Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

McMaster University

Cryptococcus neoformans is a basidiomycete fungus capable of causing deadly meningoenchephilitis, primarily in immunocompromised individuals. Formerly, C. neoformans was composed of two divergent lineages, but these have recently been elevated to species status, now C. neoformans (formerly C. neoformans var. grubii) and C. deneoformans (formerly C. neoformans var. neoformans). While both species can cause deadly infections in humans, C. neoformans is much more prevalent in clinical settings than C. deneoformans However, the genetic factors contributing to their significant differences in virulence remain largely unknown. Quantitative trait locus (QTL) mapping is a powerful tool that can be used to identify genomic regions associated with phenotypic differences between strains. Here, we analyzed a hybrid cross between these two species and identified a total of 23 QTL, including five for melanin production, six for cell size, one for cell wall thickness, five for the frequency of capsule production, three for minimal inhibitory concentration (MIC) of fluconazole in broth, and three for MIC on solid medium. For the fluconazole resistance-associated QTL, three showed environment and/or concentration-specific effects. Our results provide a large number of candidate gene regions from which to explore the molecular bases for phenotypic differences between C. neoformans and C. deneoformans.

Identification of QTLs Associated with Virulence Related Traits and Drug Resistance in Cryptococcus neoformans.

Cryptococcosis is a common opportunistic fungal infection that often disseminates into the central nervous system, leading to meningitis. Production of melanin pigments during infections is one of the most important virulence factors of its causal agent, the human pathogenic yeast Cryptococcus neoformans species complex. However, almost nothing is known about the patterns of variation in melanin production among clinical and environmental strains and the potential effects of such variations on virulence. In this study, we assembled a global collection of C. neoformans var. neoformans strains and investigated their patterns of melanin variation and potential contributors to such variations. Our analyses revealed that genetic differences and genotype-environment interactions explained up to 59% and 43% of the population's melanin variance respectively, depending on the tested environments. In comparison, environmental factors alone contributed relatively little to melanin variance. We also identified specific changes within the LAC1 gene, whose protein product catalyzes melanin synthesis, to be associated with variable melanin levels. This study provides fresh insights into the origin and evolution of virulence traits in fungal pathogens while highlighting the complex interplay between genetic and environmental factors that lead to phenotypic variance.

Genetic Factors and Genotype-Environment Interactions Contribute to Variation in Melanin Production in the Fungal Pathogen Cryptococcus neoformans.

Amphotericin B (AMB) is one of the major antifungal drugs used in the management of aspergillosis and is especially recommended for treating triazole-resistant strains of . However, relatively little is known about the AMB susceptibility patterns of  in many parts of the world. This study aims to describe the AMB susceptibility patterns in Hamilton, Ontario, Canada.

Widespread amphotericin B-resistant strains of  in Hamilton, Canada.

The basidiomycetous yeasts of the Cryptococcus neoformans and Cryptococcus gattii species complexes (CNSC and CGSC respectively) are the causative agents of cryptococcosis, a set of life-threatening diseases affecting the central nervous system, lungs, skin, and other body sites of humans and other mammals. Both the CNSC and CGSC can be subdivided into varieties, serotypes, molecular types, and lineages based on structural variations, molecular characteristics and genetic sequences. Hybridization between the haploid lineages within and between the two species complexes is known to occur in natural and clinical settings, giving rise to intraspecific and interspecific diploid/aneuploid hybrid strains. Since their initial discovery in 1977, cryptococcal hybrids have been increasingly discovered in both clinical and environmental settings with over 30% of all cryptococcal infections in some regions of Europe being caused by hybrid strains. This review summarizes the major findings to date on cryptococcal hybrids, including their possible origins, prevalence, genomic profiles and phenotypic characteristics. Our analyses suggest that CNSC and CGSC can be an excellent model system for studying fungal hybridization.

Hybrids and hybridization in the Cryptococcus neoformans and Cryptococcus gattii species complexes.

Yeasts are unicellular fungi; they are found in a diverse range of natural habitats, including soil, aquatic environments, the surface of plants, and the skin and mucosal surfaces of animal hosts. A variety of yeasts have been found in the soil environment. However, most studies of soil yeasts have come from developed countries, and there is a dearth of research on soil yeasts in Africa. In this study, we analyzed 493 soil samples from nine geographical locations in Cameroon for yeasts, using a culture - based method. A total of 110 yeast isolates were obtained. Based on their sequences at the fungal barcode locus, the Internal Transcribed Spacer (ITS) regions of the nuclear ribosomal RNA gene cluster, the 110 yeast isolates were putatively identified as belonging to 16 yeast species, including 15 Ascomycetes and one Basidiomycete. Differences in yeast species distribution were observed among the analyzed geographic regions. PCR fingerprinting analyses identified a large number of genotypes among strains within each of the obtained yeast species. Significantly, there was little evidence of geographic clustering among yeast strains from any of the yeast species. Our results suggest that Cameroon contains significant yeast diversity and that gene flow is common among local and regional soil yeast populations.

Diversity and relationships among strains of culturable yeasts in agricultural soils in Cameroon.

Cryptococcus deneoformans is an opportunist yeast pathogen and causative agent of meningoencephalitis in humans. It is known to be mainly distributed in temperate climates. Most of our current understanding of this species has come from clinical isolates, leaving environmental populations largely unexplored. The Middle East remains one such underexplored area with no published study to date investigating cryptococcal diversity in soil. In this study, we identified 76 C. deneoformans isolates from a survey of 562 soil samples collected from six cities in Saudi Arabia. Multilocus sequence typing revealed the presence of two major sequence types (STs), ST160 (n = 63) and ST294 (n = 9), along with four singleton STs, three of which were novel. One novel ST, ST613, was likely a recombinant product between ST160 and ST294. Among the 76 isolates, 75 belonged to mating type (MAT)α while one isolate was MATa. Our analyses suggest that the Saudi Arabian C. deneoformans population likely reproduces both asexually and sexually in nature. Our study is the first to report the occurrence of C. deneoformans in a desert climate, representing a novel expansion to this species' currently known ecological niche.

Fantastic yeasts and where to find them: the discovery of a predominantly clonal Cryptococcus deneoformans population in Saudi Arabian soils.

The sister yeast species Cryptococcus neoformans (serotype A) and Cryptococcus deneoformans (serotype D) are causative agents of deadly cryptococcosis and fungal meningoencephalitis. These haploid yeasts can hybridise in nature, giving rise to AD hybrids that are predominantly diploid or aneuploid. Despite their increasing prevalence in clinical settings, much remains unknown about the allelic distribution patterns in AD hybrid strains.

Patterns of allele distribution in a hybrid population of the Cryptococcus neoformans species complex.

Hybridization is increasingly recognized as an important force impacting adaptation and evolution in many lineages of fungi. During hybridization, divergent genomes and alleles are brought together into the same cell, potentiating adaptation by increasing genomic plasticity. Here, we review hybridization in fungi by focusing on two fungal pathogens of animals. Hybridization is common between the basidiomycete yeast species   ×  , and hybrid genotypes are frequently found in both environmental and clinical settings. The two species show 10-15% nucleotide divergence at the genome level, and their hybrids are highly heterozygous. Though largely sterile and unable to mate, these hybrids can propagate asexually and generate diverse genotypes by nondisjunction, aberrant meiosis, mitotic recombination, and gene conversion. Under stress conditions, the rate of such genetic changes can increase, leading to rapid adaptation. Conversely, in hybrids formed between lineages of the chytridiomycete frog pathogen   (), the parental genotypes are considerably less diverged (0.2% divergent).  hybrids are formed from crosses between lineages that rarely undergo sex. A common theme in both species is that hybrids show genome plasticity via aneuploidy or loss of heterozygosity and leverage these mechanisms as a rapid way to generate genotypic/phenotypic diversity. Some hybrids show greater fitness and survival in both virulence and virulence-associated phenotypes than parental lineages under certain conditions. These studies showcase how experimentation in model species such as  can be a powerful tool in elucidating the genotypic and phenotypic consequences of hybridization.

Hybridization Facilitates Adaptive Evolution in Two Major Fungal Pathogens.

Author Correction: Genetic Factors and Genotype-Environment Interactions Contribute to Variation in Melanin Production in the Fungal Pathogen Cryptococcus neoformans.

Yeasts, broadly defined as unicellular fungi, fulfill essential roles in soil ecosystems as decomposers and nutrition sources for fellow soil-dwellers. Broad-scale investigations of soil yeasts pose a methodological challenge as metagenomics are of limited use for identifying this group of fungi. Here we characterize global soil yeast diversity using fungal DNA barcoding on 1473 yeasts cultured from 3826 soil samples obtained from nine countries in six continents. We identify mean annual precipitation and international air travel as two significant correlates with soil yeast community structure and composition worldwide. Evidence for anthropogenic influences on soil yeast communities, directly via travel and indirectly via altered rainfall patterns resulting from climate change, is concerning as we found common infectious yeasts frequently distributed in soil in several countries. Our discovery of 41 putative novel species highlights the continued need for culture-based studies to advance our knowledge of environmental yeast diversity.

Himeshi Samarasinghe

Department of Biology

McMaster University

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Himeshi Samarasinghe

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

McMaster University

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Department of Biology