Biology subjects

Heitman, J.

Publications and source records attributed to Heitman, J..

At least 73 records · Page 4Linked to original sources

bioRxiv2020

Expression of a Malassezia codon optimized mCherry fluorescent protein in a bicistronic vector

The use of fluorescent proteins allows a multitude of approaches from live imaging and fixed cells to labelling of whole organisms, making it a foundation of diverse experiments. Tagging a protein of interest or specific cell type allows visualization and studies of cell localization, cellular dynamics, physiology, and structural characteristics. In specific instances fluorescent fusion proteins may not be properly functional as a result of structural changes that hinder protein function, or when overexpressed may be cytotoxic and disrupt normal biological processes. In our study, we describe application of a bicistronic vector incorporating a Picornavirus 2A peptide sequence between a NAT antibiotic selection marker and mCherry. This allows expression of multiple genes from a single open reading frame and production of discrete protein products through a cleavage event within the 2A peptide. We demonstrate integration of this bicistronic vector into a model Malassezia species, the haploid strain M. furfur CBS 14141, with both active selection, high fluorescence, and proven proteolytic cleavage. Potential applications of this technology can include protein functional studies, Malassezia cellular localization, and co-expression of genes required for targeted mutagenesis.

molecular biology↗

bioRxiv2020

Designing Selective and Non-Immunosuppressive Antifungal FK506 Analogs: Structures, Biophysics and Dynamics of Fungal and Human Calcineurin-Inhibitor Complexes

Calcineurin is a critical enzyme in fungal pathogenesis and antifungal drug tolerance and, therefore, an attractive antifungal target. Current clinically-accessible calcineurin inhibitors, such as FK506, are immunosuppressive to humans, so exploiting calcineurin inhibition as an antifungal strategy necessitates fungal-specificity in order to avoid inhibiting the human pathway. Harnessing fungal calcineurin-inhibitor crystal structures, we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and demonstrable efficacy in a murine model of invasive fungal infection. Our overarching goal is to better understand, at a molecular level, the interaction determinants of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition in order to guide the design of fungal-selective, non-immunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of the M. circinelloides FKBP12 bound to FK506, and of the A. fumigatus, M. circinelloides and human FKBP12 proteins bound to the FK506 analog, APX879, which exhibits enhanced selectivity for fungal pathogens. Combining structural, genetic and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12-ligand complexes that will guide the rational design of inhibitors with enhanced fungal-selectivity. Significance statementInvasive fungal infections are a leading cause of death in the immunocompromised patient population. The rise in drug resistance to current antifungals highlights the urgent need to develop more efficacious and highly selective agents. Numerous investigations of major fungal pathogens have confirmed the critical role of the calcineurin pathway for fungal virulence, making it an attractive target for antifungal development. Although FK506 inhibits calcineurin, it is immunosuppressive in humans and cannot be used as an antifungal. By combining structural, genetic, biophysical, and in silico methodologies, we pinpoint regions of FK506 and a less immunosuppressive analog, APX879, that could be altered to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.

biochemistry↗

bioRxiv2020

Tornadic shear stress induces a transient, calcineurin-dependent hyper-virulent phenotype in Mucorales molds

Trauma-related necrotizing myocutaneous mucormycosis (NMM) has a high morbidity and mortality in victims of combat-related injuries, geo-meteorological disasters, and severe burns. Inspired by the observation that several recent clusters of NMM have been associated with extreme mechanical forces (e.g. during tornados), we studied the impact of mechanical stress on Mucoralean biology and virulence in a Drosophila melanogaster infection model. In contrast to other experimental procedures to exert mechanical stress, tornadic shear challenge (TSC) by magnetic stirring induced a hyper-virulent phenotype in several clinically relevant Mucorales species but not in Aspergillus or Fusarium. Whereas fungal growth rates, morphogenesis, and susceptibility to noxious environments or phagocytes were not altered by TSC, soluble factors released in the supernatant of shear-challenged R. arrhizus spores rendered static spores hyper-virulent. Consistent with a rapid decay of TSC-induced hyper-virulence, minimal transcriptional changes were revealed by comparative RNA sequencing analysis of static and shear-challenged Rhizopus arrhizus. However, inhibition of the calcineurin/heat shock protein 90 (hsp90) stress response circuitry by cyclosporine A (CsA) and tanespimycin abrogated the increased pathogenicity of R. arrhizus spores following TSC. Similarly, calcineurin loss-of-function mutants of Mucor circinelloides displayed no increased virulence capacity in flies after undergoing TSC. Collectively, these results establish that TSC induces hyper-virulence specifically in Mucorales and point out the calcineurin/hsp90 pathway as a key orchestrator of this phenotype. Our findings invite future studies of topical calcineurin inhibitor treatment of wounds as an adjunct mitigation strategy for NMM following high-energy trauma. SignificanceGiven the limited efficacy of current medical treatments in trauma-related necrotizing mucormycosis, there is a dire need to better understand the Mucoralean pathophysiology in order to develop novel strategies to counteract fungal tissue invasion following severe trauma. Here, we describe that tornadic shear stress challenge transiently induces a hyper-virulent phenotype in various pathogenic Mucorales species but not in other molds known to cause wound infections. Our data support a model whereby shear stress-induced hyper-virulence is primarily driven by soluble factors and orchestrated by the calcineurin/hsp90 pathway. Importantly, pharmacological and genetic inhibition of calcineurin signaling abrogated hyper-virulence in shear stress-challenged Mucorales, encouraging further evaluation of (topical) calcineurin inhibitors to improve therapeutic outcomes of NMM after combat-related blast injuries or violent storms.

microbiology↗

bioRxiv2020

Transposon mobilization during infection promotes drug resistance in the human fungal pathogen Cryptococcus deneoformans

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeast-like fungi that cause lethal infections, especially in HIV-infected patients. Using Cryptococcus deneoformans in a murine model of infection, we examined contributors to drug resistance and demonstrated that transposon mutagenesis drives the development of 5-fluoroorotic acid (5FOA) resistance. Inactivation of target genes URA3 or URA5 primarily reflected the insertion of two transposable elements (TEs): the T1 DNA transposon and the TCN12 retrotransposon. Consistent with in vivo results, increased rates of mutagenesis and resistance to 5FOA and the antifungal drugs rapamycin/FK506 and 5-fluorocytosine (5FC) were found when Cryptococcus was incubated at 37{degrees} compared to 30{degrees} in vitro, a condition that mimics the temperature shift that occurs during the environment-to-host transition. Inactivation of the RNAi pathway, which suppresses TE movement in many organisms, was not sufficient to elevate TE movement at 30{degrees} to the level observed at 37{degrees}. We propose that temperature-dependent TE mobilization in Cryptococcus is an important mechanism that enhances microbial adaptation and promotes pathogenesis and drug resistance in the human host. SIGNIFICANCE STATEMENTThe incidence of infections due to fungal pathogens has dramatically increased in the past few decades with similar increases in human populations with weakened or suppressed immune systems. Understanding the mechanisms by which organisms rapidly adapt during human infection to enhance virulence and evolve drug resistance is important for developing effective treatments. We find that transposon mobilization in the human pathogen Cryptococcus causes genomic mutations in a murine model of infection and promotes resistance to antifungal drugs in vitro. Thermotolerance is a key virulence determinant for pathogenic fungi during the environment-to-host transition, and we demonstrate that a temperature increase is sufficient to trigger transposon mobilization in vitro. The link between temperature stress and transposon-associated mutations may significantly impact adaptation to the host during infection, including the acquisition of drug resistance.

microbiology↗

bioRxiv2020

Horizontal gene transfer in the human and skin commensal Malassezia: a bacterially-derived flavohemoglobin is required for NO resistance and host interaction

The skin of humans and animals is colonized by commensal and pathogenic fungi and bacteria that share this ecological niche and have established microbial interactions. Malassezia are the most abundant fungal skin inhabitant of warm-blooded animals, and have been implicated in skin diseases and systemic disorders, including Crohns disease and pancreatic cancer. Flavohemoglobin is a key enzyme involved in microbial nitrosative stress resistance and nitric oxide degradation. Comparative genomics and phylogenetic analyses within the Malassezia genus revealed that flavohemoglobin-encoding genes were acquired through independent horizontal gene transfer events from different donor bacteria that are part of the mammalian microbiome. Through targeted gene deletion and functional complementation in M. sympodialis, we demonstrated that bacterially-derived flavohemoglobins are cytoplasmic proteins required for nitric oxide detoxification and nitrosative stress resistance under aerobic conditions. RNAseq analysis revealed that endogenous accumulation of nitric oxide resulted in upregulation of genes involved in stress response, and downregulation of the MalaS7 allergen-encoding genes. Solution of the high-resolution X-ray crystal structure of Malassezia flavohemoglobin revealed features conserved with both bacterial and fungal flavohemoglobins. In vivo pathogenesis is independent of Malassezia flavohemoglobin. Lastly, we identified additional 30 genus- and species-specific horizontal gene transfer candidates that might have contributed to the evolution of this genus as the most common inhabitants of animal skin. Significance statementMalassezia species are the main fungal components of the mammalian skin microbiome and are associated with a number of skin disorders. Recently, Malassezia has also been found in association with Crohns Disease and with pancreatic cancer. The elucidation of the molecular bases of skin adaptation by Malassezia is critical to understand its role as commensal and pathogen. In this study we employed evolutionary, molecular, biochemical, and structural analyses to demonstrate that the bacterially-derived flavohemoglobins acquired by Malassezia through horizontal gene transfer resulted in a gain of function critical for nitric oxide detoxification and resistance to nitrosative stress. Our study underscores horizontal gene transfer as an important force modulating Malassezia evolution and niche adaptation.

microbiology↗

bioRxiv2019

A novel mycovirus evokes transcriptional rewiring in Malassezia and provokes host inflammation and an immunological response

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 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, 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, interferon-{beta} 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. ImportanceMalassezia 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 Crohns disease and pancreatic cancer, further implicating this fungal genus in inflammatory and neoplastic disease states. Because M. sympodialis has lost genes involved in 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 interferon-{beta} expression in cultured macrophages. This study sheds light on the mechanisms of pathogenicity of Malassezia, focusing on a previously unidentified novel mycovirus.

microbiology↗

bioRxiv2019

A novel resistance pathway for calcineurin inhibitors in the human pathogenic Mucorales Mucor circinelloides

Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides is a causal agent of mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conserved virulence factor in many pathogenic fungi and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast growth. We analyzed thirty-six calcineurin inhibitor resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbR{Delta} mutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named here bycA (bypass of calcineurin A). bycA encodes an amino acid permease. We verified that both bycA{Delta}, and the bycA{Delta} cnbR{Delta} double mutant are resistant to the calcineurin inhibitor FK506, thereby demonstrating a novel resistance mechanism against calcineurin inhibitors. We also found that the expression of bycA was significantly higher in the wild type strain treated with FK506 and in the cnbR{Delta} mutants, but significantly lower in the wild type without FK506. These findings suggest that bycA is a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor and calcineurin suppresses the bycA gene at the mRNA level to promote hyphal growth. BycA is involved in the Mucor hyphal-yeast transition as our data demonstrates a positive correlation between bycA expression, protein kinase A activity, and Mucor yeast-growth. Also calcineurin, independent of its role in morphogenesis, contributes to virulence traits including phagosome maturation blockade, host cell damages, and pro-angiogenic growth factor induction during interactions with hosts. ImportanceMucor is intrinsically resistant to most known antifungals, which makes mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase widely conserved across eukaryotes. When calcineurin function is inhibited in Mucor, growth shifts to a less-virulent yeast growth form which makes calcineurin an attractive target for development of new antifungal drugs. Previously we identified two distinct mechanisms through which Mucor can become resistant to calcineurin inhibitors involving Mendelian mutations in the gene for FKBP12, calcineurin A or B subunits and epimutations silencing the FKBP12 gene. Here, we identified a third novel mechanism where loss of function mutations in the amino acid permease encoding the bycA gene contribute to resistance against calcineurin inhibitors. When calcineurin activity is absent, BycA can activate PKA to promote yeast growth via a cAMP-independent pathway. Our data also shows that calcineurin activity, primarily contributes to host - pathogen interactions in the pathogenesis of Mucor.

microbiology↗

bioRxiv2019

Centromere scission drives chromosome shuffling and reproductive isolation

A fundamental characteristic of eukaryotic organisms is the generation of genetic variation via sexual reproduction. Conversely, significant large-scale genome structure variations could hamper sexual reproduction, causing reproductive isolation and promote speciation. The underlying processes behind large-scale genome rearrangements are not well understood and include chromosome translocations involving centromeres. Recent genomic studies in the Cryptococcus species complex revealed that chromosome translocations generated via centromere recombination have reshaped the genomes of different species. In this study, multiple DNA double-strand breaks (DSBs) were generated via the CRISPR/Cas9 system at centromere-specific retrotransposons in the human fungal pathogen Cryptococcus neoformans. The resulting DSBs were repaired in a complex manner, leading to the formation of multiple inter-chromosomal rearrangements and new telomeres, similar to chromothripsis-like events. The newly generated strains harboring chromosome translocations exhibited normal vegetative growth but failed to undergo successful sexual reproduction with the parental wild-type strain. One of these strains failed to produce any spores, while another produced [~]3% viable progeny. The germinated progeny exhibited aneuploidy for multiple chromosomes and showed improved fertility with both parents. All chromosome translocation events were accompanied without any detectable change in gene sequences and thus, suggest that chromosomal translocations alone may play an underappreciated role in the onset of reproductive isolation and speciation.

genetics↗

bioRxiv2019

Uniparental mitochondrial inheritance in Cryptococcus is controlled by the pheromone and pheromone receptor mating-type locus

Mitochondria are inherited uniparentally during sexual reproduction in the majority of eukaryotic species studied, including humans, mice, nematodes, as well as many fungal species. Mitochondrial uniparental inheritance (mito-UPI) could be beneficial in that it avoids possible genetic conflicts between organelles with different genetic backgrounds, as recently shown in mice; and it could prevent the spread of selfish genetic elements in the mitochondrial genome. Despite the prevalence of observed mito-UPI, the underlying mechanisms and the genes involved in controlling this non-mendelian inheritance are poorly understood in many species. In Cryptococcus neoformans, a human pathogenic basidiomyceteous fungus, mating types (MAT and MATa) are defined by alternate alleles at the single MAT locus that evolved from fusion of the two MAT loci (P/R encoding pheromones and pheromone receptors, HD encoding homeodomain transcription factors) that are the ancestral state in the basidiomycota. Mitochondria are inherited uniparentally from the MATa parent in C. neoformans and this requires the SXI1 and SXI2a HD factors encoded by MAT. However, there is evidence additional genes contribute to control of mito-UPI in Cryptococcus. Here we show that in Cryptococcus amylolentus, a sibling species of C. neoformans with unlinked P/R and HD MAT loci, mitochondrial uniparental inheritance is controlled by the P/R locus, and is independent of the HD locus. Consistently, by replacing the MAT alleles of the pheromones (MF) and pheromone receptor (STE3) with the MATa alleles, we show that these P/R locus defining genes indeed affect mito-UPI in C. neoformans during sexual reproduction. Additionally, we show that during early stages of C. neoformans sexual reproduction, conjugation tubes are always produced by the MAT cells, resulting in unidirectional migration of the MAT nucleus into the MATa cell during zygote formation. This process is controlled by the P/R locus and could serve to physically restrict movement of MAT mitochondria in the zygotes, and thereby contribute to mito-UPI. We propose a model in which both physical and genetic mechanisms function in concert to prevent the coexistence of mitochondria from the two parents in the zygote and subsequently in the meiotic progeny, thus ensuring mito-UPI in pathogenic Cryptococcus, as well as in closely related non-pathogenic species. The implications of these findings are discussed in the context of the evolution of mito-UPI in fungi and other more diverse eukaryotes.

genetics↗

bioRxiv2019

Mating-type specific divergent RPL22 paralogs are essential, differentially regulated, and control aspects of sexual reproduction in Cryptococcus neoformans

The MAT locus of Cryptococcus neoformans has a bipolar organization characterized by an unusually large structure, spanning over 100 kb. MAT genes have been characterized by functional genetics as being involved in sexual reproduction and virulence. However, classical gene replacement failed to achieve mutants for five MAT genes (RPL22, RPO41, MYO2, PRT1, RPL39), indicating that they are likely essential. In the present study, targeted gene replacement was performed in a diploid strain for both the and a alleles of the ribosomal genes RPL22 and RPL39. Mendelian analysis of the progeny confirmed that both RPL22 and RPL39 are essential for viability. Ectopic integration of the RPL22 allele of opposite MAT identity in the heterozygous RPL22a/rpl22{Delta} or RPL22/rpl22a{Delta} mutant strains failed to complement their essential phenotype. Evidence suggests that this is due to differential expression of the RPL22 genes, and an RNAi-dependent mechanism that contributes to control RPL22a expression. Furthermore, via CRISPR/Cas9 technology the RPL22 alleles were exchanged in haploid MAT and MATa strains of C. neoformans. These RPL22 exchange strains displayed morphological and genetic defects during bilateral mating. These results contribute to elucidate functions of C. neoformans essential mating type genes that may constitute a type of imprinting system to promote inheritance of nuclei of both mating types.

genetics↗

bioRxiv2019

Long transposon-rich centromeres in an oomycete reveal divergence of centromere features in Stramenopila-Alveolata-Rhizaria lineages

Centromeres are chromosomal regions that serve as platforms for kinetochore assembly and spindle attachments, ensuring accurate chromosome segregation during cell division. Despite functional conservation, centromere DNA sequences are diverse and often repetitive, making them challenging to assemble and identify. Here, we describe centromeres in an oomycete Phytophthora sojae by combining long-read sequencing-based genome assembly and chromatin immunoprecipitation for the centromeric histone CENP-A followed by high-throughput sequencing (ChIP-seq). P. sojae centromeres cluster at a single focus at different life stages and during nuclear division. We report an improved genome assembly of the P. sojae reference strain, which enabled identification of 15 enriched CENP-A binding regions as putative centromeres. By focusing on a subset of these regions, we demonstrate that centromeres in P. sojae are regional, spanning 211 to 356 kb. Most of these regions are transposon-rich, poorly transcribed, and lack the histone modification H3K4me2 but are embedded within regions with the heterochromatin marks H3K9me3 and H3K27me3. Strikingly, we discovered a Copia-like transposon (CoLT) that is highly enriched in the CENP-A chromatin. Similar clustered elements are also found in oomycete relatives of P. sojae, and may be applied as a criterion for prediction of oomycete centromeres. This work reveals a divergence of centromere features in oomycetes as compared to other organisms in the Stramenopila-Alveolata-Rhizaria (SAR) supergroup including diatoms and Plasmodium falciparum that have relatively short and simple regional centromeres. Identification of P. sojae centromeres in turn also advances the genome assembly. Author summaryOomycetes are fungal-like microorganisms that belong to the stramenopiles within the Stramenopila-Alveolata-Rhizaria (SAR) supergroup. The Phytophthora oomycetes are infamous as plant killers, threatening crop production worldwide. Because of the highly repetitive nature of their genomes, assembly of oomycete genomes presents challenges that impede identification of centromeres, which are chromosomal sites mediating faithful chromosome segregation. We report long-read sequencing-based genome assembly of the Phytophthora sojae reference strain, which facilitated the discovery of centromeres. P. sojae harbors large regional centromeres fully embedded in heterochromatin, and enriched for a Copia-like transposon that is also found in discrete clusters in other oomycetes. This study provides insight into the oomycete genome organization, broadens our knowledge of the centromere structure, function and evolution in eukaryotes, and may help elucidate the high frequency of aneuploidy during oomycete reproduction.

microbiology↗

bioRxiv2019

Cryptococcus neoformans recovered from olive trees (Olea europaea) in Turkey reveal allopatry with African and South American lineages

Cryptococcus species are life-threatening human fungal pathogens that cause cryptococcal meningoencephalitis in both immunocompromised and healthy hosts. The natural environmental niches of Cryptococcus include pigeon (Columba livia) guano, soil, and a variety of tree species such as Eucalyptus camaldulensis, Ceratonia siliqua, Platanus orientalis, and Pinus spp. Genetic and genomic studies of extensive sample collections have provided insights into the population distribution and composition of different Cryptococcus species in geographic regions around the world. However, few such studies examined Cryptococcus in Turkey. We sampled 388 Olea europaea (olive) and 132 E. camaldulensis trees from 7 locations in coastal and inland areas of the Aegean region of Anatolian Turkey in September 2016 to investigate the distribution and genetic diversity present in the natural Cryptococcus population. We isolated 84 Cryptococcus neoformans strains (83 MAT and 1 MATa) and 3 Cryptococcus deneoformans strains (all MATa) from 87 (22.4% of surveyed) O. europaea trees; a total of 32 C. neoformans strains were isolated from 32 (24.2%) of the E. camaldulensis trees, all of which were MAT. A statistically significant difference was observed in the frequency of C. neoformans isolation between coastal and inland areas (P < 0.05). Thus, O. europaea trees could represent a novel niche for C. neoformans. Interestingly, the MATa C. neoformans isolate was fertile in laboratory crosses with VNI and VNB MAT tester strains and produced robust hyphae, basidia, and basidiospores, thus suggesting potential sexual reproduction in the natural population. Sequencing analyses of the URA5 gene identified at least 5 different genotypes among the isolates. Population genetics and genomic analyses revealed that most of the isolates in Turkey belong to the VNBII lineage of C. neoformans, which is predominantly found in southern Africa; these isolates are part of a distinct minor clade within VNBII that includes several isolates from Zambia and Brazil. Our study provides insights into the geographic distribution of different C. neoformans lineages in the Mediterranean region and highlights the need for wider geographic sampling to gain a better understanding of the natural habitats, migration, epidemiology, and evolution of this important human fungal pathogen.

genetics↗

bioRxiv2019

Early diverging fungus Mucor circinelloides lacks centromeric histone CENP-A and displays a mosaic of point and regional centromeres

Centromeres are rapidly evolving across eukaryotes, despite performing a conserved function to ensure high fidelity chromosome segregation. CENP-A chromatin is a hallmark of a functional centromere in most organisms. Due to its critical role in kinetochore architecture, the loss of CENP-A is tolerated in only a few organisms, many of which possess holocentric chromosomes. Here, we characterize the consequence of the loss of CENP-A in the fungal kingdom. Mucor circinelloides, an opportunistic human pathogen, lacks CENP-A along with the evolutionarily conserved CENP-C, but assembles a monocentric chromosome with a localized kinetochore complex throughout the cell cycle. Mis12 and Dsn1, two conserved kinetochore proteins were found to bind nine short overlapping regions, each comprising an [~]200-bp AT-rich sequence followed by a centromere-specific conserved motif that echoes the structure of budding yeast point centromeres. Resembling fungal regional centromeres, these core centromere regions are embedded in large genomic expanses devoid of genes yet marked by Grem-LINE1s, a novel retrotransposable element silenced by the Dicer-dependent RNAi pathway. Our results suggest that these hybrid features of point and regional centromeres arose from the absence of CENP-A, thus defining novel mosaic centromeres in this early-diverging fungus.

genetics↗

bioRxiv2019

Advancing functional genetics through Agrobacterium-mediated insertional mutagenesis and CRISPR/Cas9 in the commensal and pathogenic yeast Malassezia furfur

Malassezia encompasses a monophyletic group of basidiomycetous yeasts naturally found on the skin of humans and other animals. Malassezia species have lost genes for lipid biosynthesis, and are therefore lipid-dependent and difficult to manipulate under laboratory conditions. In this study we applied a recently-developed Agrobacterium tumefaciens-mediated transformation protocol to perform T-DNA random insertional mutagenesis in Malassezia furfur. A total of 767 transformants were screened after exposure to 10 different stresses, and the 19 mutants that exhibited a phenotype different from the wild type were further characterized. The majority of these strains had single T-DNA insertions, which were identified within the open reading frames of genes, within untranslated regions, and in intergenic regions. Some T-DNA insertions generated chromosomal rearrangements, and others could not be characterized. To validate the findings of the forward genetic screen, a novel CRISPR/Cas9 system was developed to generate targeted deletion mutants for 2 genes identified in the screen: CDC55 and PDR10. This system is based on co-transformation of M. furfur mediated by A. tumefaciens to deliver both a CAS9-gRNA construct that induces double-strand DNA breaks, and a gene replacement allele that serves as a homology directed repair template. Targeted deletion mutants for both CDC55 and PDR10 were readily generated with this method. This study demonstrates the feasibility and reliability of A. tumefaciens-mediated transformation to aid in the identification of gene functions in M. furfur through both insertional mutagenesis and CRISPR/Cas9-mediated targeted gene deletion.

microbiology↗

bioRxiv2019

Drug-resistant epimutants exhibit organ-specific stability and induction during murine infections caused by the human fungal pathogen Mucor circinelloides

The environmentally ubiquitous fungus Mucor circinelloides is a primary cause of the emerging disease mucormycosis. Mucor infection is notable for causing high morbidity and mortality, especially in immunosuppressed patients, while being inherently resistant to the majority of clinically available antifungal drugs. A new, RNAi-dependent, and reversible epigenetic mechanism of antifungal resistance - epimutation - was recently discovered in M. circinelloides. However, the effects of epimutation in a host-pathogen setting were unknown. We employed a systemic, intravenous murine model of Mucor infection to elucidate the potential impact of epimutation in vivo. Infection with an epimutant strain resistant to the antifungal agents FK506 and rapamycin revealed that the epimutant-induced drug resistance was stable in vivo in a variety of different organs and tissues. Reversion of the epimutant-induced drug resistance was observed to be more rapid in isolates from the brain, as compared to those recovered from the liver, spleen, kidney, or lungs. Importantly, infection with a wild-type strain of Mucor led to increased rates of epimutation after strains were recovered from organs and exposed to FK506 stress in vitro. Once again, this effect was more pronounced in strains recovered from the brain than from other organs. In summary, we report the rapid induction and reversion of RNAi-dependent drug resistance after in vivo passage through a murine model, with pronounced impact in strains recovered from brain. Defining the role played by epimutation in drug resistance and infection advances our understanding of Mucor and other fungal pathogens, and may have implications for antifungal therapy.\n\nIMPORTANCEThe emerging fungal pathogen Mucor circinelloides causes a severe infection, mucormycosis, which leads to considerable morbidity and mortality. Treatment of Mucor infection is challenging because Mucor is inherently resistant to nearly all clinical antifungal agents. An RNAi-dependent and reversible mechanism of antifungal resistance, epimutation, was recently described in Mucor. Epimutation has not been studied in vivo and it was unclear whether it would contribute to antifungal resistance observed clinically. We demonstrate that epimutation can be both induced and reverted after in vivo passage through a mouse model; rates of both induction and reversion are higher after brain infection than after infection of other organs (liver, spleen, kidneys, or lungs). Elucidating the roles played by epimutation in drug resistance and infection will improve our understanding of Mucor and other fungal pathogens, and may have implications for antifungal treatment.

microbiology↗

bioRxiv2019

Hypermutation in Cryptococcus reveals anovel pathway to 5-fluorocytosine (5FC) resistance

Drug resistance is a critical challenge in treating infectious disease. For fungal infections, this issue is exacerbated by the limited number of available and effective antifungal agents. Patients infected with the fungal pathogen Cryptococcus are most effectively treated with a combination of amphotericin B and 5-fluorocytosine (5FC). Isolates causing infections frequently develop resistance to 5FC although the mechanism of this resistance is poorly understood. Here we show that resistance is acquired more frequently in isolates with defects in DNA mismatch repair that confer an elevated mutation rate. Natural isolates of Cryptococcus with mismatch repair defects have recently been described and defective mismatch repair has been reported in other pathogenic fungi. In addition, whole genome sequencing was utilized to identify mutations associated with 5FC resistance in vitro. Using a combination of candidate-based Sanger and whole genome Illumina sequencing, the presumptive genetic basis of resistance in 16 independent isolates was identified, including mutations in the known resistance genes FUR1 and FCY2, as well as a novel gene, UXS1. Mutations in UXS1 lead to accumulation of a metabolic intermediate that appears to suppress toxicity of both 5FC and its toxic derivative 5FU. Interestingly, while a UXS1 ortholog has not been identified in other fungi like Saccharomyces cerevisiae, where the mechanisms underlying 5FC and 5FU resistance were elucidated, a UXS1 ortholog is found in humans, suggesting that mutations in UXS1 in cancer cells may also play a role in resistance to 5FU when used during cancer chemotherapy in humans.

microbiology↗

bioRxiv2019

Convergent evolution of linked mating-type loci in basidiomycetes: an ancient fusion event that has stood the test of time

Sexual development is a key evolutionary innovation of eukaryotes. In many species, mating involves interaction between compatible mating partners that can undergo cell and nuclear fusion and subsequent steps of development including meiosis. Mating compatibility in fungi is governed by mating type determinants, which are localized at mating type (MAT) loci. In basidiomycetes, the ancestral state is hypothesized to be tetrapolar (bifactorial), with two genetically unlinked MAT loci containing homeodomain transcription factor genes (HD locus) and pheromone and pheromone receptor genes (P/R locus), respectively. Alleles at both loci must differ between mating partners for completion of sexual development. However, there are also basidiomycete species with bipolar (unifactorial) mating systems, which can arise through genomic linkage of the HD and P/R loci. In the order Tremellales, which is comprised of mostly yeast-like species, bipolarity is found only in the human pathogenic Cryptococcus species. Here, we describe the analysis of MAT loci from the Trichosporonales, a sister order to the Tremellales. We analyzed genome sequences from 29 strains that belong to 24 species, including two new genome sequences generated in this study. Interestingly, in all of the species analyzed, the MAT loci are fused and a single HD gene is present in each mating type. This is similar to the organization in the pathogenic Cryptococci, which also have linked MAT loci and carry only one HD gene per MAT locus instead of the usual two HD genes found in the vast majority of basidiomycetes. However, the HD and P/R allele combinations in the Trichosporonales are different from those in the pathogenic Cryptococcus species. The differences in allele combinations compared to the bipolar Cryptococci as well as the existence of tetrapolar Tremellales sister species suggest that fusion of the HD and P/R loci and differential loss of one of the two HD genes per MAT allele occurred independently in the Trichosporonales and pathogenic Cryptococci. This finding supports the hypothesis of convergent evolution at the molecular level towards fused mating-type regions in fungi, similar to previous findings in other fungal groups. Unlike the fused MAT loci in several other basidiomycete lineages though, the gene content and gene order within the fused MAT loci are highly conserved in the Trichosporonales, and there is no apparent suppression of recombination extending from the MAT loci to adjacent chromosomal regions, suggesting different mechanisms for the evolution of physically linked MAT loci in these groups.\n\nAuthor summarySexual development in fungi is governed by genes located within a single mating type (MAT) locus or at two unlinked MAT loci. While the latter is thought to be the ancestral state in basidiomycetes, physical linkage of the two MAT loci has occurred multiple times during basidiomycete evolution. Here, we show that physically linked MAT loci are present in all analyzed species of the basidiomycete order Trichosporonales. In contrast to previously studied basidiomycetes, the fused MAT loci in the Trichosporonales have highly conserved gene order, suggesting that this fusion might date back to the common ancestor of this lineage.

evolutionary biology↗

bioRxiv2019

Genetic and genomic analyses reveal boundaries between species closely related to Cryptococcus pathogens

Speciation is a central mechanism of biological diversification. While speciation is well studied in plants and animals, in comparison, relatively little is known about speciation in fungi. One fungal model is the Cryptococcus genus, which is best known for the pathogenic Cryptococcus neoformans/Cryptococcus gattii species complex that causes over 200,000 new infections in humans annually. The closest non-human pathogenic relatives are the sibling species, Cryptococcus amylolentus and Tsuchiyaea wingfieldii. However, because relatively few isolates of each species are available, it is unclear whether they represent divergent lineages of the same species or different biological species. The recent isolation of an additional strain, preliminarily identified as T. wingfieldii, prompted us to reexamine this group as it may inform about the evolutionary processes underlying the diversification of both non-pathogenic and pathogenic Cryptococcus lineages. Using genomic data, we reappraised the phylogenetic relationship of the four available strains and confirmed the genetic separation of C. amylolentus and T. wingfieldii (now Cryptococcus wingfieldii), and revealed an additional cryptic species, for which the name Cryptococcus floricola is proposed. Comparison of full-length chromosome assemblies revealed approximately 6% pairwise sequence divergence between the three species, and identified significant genomic changes, including inversions as well as a reciprocal translocation that involved inter-centromeric ectopic recombination, which together likely impose significant barriers to genetic exchange. Using genetic crosses, we show that while C. wingfieldii cannot interbreed with any of the other strains, C. floricola can undergo sexual reproduction with C. amylolentus. However, most of the spores resulting from this cross were inviable, and many were sterile, indicating that the two species are genetically isolated through intrinsic post-zygotic barriers and possibly due to niche differentiation. Genome sequencing and analysis of the progeny demonstrated decreased recombination frequency during meiosis in heterospecific crosses compared to C. amylolentus conspecific crosses. This study advances our understanding of speciation in fungi and highlights the power of genomics in assisting our ability to correctly identify and discriminate fungal species. Author SummaryThe idea of species as discrete natural units seems rather intuitive for most people, just as cells are the basic units of life. However, when observing variation across a species range, boundaries can become blurred making it less than obvious when different populations evolve into separate species. Additionally, separate species can still interbreed, such as lions breeding with tigers to produce a liger or a tigon (depending on the paternal and maternal species of origin), but the resulting offspring is usually inviable or sterile, which in turn is evidence that the parents involved are distinct species. Therefore, what species are and how they originate is still an open question in evolutionary biology. While recent advances have been made in the fields of animal and plant speciation, many other important components of biological diversity, such as fungi, are still understudied. Genome sequencing is now providing new tools to address the genetic mechanisms that drive divergence and reproductive isolation between populations, including genetic incompatibilities, sequence divergence, and chromosomal rearrangements. Here we focus on the Cryptococcus amylolentus species complex, a non-pathogenic fungal lineage closely related to the human pathogenic Cryptococcus neoformans/Cryptococcus gattii complex. Using genetic and genomic analysis we reexamined the species boundaries of four available isolates within the C. amylolentus complex and revealed three genetically isolated species. The genomes of these species are ~6% divergent and exhibit chromosome rearrangements, including translocations and small-scale inversions. Although two of the species (C. amylolentus and newly described C. floricola) are still able to interbreed, the resulting hybrid progeny were mostly inviable, and many were sterile, indicating that barriers to reproduction have already been established. Our results will foster additional studies addressing the transitions between non-pathogenic and pathogenic Cryptococcus lineages.

evolutionary biology↗