic serotype D strains. Our studies highlight a role for sexual recombination in the evolution of a human fungal pathogen and provide a robust genetic platform to establish the molecular determinants of virulence.
Project description:Cryptococcus neoformans var. grubii is the causative agent of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals, typically human immunodeficiency virus/AIDS patients from developing countries. Despite the worldwide emergence of this ubiquitous infection, little is known about the global molecular epidemiology of this fungal pathogen. Here we sequence the genomes of 188 diverse isolates and characterize the major subdivisions, their relative diversity, and the level of genetic exchange between them. While most isolates of C. neoformans var. grubii belong to one of three major lineages (VNI, VNII, and VNB), some haploid isolates show hybrid ancestry including some that appear to have recently interbred, based on the detection of large blocks of each ancestry across each chromosome. Many isolates display evidence of aneuploidy, which was detected for all chromosomes. In diploid isolates of C. neoformans var. grubii (serotype AA) and of hybrids with C. neoformans var. neoformans (serotype AD) such aneuploidies have resulted in loss of heterozygosity, where a chromosomal region is represented by the genotype of only one parental isolate. Phylogenetic and population genomic analyses of isolates from Brazil reveal that the previously "African" VNB lineage occurs naturally in the South American environment. This suggests migration of the VNB lineage between Africa and South America prior to its diversification, supported by finding ancestral recombination events between isolates from different lineages and regions. The results provide evidence of substantial population structure, with all lineages showing multi-continental distributions; demonstrating the highly dispersive nature of this pathogen.
Project description:Iron, copper, and zinc are essential for all living organisms. Moreover, the homeostasis of these metals is vital to microorganisms during pathogenic interactions with a host. Most pathogens have developed specific mechanisms for the uptake of micronutrients from their hosts in order to counteract the low availability of essential ions in infected tissues. We report here an analysis of genes potentially involved in iron, copper, and zinc uptake and homeostasis in the fungal pathogens Paracoccidioides brasiliensis, Cryptococcus neoformans var. grubii, and Cryptococcus gattii. Although prior studies have identified certain aspects of metal regulation in Cryptococcus species, little is known regarding the regulation of these elements in P. brasiliensis. We also present amino acid sequences analyses of deduced proteins in order to examine possible conserved domains. The genomic data reveals, for the first time, genes associated to iron, copper, and zinc assimilation and homeostasis in P. brasiliensis. Furthermore, analyses of the three fungal species identified homologs to genes associated with high-affinity uptake systems, vacuolar and mitochondrial iron storage, copper uptake and reduction, and zinc assimilation. However, homologs to genes involved in siderophore production were only found in P. brasiliensis. Interestingly, in silico analysis of the genomes of P. brasiliensisPb01, Pb03, and Pb18 revealed significant differences in the presence and/or number of genes involved in metal homeostasis, such as in genes related to iron reduction and oxidation. The broad analyses of the genomes of P. brasiliensis, C. neoformans var. grubii, and C. gattii for genes involved in metal homeostasis provide important groundwork for numerous interesting future areas of investigation that are required in order to validate and explore the function of the identified genes and gene pathways.
Project description:The basidiomycetous yeast Cryptococcus neoformans is an important human fungal pathogen. Two varieties, C. neoformans var. neoformans and C. neoformans var. gattii, have been identified. Both are heterothallic with two mating types, MATa and MATalpha. Some rare isolates are self-fertile and are considered occasional diploid or aneuploid strains. In the present study, 133 isolates, mostly from Italian patients, were investigated to detect the presence of diploid strains in the Igiene Università Milano culture collection. All of the diploid isolates were further investigated by different methods to elucidate their origins. Forty-nine diploid strains were identified by flow cytometry. PCR fingerprinting using the (GACA)(4) primer showed that the diploid state was associated with two specific genotypes identified as VN3 and VN4. Determination of mating type on V8 juice medium confirmed that the majority of the strains were sterile. PCR and dot blotting using the two pheromone genes (MFa and MFalpha) as probes identified 36 of the 49 diploid isolates as MATa/alpha. The results of pheromone gene sequencing showed that two allelic MFalpha genes exist and are distinct for serotypes A and D. In contrast, the MFa gene sequence was conserved in both serotype alleles. Amplification of serotype-specific STE20 alleles demonstrated that the diploid strains contained one mating locus inherited from a serotype A parent and one inherited from a serotype D parent. The present results suggest that diploid isolates may be common among the C. neoformans population and that in Italy and other European countries serotype A and D populations are not genetically isolated but are able to recombine by sexual reproduction.