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Heitman, J.

Publications and source records attributed to Heitman, J..

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bioRxiv2019

Centromere-mediated chromosome break drives karyotype evolution in closely related Malassezia species

Intra-chromosomal or inter-chromosomal genomic rearrangements often lead to speciation (1). Loss or gain of a centromere leads to alterations in chromosome number in closely related species. Thus, centromeres can enable tracing the path of evolution from the ancestral to a derived state (2). The Malassezia species complex of the phylum Basiodiomycota shows remarkable diversity in chromosome number ranging between six and nine chromosomes (3-5). To understand these transitions, we experimentally identified all eight centromeres as binding sites of an evolutionarily conserved outer kinetochore protein Mis12/Mtw1 in M. sympodialis. The 3 to 5 kb centromere regions share an AT-rich, poorly transcribed core region enriched with a 12 bp consensus motif. We also mapped nine such AT-rich centromeres in M. globosa and the related species Malassezia restricta and Malassezia slooffiae. While eight predicted centromeres were found within conserved synteny blocks between these species and M. sympodialis, the remaining centromere in M. globosa (MgCEN2) or its orthologous centromere in M. slooffiae (MslCEN4) and M. restricta (MreCEN8) mapped to a synteny breakpoint compared with M. sympodialis. Taken together, we provide evidence that breakage and loss of a centromere (CEN2) in an ancestral Malassezia species possessing nine chromosomes resulted in fewer chromosomes in M. sympodialis. Strikingly, the predicted centromeres of all closely related Malassezia species map to an AT-rich core on each chromosome that also shows enrichment of the 12 bp sequence motif. We propose that centromeres are fragile AT-rich sites driving karyotype diversity through breakage and inactivation in these and other species. Significance statementThe number of chromosomes can vary between closely related species. Centromere loss destabilizes chromosomes and results in reduced number of chromosomes to drive speciation. A series of evidence from studies on various cancers suggest that an imbalance in kinetochore-microtubule attachments results in breaks at the centromeres. To understand if such events can cause chromosome number changes in nature, we studied six species of Malassezia, of which three possess eight chromosomes and others have nine chromosomes each. We find signatures of chromosome breakage at the centromeres in organisms having nine chromosomes. We propose that the break at the centromere followed by fusions of acentric chromosomes to other chromosomes could be a plausible mechanism shaping the karyotype of Malassezia and related organisms. ClassificationBiological sciences, Genetics

genomics↗

bioRxiv2019

Centromere deletion in Cryptococcus deuterogattii leads to neocentromere formation and chromosome fusions

The human fungal pathogen Cryptococcus deuterogattii is RNAi-deficient and lacks active transposons in its genome. C. deuterogattii has regional centromeres that contain only transposon relics. To investigate impact of centromere loss on the C. deuterogattii genome, either centromere 9 or 10 was deleted. Deletion of either centromere resulted in neocentromere formation and interestingly, the genes covered by these neocentromeres maintained wild-type expression levels. In contrast to cen9{Delta} mutants, cen10{Delta} mutant strains exhibited growth defects and were aneuploid for chromosome 10. At an elevated growth temperature (37{degrees}C), the cen10{Delta} chromosome was found to have undergone fusion with another native chromosome in some isolates and this fusion restored wild-type growth. Following chromosomal fusion, the neocentromere was inactivated, and the native centromere of the fused chromosome served as the active centromere. The neocentromere formation and chromosomal fusion events observed in this study in C. deuterogattii may be similar to events that triggered genomic changes within the Cryptococcus/Kwoniella species complex and may contribute to speciation throughout the eukaryotic domain.

genetics↗

bioRxiv2019

Broad antifungal resistance mediated by RNAi-dependent epimutation in the basal human fungal pathogen Mucor circinelloides

Mucormycosis - an emergent, deadly fungal infection - is difficult to treat, in part because the causative species demonstrate broad clinical antifungal resistance. However, the mechanisms underlying drug resistance in these infections remain poorly understood. Our previous work demonstrated that one major agent of mucormycosis, Mucor circinelloides, can develop resistance to the antifungal agents FK506 and rapamycin through a novel, transient RNA interference-dependent mechanism known as epimutation. Epimutations silence the drug target gene and are selected by drug exposure; the target gene is re-expressed and sensitivity is restored following passage without drug. This silencing process involves generation of small RNA (sRNA) against the target gene via core RNAi pathway proteins. To further elucidate the role of epimutation in the broad antifungal resistance of Mucor, epimutants were isolated that confer resistance to another antifungal agent, 5-fluoroorotic acid (5-FOA). We identified epimutant strains that exhibit resistance to 5-FOA without mutations in PyrF or PyrG, enzymes which convert 5-FOA into the active toxic form. Using sRNA hybridization as well as sRNA library analysis, we demonstrate that these epimutants harbor sRNA against either pyrF or pyrG, and further show that this sRNA is lost after reversion to drug sensitivity. We conclude that epimutation is a mechanism capable of targeting multiple genes, enabling Mucor to develop resistance to a variety of antifungal agents. Elucidation of the role of RNAi in epimutation affords a fuller understanding of mucormycosis. Furthermore, it improves our understanding of fungal pathogenesis and adaptation to stresses, including the evolution of drug resistance.

microbiology↗

bioRxiv2019

Unisexual reproduction promotes foraging for mating partners in the global human fungal pathogen Cryptococcus deneoformans

Courtship is pivotal for successful mating. However, courtship is challenging for the Cryptococcus neoformans species complex, comprised of opportunistic fungal pathogens, as the majority of isolates are mating type. In the absence of mating partners of the opposite mating type, C. deneoformans can undergo unisexual reproduction, during which a yeast-to-hyphal morphological transition occurs. Hyphal growth during unisexual reproduction is a quantitative trait, which reflects a strains ability to undergo unisexual reproduction. In this study, we determined whether unisexual reproduction confers an ecological benefit by promoting foraging for mating partners. Through competitive mating assays using strains with different abilities to produce hyphae, we showed that unisexual reproduction potential did not enhance competition for mating partners of the same mating type, but when cells of the opposite mating type were present, cells with enhanced hyphal growth were more competitive for mating partners of either the same or opposite mating type. Enhanced mating competition was also observed in a strain with increased hyphal production that lacks the mating repressor gene GPA3, which contributes to the pheromone response. Hyphal growth in unisexual strains also enables contact between adjacent colonies and enhances mating efficiency during mating confrontation assays. The pheromone response pathway activation positively correlated with unisexual reproduction hyphal growth during bisexual mating and exogenous pheromone promoted bisexual cell fusion. Despite the benefit in competing for mating partners, unisexual reproduction conferred a fitness cost. Taken together, these findings suggest C. deneoformans employs hyphal growth to facilitate contact between colonies at long distances and utilizes pheromone sensing to enhance mating competition.\n\nAuthor SummarySexual reproduction plays a pivotal role in shaping fungal population structure and diversity in nature. The global human fungal pathogen Cryptococcus neoformans species complex evolved distinct sexual cycles: bisexual reproduction between mating partners of the opposite mating types, and unisexual reproduction with only one mating type. During both sexual cycles, cells undergo a yeast-to-hyphal morphological transition and nuclei diploidize through either cell-cell fusion followed by nuclear fusion during bisexual reproduction or endoreplication during unisexual reproduction. Despite the complex sexual life cycle, the majority of Cryptococcal isolates are mating type. Albeit the scarcity of MATa cells in the environment, meiotic recombination is prevalent. To decipher this conundrum, we ask whether there is an underlying mechanism in which Cryptococcus species increase their mating opportunities. In this study, we showed that the undirected hyphal growth during unisexual reproduction enables MAT cells to forage for mating partners over a larger surface area, and when MAT hyphae come into close proximity of rare MATa cells, pheromone response pathway activation in both MAT and MATa cells can further enhance mating. This mating enhancement could promote outcrossing and facilitate genome reshuffling via meiotic recombination.

microbiology↗

bioRxiv2019

Cryptococcus deuterogattii VGIIa infection associated with travel to the Pacific Northwest outbreak region in an anti-GM-CSF autoantibody positive patient in the United States

The Pacific Northwest (PNW), Vancouver Island, Oregon, and Washington have been the location of an ongoing Cryptococcus gattii 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 three years earlier and Costa Rica four months prior to presentation. Phenotypic evidence shows the fungal mass isolated from the patients brain is C. gattii. In agreement with the phenotypic results, MLST provides genotypic evidence that assigns the infecting organism within in the C. gattii species complex and belonging to the C. deuterogattii VGIIa clade. Whole genome sequencing revealed >99.99% identity to the C. deuterogattii reference strain R265, indicating that the infecting strain is derived from the highly clonal outbreak strains in the PNW. We conclude the patient acquired the C. gattii infection during his travel to region three years prior and 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 a risk factor associated with C. gattii infection.\n\nImportanceMortality rates associated with C. gattii infections are estimated to be between 13% and 33% depending on an individuals predisposition, and C. gattii has caused more 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 study describes the first North American travel who acquired C. deuterogattii infection presenting within the United States, and the first case of a C. deuterogattii outbreak infection associated with anti-GM-CSF autoantibodies. Early and accurate diagnoses are important for disease prevention, treatment, and control of infectious diseases. Continual reporting of C. deuterogattii infections is necessary to raise awareness of the ongoing outbreak in the PNW and alert travelers and physicians to the endemic areas with potential risks.

microbiology↗

bioRxiv2018

Whole genome analysis illustrates global clonal population structure of the ubiquitous dermatophyte pathogen Trichophyton rubrum

Dermatophytes include fungal species that infect humans, as well as those which also infect other animals or only grow in the environment. The dermatophyte species Trichophyton rubrum is a frequent cause of skin infection in immunocompetent individuals. While members of the T. rubrum species complex have been further categorized based on various morphologies, the population structure and ability to undergo sexual reproduction are not well understood. In this study, we analyze a large set of T. rubrum and Trichophyton interdigitale isolates to examine mating types, evidence of mating, and genetic variation. We find that nearly all isolates of T. rubrum are of a single mating type, and that incubation with T. rubrum morphotype megninii isolates of the other mating type failed to induce sexual development. While the region around the mating type locus is characterized by a higher frequency of SNPs compared to other genomic regions, we find that the population is remarkably clonal, with highly conserved gene content, low levels of variation, and little evidence of recombination These results support a model of recent transition to asexual growth when this species specialized to growth on human hosts.\n\nData access: Genome sequence data is available in NCBI under the Umbrella BioProject PRJNA186851.

genetics↗

bioRxiv2017

A High Resolution Map of Meiotic Recombination in Cryptococcus Demonstrates Decreased Recombination in Unisexual Reproduction

Multiple species within the basidiomycete genus, Cryptococcus, cause cryptococcal disease. These species are estimated to affect nearly a quarter of a million people leading to approximately 180,000 mortalities, annually. Sexual repro-duction, which can occur between haploid yeasts of the same or opposite mating type, is a potentially important contributor to pathogenesis as recombination can generate novel genotypes and transgressive phenotypes. However, our quantitative understanding of recombination in this clinically important yeast is limited. Here we describe genome-wide estimates of recombination rates in Cryptococcus deneoformans and compare recombination between progeny from - unisexual and a- bisexual crosses. We find that offspring from bisexual crosses have modestly higher average rates of recombination than those derived from unisexual crosses. Recombination hot and cold spots across the C. deneoformans genome are also identified and are associated with increased GC content. Finally, we observed regions genome-wide with allele frequencies deviating from the expected parental ratio. These findings and observations advance our quantitative understanding of the genetic events that occur during sexual reproduction in C. deneoformans, and the impact that different forms of sexual reproduction are likely to have on genetic diversity in this important fungal pathogen.

genetics↗

bioRxiv2017

Outbreak of invasive wound mucormycosis in a burn unit due to multiple strains of Mucor circinelloides f. circinelloides resolved by whole genome sequencing

Mucorales are ubiquitous environmental molds responsible for mucormycosis in diabetic, immunocompromised, and severely burned patients. Small outbreaks of invasive wound mucormycosis (IWM) have already been reported in burn units without extensive microbiological investigations. We faced an outbreak of IWM in our center and investigated the clinical isolates with whole genome sequencing (WGS) analysis.\n\nWe analyzed M. circinelloides isolates from patients in our burn unit (BU1) together with non-outbreak isolates from burn unit 2 (BU2, Paris area) and from France over a two-year period (2013-2015). For each isolate, WGS and a de novo genome assembly was performed from read data extracted from the aligned contig sequences of the reference genome (1006PhL).\n\nA total of 21 isolates were sequenced including 14 isolates from six BU1 patients. Phylogenetic classification showed that the clinical isolates clustered in four highly divergent clades. Clade1 contained at least one of the strains from the six epidemiologically-linked BU1 patients. The clinical isolates seemed specific to each patient. Two patients were infected with more than two strains from different clades suggesting that an environmental reservoir of clonally unrelated isolates was the source of contamination. Only two patients shared one strain in BU1, suggesting direct transmission or contamination with the same environmental source.\n\nWGS coupled with precise epidemiological data and analysis of several isolates per patients revealed in our study a complex situation with both potential cross-transmission and multiple contaminations with a heterogeneous pool of strains from a cryptic environmental reservoir.\n\nImportanceInvasive wound mucormycosis (IWM) is a severe infection due to the environmental molds belonging to the order Mucorales. Severely burned patients are particularly at risk for IWM. Here, we used Whole Genome Sequencing (WGS) analysis to resolve an outbreak of IWM due to Mucor circinelloides that occurred in our hospital (BU1). We sequenced 21 clinical isolates, including 14 from BU1 and 7 unrelated isolates, and compared them to the reference genome (1006PhL). This analysis revealed that the outbreak was mainly due to multiple strains that seemed patient-specific, suggesting that the patients were more likely infected from a pool of diverse strains from the environment rather than from direct transmission between the patients. This study revealed the complexity of a Mucorales outbreak in the settings of IWM in burn patients, which has been highlighted based on whole genome sequencing and careful sampling.

microbiology↗

bioRxiv2017

An atlas of genetic variation for linking pathogen-induced cellular traits to human disease

Genome-wide association studies (GWAS) have identified thousands of genetic variants associated with disease. To facilitate moving from associations to disease mechanisms, we leveraged the role of pathogens in shaping human evolution with the Hi-HOST Phenome Project (H2P2): a catalog of cellular GWAS comprised of 79 phenotypes in response to 8 pathogens in 528 lymphoblastoid cell lines. Seventeen loci surpass genome-wide significance (p<5x10-8) for phenotypes ranging from pathogen replication to cytokine production. Combining H2P2 with clinical association data from the eMERGE Network and experimental validation revealed evidence for mechanisms of action and connections with diseases. We identified a SNP near CXCL10 as a cis-cytokine-QTL and a new risk factor for inflammatory bowel disease. A SNP in ZBTB20 demonstrated pleiotropy, partially mediated through NF{kappa}B signaling, and was associated with viral hepatitis. Data are available in an H2P2 web portal to facilitate further interpreting human genome variation through the lens of cell biology.

genetics↗

bioRxiv2017

Natural mismatch repair mutations mediate phenotypic diversity and drug resistance in Cryptococcus deuterogattii

Pathogenic microbes confront a constant evolutionary conflict between the pressure to maintain genome stability and the need to adapt to mounting external stresses. Prokaryotes often respond with elevated mutation rates, but to date little evidence exists of stable eukaryotic hypermutators in nature. Whole genome resequencing of the human fungal pathogen Cryptococcus deuterogattii identified an outbreak lineage characterized by a nonsense mutation in MSH2. This defect in mismatch repair 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 FRR1, 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.

genomics↗

bioRxiv2017

Population Genomics Of Cryptococcus neoformans var. grubii Reveals New Biogeographic Relationships And Finely Maps Hybridization

Cryptococcus neoformans var. grubii is the causative agent of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals, typically HIV/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 characterized 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 A/A) and of hybrids with C. neoformans var. neoformans (serotype A/D) 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 revealed 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.\n\nAuthor SummaryCryptococcus neoformans var. grubii is a human fungal pathogen of immunocompromised individuals that has global clinical impact, causing half a million deaths per year. Substantial genetic substructure exists for this pathogen, with two lineages found globally (VNI, VNII) whereas a third has appeared confined to sub-Saharan Africa (VNB). Here, we utilized genome sequencing of a large set of global isolates to examine the genetic diversity, hybridization, and biogeography of these lineages. We found that while the three major lineages are well separated, recombination between the lineages has occurred, notably resulting in hybrid isolates with segmented ancestry across the genome. In addition, we showed that isolates from South America are placed within the VNB lineage, formerly thought to be confined to Africa, and that there is phylogenetic separation between these geographies that substantially expands the diversity of these lineages. Our findings provide a new framework for further studies of the dynamics of natural populations of C. neoformans var. grubii.

genomics↗

bioRxiv2017

Population Genomics And The Evolution Of Virulence In The Fungal Pathogen Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes approximately 625,000 deaths per year from nervous system infections. Here, we leveraged a unique, genetically diverse population of C. neoformans from sub-Saharan Africa, commonly isolated from mopane trees, to determine how selective pressures in the environment coincidentally adapted C. neoformans for human virulence. Genome sequencing and phylogenetic analysis of 387 isolates, representing the global VNI and African VNB lineages, highlighted a deep, non-recombining split in VNB (herein VNBI and VNBII). VNBII was enriched for clinical samples relative to VNBI, while phenotypic profiling of 183 isolates demonstrated that VNBI isolates were significantly more resistant to oxidative stress and more heavily melanized than VNBII isolates. Lack of melanization in both lineages was associated with loss-of-function mutations in the BZP4 transcription factor. A genome-wide association study across all VNB isolates revealed sequence differences between clinical and environmental isolates in virulence factors and stress response genes. Inositol transporters and catabolism genes, which process sugars present in plants and the human nervous system, were identified as targets of selection in all three lineages. Further phylogenetic and population genomic analyses revealed extensive loss of genetic diversity in VNBI, suggestive of a history of population bottlenecks, along with unique evolutionary trajectories for mating type loci. These data highlight the complex evolutionary interplay between adaptation to natural environments and opportunistic infections, and that selection on specific pathways may predispose isolates to human virulence.

genomics↗