Patients infected with the fungal pathogen Cryptococcus are most effectively treated with a combination of 5-fluorocytosine (5FC) and amphotericin B. 5FC acts as a prodrug, which is converted into ...toxic 5-fluorouracil (5FU) upon uptake into fungal cells. However, the pathogen frequently develops resistance through unclear mechanisms. Here we show that resistance to 5FC in Cryptococcus deuterogattii is acquired more frequently in isolates with defects in DNA mismatch repair that confer an elevated mutation rate. We use whole genome sequencing of 16 independent isolates to identify mutations associated with 5FC resistance in vitro. We find mutations in known resistance genes (FUR1 and FCY2) and in a gene UXS1, previously shown to encode an enzyme that converts UDP-glucuronic acid to UDP-xylose for capsule biosynthesis, but not known to play a role in 5FC metabolism. Mutations in UXS1 lead to accumulation of UDP-glucuronic acid and alterations in nucleotide metabolism, which appear to suppress toxicity of both 5FC and its toxic derivative 5FU.
Pathogenic microbes confront an evolutionary conflict between the pressure to maintain genome stability and the need to adapt to mounting external stresses. Bacteria often respond with elevated ...mutation rates, but little evidence exists of stable eukaryotic hypermutators in nature. Whole genome resequencing of the human fungal pathogen
identified an outbreak lineage characterized by a nonsense mutation in the mismatch repair component
This defect results in a moderate mutation rate increase in typical genes, and a larger increase in genes containing homopolymer runs. This allows facile inactivation of genes with coding homopolymer runs including
, which encodes the target of the immunosuppresive antifungal drugs FK506 and rapamycin. Our study identifies a eukaryotic hypermutator lineage spread over two continents and suggests that pathogenic eukaryotic microbes may experience similar selection pressures on mutation rate as bacterial pathogens, particularly during long periods of clonal growth or while expanding into new environments.
Malassezia
species represent the most common fungal inhabitant of the mammalian skin microbiome and are natural skin commensal flora. However, these fungi are also associated with a variety of ...clinical skin disorders. Recent studies have reported associations of
Malassezia
with Crohn’s disease and pancreatic cancer, further implicating this fungal genus in inflammatory and neoplastic disease states. Because
M. sympodialis
has lost genes involved in RNA interference (RNAi), we hypothesized
Malassezia
could harbor dsRNA mycoviruses. Indeed, we identified a novel mycovirus of the totivirus family in several
Malassezia
species and characterized the MsMV1 mycovirus of
M. sympodialis
. We found conditions that lead to curing of the virus and analyzed isogenic virus-infected/virus-cured strains to determine MsMV1 genetic and pathogenic impacts. MsMV1 induces a strong overexpression of transcription factors and ribosomal genes, while downregulating cellular metabolism. Moreover, MsMV1 induced a significantly higher level of beta interferon expression in cultured macrophages. This study sheds light on the mechanisms of pathogenicity of
Malassezia
, focusing on a previously unidentified novel mycovirus.
ABSTRACT
Mycoviruses infect fungi, and while most persist asymptomatically, there are examples of mycoviruses having both beneficial and detrimental effects on their host. Virus-infected
Saccharomyces
and
Ustilago
strains exhibit a killer phenotype conferring a growth advantage over uninfected strains and other competing yeast species, whereas hypovirus-infected
Cryphonectria parasitica
displays defects in growth, sporulation, and virulence. In this study, we identify a double-stranded RNA (dsRNA) mycovirus in five
Malassezia
species. Sequence analysis reveals it to be a totivirus with two dsRNA segments: a larger 4.5-kb segment with genes encoding components for viral replication and maintenance, and a smaller 1.4-kb segment encoding a novel protein. Furthermore, transcriptome sequencing (RNA-seq) of virus-infected versus virus-cured
Malassezia sympodialis
revealed an upregulation of dozens of ribosomal components in the cell, suggesting the virus modifies the transcriptional and translational landscapes of the cell. Given that
Malassezia
is the most abundant fungus on human skin, we assessed the impact of the mycovirus in a murine epicutaneous infection model. Although infection with virus-infected strains was not associated with an increased inflammatory response, we did observe enhanced skin colonization in one of two virus-infected
M. sympodialis
strains. Noteworthy, beta interferon expression was significantly upregulated in bone marrow-derived macrophages when challenged with virus-infected, compared to virus-cured,
M. sympodialis
, suggesting that the presence of the virus can induce an immunological response. Although many recent studies have illuminated how widespread mycoviruses are, there are relatively few in-depth studies about their impact on disease caused by the host fungus. We describe here a novel mycovirus in
Malassezia
and its possible implications in pathogenicity.
IMPORTANCE
Malassezia
species represent the most common fungal inhabitant of the mammalian skin microbiome and are natural skin commensal flora. However, these fungi are also associated with a variety of clinical skin disorders. Recent studies have reported associations of
Malassezia
with Crohn’s disease and pancreatic cancer, further implicating this fungal genus in inflammatory and neoplastic disease states. Because
M. sympodialis
has lost genes involved in RNA interference (RNAi), we hypothesized
Malassezia
could harbor dsRNA mycoviruses. Indeed, we identified a novel mycovirus of the totivirus family in several
Malassezia
species and characterized the MsMV1 mycovirus of
M. sympodialis
. We found conditions that lead to curing of the virus and analyzed isogenic virus-infected/virus-cured strains to determine MsMV1 genetic and pathogenic impacts. MsMV1 induces a strong overexpression of transcription factors and ribosomal genes, while downregulating cellular metabolism. Moreover, MsMV1 induced a significantly higher level of beta interferon expression in cultured macrophages. This study sheds light on the mechanisms of pathogenicity of
Malassezia
, focusing on a previously unidentified novel mycovirus.
RNAi is a ubiquitous pathway that serves central functions throughout eukaryotes, including maintenance of genome stability and repression of transposon expression and movement. However, a number of ...organisms have lost their RNAi pathways, including the model yeast Saccharomyces cerevisiae, the maize pathogen Ustilago maydis, the human pathogen Cryptococcus deuterogattii, and some human parasite pathogens, suggesting there may be adaptive benefits associated with both retention and loss of RNAi. By comparing the RNAi-deficient genome of the Pacific Northwest Outbreak C. deuterogattii strain R265 with the RNAi-proficient genomes of the Cryptococcus pathogenic species complex, we identified a set of conserved genes that were lost in R265 and all other C. deuterogattii isolates examined. Genetic and molecular analyses reveal several of these lost genes play roles in RNAi pathways. Four novel components were examined further. Znf3 (a zinc finger protein) and Qip1 (a homolog of N. crassa Qip) were found to be essential for RNAi, while Cpr2 (a constitutive pheromone receptor) and Fzc28 (a transcription factor) are involved in sex-induced but not mitosis-induced silencing. Our results demonstrate that the mitotic and sex-induced RNAi pathways rely on the same core components, but sex-induced silencing may be a more specific, highly induced variant that involves additional specialized or regulatory components. Our studies further illustrate how gene network polymorphisms involving known components of key cellular pathways can inform identification of novel elements and suggest that RNAi loss may have been a core event in the speciation of C. deuterogattii and possibly contributed to its pathogenic trajectory.
The genus
includes yeasts that are commonly found on the skin or hair of animals and humans as commensals and are associated with a number of skin disorders. We have previously developed an
...transformation system effective for both targeted gene deletion and insertional mutagenesis in
and
In the present study, these molecular resources were applied to characterize the immunophilin FKBP12 as the target of tacrolimus (FK506), ascomycin, and pimecrolimus, which are calcineurin inhibitors that are used as alternatives to corticosteroids in the treatment of inflammatory skin disorders such as those associated with
species. While
and
showed
sensitivity to these agents,
Δ mutants displayed full resistance to all three of them, confirming that FKBP12 is the target of these calcineurin inhibitors and is essential for their activity. We found that calcineurin inhibitors act additively with fluconazole through an FKBP12-dependent mechanism. Spontaneous
isolates resistant to calcineurin inhibitors had mutations in the gene encoding FKBP12 in regions predicted to affect the interactions between FKBP12 and FK506 based on structural modeling. Due to the presence of homopolymer nucleotide repeats in the gene encoding FKBP12, an
Δ hypermutator of
was engineered and exhibited an increase of more than 20-fold in the rate of emergence of resistance to FK506 compared to that of the wild-type strain, with the majority of the mutations found in these repeats.
species are the most abundant fungal components of the mammalian and human skin microbiome. Although they belong to the natural skin commensal flora of humans, they are also associated with a variety of clinical skin disorders. The standard treatment for
-associated inflammatory skin infections is topical corticosteroids, although their use has adverse side effects and is not recommended for long treatment periods. Calcineurin inhibitors have been proposed as a suitable alternative to treat patients affected by skin lesions caused by
Although calcineurin inhibitors are well-known as immunosuppressive drugs, they are also characterized by potent antimicrobial activity. In the present study, we investigated the mechanism of action of FK506 (tacrolimus), ascomycin (FK520), and pimecrolimus in
and
and found that the conserved immunophilin FKBP12 is the target of these drugs with which it forms a complex that directly binds calcineurin and inhibits its signaling activity. We found that FKBP12 is also required for the additive activity of calcineurin inhibitors with fluconazole. Furthermore, the increasing natural occurrence in fungal pathogen populations of mutator strains poses a high risk for the rapid emergence of drug resistance and adaptation to host defense. This led us to generate an engineered hypermutator
Δ mutant strain of
and genetically evaluate mutational events resulting in a substantially increased rate of resistance to FK506 compared to that of the wild type. Our study paves the way for the novel clinical use of calcineurin inhibitors with lower immunosuppressive activity that could be used clinically to treat a broad range of fungal infections, including skin disorders caused by
.
eLife digest.
Fungi are enigmatic organisms that flourish in soil, on decaying plants, or during infection of animals or plants. Growing in myriad forms, from single-celled yeast to multicellular ...molds and mushrooms, fungi have also evolved a variety of strategies to reproduce. Normally, fungi reproduce in one of two ways: either they reproduce asexually, with one individual producing a new individual identical to itself, or they reproduce sexually, with two individuals of different 'mating types' contributing to produce a new individual. However, individuals of some species exhibit 'homothallism' or self-fertility: these individuals can produce reproductive cells that are universally compatible, and therefore can reproduce sexually with themselves or with any other cell in the population.
Homothallism has evolved multiple times throughout the fungal kingdom, suggesting it confers advantage when population numbers are low or mates are hard to find. Yet some homothallic fungi been overlooked compared to heterothallic species, whose mating types have been well characterised. Understanding the genetic basis of homothallism and how it evolved in different species can provide insights into pathogenic species that cause fungal disease.
With that in mind, Passer, Clancey et al. explored the genetic basis of homothallism in
, a close relative of
a species that causes fungal infections in humans. A combination of genetic sequencing techniques and experiments were applied to analyse, compare, and manipulate
genome to see how this species evolved self-fertility.
Passer, Clancey et al. showed that
evolved the ability to reproduce sexually by itself via a unique evolutionary pathway. The result is a form of homothallism never reported in fungi before.
lost some of the genes that control mating in other species of fungi, and acquired genes from the opposing mating types of a heterothallic ancestor to become self-fertile.
Passer, Clancey et al. also found that, unlike other
species that switch between asexual and sexual reproduction,
grows only as long, branching filaments called hyphae, a sexual form. The species reproduces sexually with itself throughout its life cycle and is unable to produce a yeast (asexual) form, in contrast to other closely related species.
This work offers new insights into how different modes of sexual reproduction have evolved in fungi. It also provides another interesting case of how genome plasticity and evolutionary pressures can produce similar outcomes, homothallism, via different evolutionary paths. Lastly, assembling the complete genome of
will foster comparative studies between pathogenic and non-pathogenic
species.
The region encompassing the Pacific Northwest (PNW), Vancouver Island, Oregon, and Washington has been the location of an ongoing
outbreak since the 1990s, and there is evidence that the outbreak is ...expanding along the West Coast into California. Here we report a clinical case of a 69-year-old, HIV-negative man from North Carolina who was diagnosed with a fungal brain mass by magnetic resonance imaging (MRI) and pathology. He had traveled to Seattle and Vancouver 3 years earlier and to Costa Rica 4 months prior to presentation. Phenotypic evidence showed that the fungal mass isolated from the patient's brain represented
In agreement with the phenotypic results, multilocus sequence typing (MLST) provided genotypic evidence that assigned the infecting organism within the
species complex and to the
VGIIa clade. Whole-genome sequencing revealed >99.99% identity with the
reference strain R265, indicating that the infecting strain is derived from the highly clonal outbreak strains in the PNW. We conclude that the patient acquired the
infection during his travel to the region 3 years prior and that the infection was dormant for an extended period of time before causing disease. The patient tested positive for anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies, supporting earlier reports that implicate these autoantibodies as a risk factor associated with
infection.
Mortality rates associated with
infections are estimated to be between 13% and 33%, depending on an individual's predisposition, and
has caused at least 39 deaths in the PNW region. There have been four other international travel cases reported in patients from Europe and Asia with travel history to the PNW, but this report describes the first North American traveler who acquired
infection presenting within the United States and the first case of a
outbreak infection associated with anti-GM-CSF autoantibodies. Early and accurate diagnoses are important for disease prevention and treatment and for control of infectious diseases. Continual reporting of
infections is necessary to raise awareness of the ongoing outbreak in the PNW and to alert travelers and physicians to the areas of endemicity with potential risks.
The establishment and maintenance of higher-order structure at centromeres is essential for accurate chromosome segregation. The monopolin complex is thought to cross-link multiple kinetochore ...complexes to prevent merotelic attachments that result in chromosome missegregation. This model is based on structural analysis and the requirement that monopolin execute mitotic and meiotic chromosome segregation in Schizosaccharomyces pombe, which has more than one kinetochore-microtubule attachment/centromere, and co-orient sister chromatids in meiosis I in Saccharomyces cerevisiae. Recent data from S. pombe suggest an alternative possibility: that the recruitment of condensin is the primary function of monopolin. Here we test these models using the yeast Candida albicans. C. albicans cells lacking monopolin exhibit defects in chromosome segregation, increased distance between centromeres, and decreased stability of several types of repeat DNA. Of note, changing kinetochore-microtubule copy number from one to more than one kinetochore-microtubule/centromere does not alter the requirement for monopolin. Furthermore, monopolin recruits condensin to C. albicans centromeres, and overexpression of condensin suppresses chromosome segregation defects in strains lacking monopolin. We propose that the key function of monopolin is to recruit condensin in order to promote the assembly of higher-order structure at centromere and repetitive DNA.
Assembly of kinetochore complexes, involving greater than one hundred proteins, is essential for chromosome segregation and genome stability. Neocentromeres, or new centromeres, occur when ...kinetochores assemble de novo, at DNA loci not previously associated with kinetochore proteins, and they restore chromosome segregation to chromosomes lacking a functional centromere. Neocentromeres have been observed in a number of diseases and may play an evolutionary role in adaptation or speciation. However, the consequences of neocentromere formation on chromosome missegregation rates, gene expression, and three-dimensional (3D) nuclear structure are not well understood. Here, we used Candida albicans, an organism with small, epigenetically-inherited centromeres, as a model system to study the functions of twenty different neocentromere loci along a single chromosome, chromosome 5. Comparison of neocentromere properties relative to native centromere functions revealed that all twenty neocentromeres mediated chromosome segregation, albeit to different degrees. Some neocentromeres also caused reduced levels of transcription from genes found within the neocentromere region. Furthermore, like native centromeres, neocentromeres clustered in 3D with active/functional centromeres, indicating that formation of a new centromere mediates the reorganization of 3D nuclear architecture. This demonstrates that centromere clustering depends on epigenetically defined function and not on the primary DNA sequence, and that neocentromere function is independent of its distance from the native centromere position. Together, the results show that a neocentromere can form at many loci along a chromosome and can support the assembly of a functional kinetochore that exhibits native centromere functions including chromosome segregation accuracy and centromere clustering within the nucleus.
Hybridization has resulted in the origin and variation in extant species, and hybrids continue to arise despite pre- and post-zygotic barriers that limit their formation and evolutionary success. One ...important system that maintains species boundaries in prokaryotes and eukaryotes is the mismatch repair pathway, which blocks recombination between divergent DNA sequences. Previous studies illuminated the role of the mismatch repair component Msh2 in blocking genetic recombination between divergent DNA during meiosis. Loss of Msh2 results in increased interspecific genetic recombination in bacterial and yeast models, and increased viability of progeny derived from yeast hybrid crosses. Hybrid isolates of two pathogenic fungal Cryptococcus species, Cryptococcus neoformans and Cryptococcus deneoformans, are isolated regularly from both clinical and environmental sources. In the present study, we sought to determine if loss of Msh2 would relax the species boundary between C. neoformans and C. deneoformans. We found that crosses between these two species in which both parents lack Msh2 produced hybrid progeny with increased viability and high levels of aneuploidy. Whole-genome sequencing revealed few instances of recombination among hybrid progeny and did not identify increased levels of recombination in progeny derived from parents lacking Msh2. Several hybrid progeny produced structures associated with sexual reproduction when incubated alone on nutrient-rich medium in light, a novel phenotype in Cryptococcus. These findings represent a unique, unexpected case where rendering the mismatch repair system defective did not result in increased meiotic recombination across a species boundary. This suggests that alternative pathways or other mismatch repair components limit meiotic recombination between homeologous DNA and enforce species boundaries in the basidiomycete Cryptococcus species.
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