Biology subjects

Heitman, J.

Publications and source records attributed to Heitman, J..

At least 19 recordsLinked to original sources

bioRxiv2026

A single meiotic product dominates inheritance in Mucor circinelloides

Meiosis is a major source of genetic diversity, yet it remains poorly understood in many pathogenic fungi. In Mucorales, the causative agents of mucormycosis, sexual reproduction has been difficult to study, and reported inheritance patterns have appeared inconsistent with conventional meiosis. Here, meiosis in Mucor circinelloides was demonstrated at genome-wide resolution by generating telomere-to-telomere chromosome assemblies from compatible mating types and analyzing parental markers distributed across the genome. The results reveal meiotic recombination and independent chromosome assortment. However, the outcome of meiosis is strikingly unconventional: all sexual spores derived from a single zygospore share an identical nuclear genotype, even when germination produces distinct sporangia. This pattern suggests that the viable progeny of each zygospore is derived from a single meiotic product, rather than from the four products expected under conventional meiosis. Mitochondrial inheritance is also uniparental, with progeny mitochondria inherited from the (+) mating-type parent. Together, these findings show that sexual reproduction in M. circinelloides entails meiotic recombination, but restricts inheritance to a single viable nuclear product and a single mitochondrial lineage. This decoupling of recombination from equal meiotic contribution reveals a hidden layer of inheritance control in Mucorales, showing how pathogenic fungi can reshuffle chromosomes while producing genetically uniform sibling progeny, an outcome reminiscent of female meiosis in animals.

genetics↗

bioRxiv2026

Host brain infection induces RNAi epimutation in Mucor circinelloides and enhances blood-brain barrier invasion

Mucorales fungi cause mucormycosis, a rapidly progressive invasive fungal disease with high mortality, particularly in immunocompromised patients and in severe disseminated infections. Central nervous system involvement is among the most devastating manifestations of mucormycosis, yet the mechanisms that enable Mucorales to adapt to the brain environment remain poorly understood. In Mucor species, RNA interference (RNAi) can generate reversible heritable epimutations that silence specific genes without altering DNA sequence, providing a transient and reversible mechanism for antifungal drug resistance. Here, we show that host brain infection induces RNAi-dependent silencing of endogenous fungal loci that promote central nervous system adaptation. Small RNA sequencing of Mucor circinelloides recovered from infected mouse organs identified seven brain epimutation loci, named bepA to bepG, that accumulated small RNAs preferentially after brain infection. Among these, bepA encodes a conserved mitogen-activated protein kinase (MAPK)-like serine/threonine kinase related to cell wall integrity signaling. Deletion of the bepA gene confers resistance to the calcineurin inhibitors FK506 and cyclosporin A, allowing hyphal growth under conditions that impair calcineurin-dependent morphogenesis. BepA modulates FK506-induced MAPK signaling and controls a transcriptional program enriched in cell-wall remodeling genes. To model fungal central nervous system entry by a filamentous fungal pathogen, we established a human blood-brain barrier transwell platform that quantifies Mucor migration across brain endothelial monolayers. This model revealed that bepA mutation enhances fungal migration across brain endothelial monolayers without detectable changes in electrical barrier resistance. These findings identify brain-induced RNAi epimutation as a mechanism of organ-specific fungal adaptation and reveal reversible silencing of a MAPK-like kinase as a route to enhanced central nervous system invasion.

genetics↗

bioRxiv2026

Streptomyces induces unisexual reproduction of a major human fungal pathogen

Cryptococcus, a human pathogenic yeast, can undergo unisexual reproduction in the absence of compatible mating partners, yet this developmental program remains poorly understood. Here, we report that tunicamycin, a natural product produced by Streptomyces species, induces a complete unisexual reproductive cycle in different Cryptococcus species. Bacterial confrontation or purified tunicamycin trigger hyphal differentiation, meiotic recombination, and basidiospore formation, as confirmed by whole-genome sequencing and SNP analyses. We further demonstrate that tunicamycin-induced unisexual development requires canonical sexual regulators and meiotic machinery. Transcriptomic profiling reveals staged developmental reprogramming characterized by early induction of stress- and cell cycle-associated genes, followed by enrichment of genes in the filamentation-related pathways. This capacity varies among natural isolates of C. neoformans and was conserved in Cryptococcus gattii. Together, our findings demonstrate that a bacterial metabolite can activate meiotically competent unisexual reproduction, highlighting interkingdom chemical signaling as a potential driver of fungal evolution in natural environments.

microbiology↗

bioRxiv2026

An ancestral mitochondrial DNA insertion disrupts RNAi and enables persistence of a novel mycovirus in Cryptococcus neoformans

RNA interference (RNAi) is a widely conserved genome-defense mechanism that protects eukaryotes against viruses and transposable elements. We previously showed that RNAi loss can lead to hypermutation and antifungal drug resistance in the human fungal pathogen Cryptococcus neoformans, illuminating the potential clinical relevance of this pathway. In this study, we identified another function of RNAi in C. neoformans: mycovirus restriction. By screening known RNAi-deficient C. neoformans isolates, we identified a novel dsRNA mycovirus of the Orthototiviridae family, which we named CnTV1. We subsequently detected CnTV1 in three additional RNAi-deficient isolates. All CnTV1-positive isolates shared an ancestral nuclear mitochondrial DNA segment (NUMT) insertion that disrupts the gene encoding Argonaute (Ago1). Restoration of RNAi in sexually produced zygotes efficiently eliminated CnTV1. RNAi rescue by CRISPR-Cas9-mediated allele exchange eliminated CnTV1 during vegetative growth, further demonstrating RNAi is sufficient for mycoviral control. Loss of the RNA helicase Ski2 or the exoribonuclease Xrn1 increased CnTV1 abundance, revealing RNAi-independent restriction of the virus. Restoration of RNAi cleared the virus even in the absence of Ski2 or Xrn1, indicating a dominant role for RNAi in antiviral defense. To investigate the biological implications of CnTV1 infection, we developed a cytoplasmic-mixing approach and generated isogenic strain pairs in an RNAi-deficient background that differ only in viral infection status. Leveraging these strains, we show that CnTV1 infection leads to coordinated, low-magnitude transcriptomic changes and that strains lacking the mycovirus were moderately less virulent in a murine infection model. Our findings reveal that RNAi serves as a dominant antiviral defense system in C. neoformans and suggest that naturally occurring RNAi deficiency may be more prevalent than previously appreciated. This work highlights mycovirus persistence as an important consequence of RNAi loss in this WHO-designated critical priority fungal pathogen.

genetics↗

bioRxiv2026

Trehalose-6-phosphate synthase promotes thermotolerance by governing glycolytic flux in Cryptococcus deneoformans

Growth at physiologically relevant temperatures is essential for fungal pathogenesis and is controlled by several cellular factors. The evolution of fungal thermotolerance is concerning as warming environments may promote the emergence of new pathogens. Trehalose, a disaccharide absent in mammals, plays a central role in thermotolerance by stabilizing proteins and membranes during heat stress. Trehalose is synthesized from glucose-6-phosphate (G6P) and uridine-diphosphate-glucose (UDPG) in two steps catalyzed by trehalose-6-phosphate synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2). Here, we investigated genetic suppression of Tps1 function in Cryptococcus deneoformans, a species in the Cryptococcus pathogenic species complex. Tps1 is essential for growth at 37{degrees}C in C. deneoformans and spontaneous suppressor mutations restored the growth of tps1{Delta} mutants at 37{degrees}C. Whole-genome sequencing followed by variant calling analysis primarily identified loss-of-function mutations in the gene encoding hexokinase 1 (Hxk1). Targeted gene deletion mutants further showed that loss of either HXK1 or HXK2 can bypass tps1{Delta} in a carbon source-dependent manner. The tps1{Delta} mutant exhibited elevated hexokinase activity, accumulation of G6P and glycogen, and ATP depletion after heat shock. Deletion of HXK1 or HXK2 restored hexokinase activity and partially restored G6P and ATP levels in the tps1{Delta} mutant, while glycogen remained elevated, indicating that excess glycolytic flux underlies the tps1{Delta} high-temperature growth defect. Overall, our study uncovers a previously unappreciated mechanism of Tps1-mediated heat adaptation in C. deneoformans, by revealing that Tps1 functions as a critical metabolic gatekeeper that safeguards glycolytic flux to sustain growth at elevated temperatures. Article summaryTrehalose is crucial for fungal thermal adaptation and mutants lacking trehalose are inviable at 37{degrees}C. This study examined how genetic suppressors restore viability at 37{degrees}C in mutants lacking TPS1, which encodes trehalose-6-phosphate synthase. Through whole-genome sequencing of spontaneous suppressor isolates and variant calling analysis, mutations were identified in HXK1. Gene deletion mutants and biochemical assays show these mutations alter glycolytic flux. We show that tps1{Delta} mutants exhibit unbridled glycolysis, and their growth at 37{degrees}C is restored by hxk1{Delta} mutations that reduce glycolytic flux. This study highlights the interdependence between Tps1 and Hxk1, which may have broader relevance across organisms.

genetics↗

bioRxiv2026

Host skin lipids trigger MAT-dependent mating, pathogenic hyphal growth, and parasexual reproduction of Malassezia furfur

The lipophilic yeast genus Malassezia dominates the human skin mycobiome and, while typically commensal, is associated with skin disorders in which hyphal cells are frequently observed. However, the mechanisms underlying hyphal differentiation and its contribution to pathogenesis remain poorly understood. Here, we show that hyphal growth in Malassezia furfur requires a complete mating-type system and is promoted by noncanonical reproductive interactions. Interspecific mating generates stable diploid hybrids with evidence of recombination, whereas intraspecific mating produces cell-fusion products that undergo recombination and haploidization, consistent with a parasexual cycle. Functional analyses reveal that the pheromone/receptor (P/R) locus governs cell recognition and fusion, while the homeodomain (HD) locus is required for hyphal development despite having lost canonical mating-type determination. Leveraging in vitro and in vivo skin models, we show that hyphal cells invade the epidermis more efficiently and elicit stronger inflammatory responses than yeast cells. Together, these findings link parasexual-like reproduction, mating-type loci function, and morphogenesis in Malassezia to pathogenic interactions with the host.

microbiology↗

bioRxiv2026

Variable ectopic heterochromatin islands provide an alternative route to antifungal heteroresistance in Cryptococcus neoformans

The fungal pathogen Cryptococcus neoformans is estimated to cause approximately 180,000 deaths annually, primarily among immunocompromised HIV patients in resource-limited countries in central and southern Africa. Economic constraints regularly restrict treatment to fluconazole monotherapy. Unstably resistant cells arise in any otherwise fluconazole-sensitive C. neoformans population, with such heteroresistance often involving chromosome 1 disomy. Here we uncover a form of heteroresistance that occurs by a mechanism distinct from aneuploidy. Transient islands of histone H3 lysine 9 methylation-dependent heterochromatin occur at several locations across the genomes of clinical isolates from African patients. Some islands dissipate in the absence of fluconazole selection but are restored upon re-exposure. Deletion of specific heterochromatin island-located genes increased fluconazole resistance in an otherwise wild-type background, suggesting that heterochromatin-mediated alterations in gene expression elicit unstable resistance. Thus, in addition to aneuploidy, transient heterochromatin islands also appear to contribute to C. neoformans fluconazole heteroresistance. Heterochromatin islands have recently been shown to confer unstable antifungal resistance in the ascomycete fungus Schizosaccharomyces pombe (fission yeast) and fungal species of the Mucor circinelloides complex. Thus, similar unstable resistant epimutations may be widespread in pathogenic fungi, however, their instability and inaccessibility to detection by direct sequencing poses challenges for monitoring this form of heteroresistance in clinical settings.

microbiology↗

bioRxiv2026

Genomic hallmarks of parasexual reproduction in three hybrid groups of the human pathogen Cryptococcus neoformans

Hybridization is a major driver of fungal evolution, yet knowledge of the molecular mechanisms underpinning hybridization and its genomic impact remain limited. Here, we analyse 197 Cryptococcus neoformans genomic sequences, including 13 newly sequenced strains, identifying three genetically clustered and distinct hybrid groups (H1, H2 and H3) each with unique parental origins and ecological associations. Using phylogenomics, population structure analyses, and long-read genome assemblies, we identified hybrid genomes with chromosome-wide loss of heterozygosity (LOH), inheritance of large intact parental haplotype blocks and widespread aneuploidy within and across genomes. These patterns are also observed when progeny were generated with spo11{Delta} parents, indicating these features are a result of a meiotic-independent process such as parasex. This is further underscored by discovery of haploid and near-haploid recombinants in both spo11{Delta} mutant progeny and reanalysed wild-type hybrid progeny. We hypothesise these haploid and near-haploid recombinants are generated through ploidy reduction via independent chromosome assortment because of concerted chromosomal loss, which is a key feature of the parasexual cycle. Phenotypic assays demonstrated that several hybrid isolates have diverse growth and virulence patterns, underscoring functional consequences of genome plasticity. Together, our work suggests a non-meiotic reproductive process contributes to shape the genotypic and phenotypic diversity of Cryptococcus neoformans.

genomics↗

bioRxiv2026

Systematic Profiling of Essential Fungal Transcription Factors Uncovers Ezt1 as a Central Pathobiological and Morphogenic Regulator in Cryptococcus neoformans

Cryptococcus neoformans is a global fungal pathogen that causes fatal cryptococcosis, and the limitations of current antifungals underscore the urgent need for new therapeutics. Here we systematically investigate essential transcriptional regulators in C. neoformans as potential antifungal targets, developing experimental pipelines that assess growth requirement, essentiality and function through conditional gene expression, constitutive overexpression, and meiotic spore analysis. We identify one quasi-essential (growth-required but non-essential) transcription factor, Fhl1, and 13 essential transcriptional regulators, three of which are transcription factors (Ezt1, Ezt2 and Cbf1) highly divergent from counterparts in other eukaryotes. Notably, Ezt1 modulates the expression of more than 1,200 genes, controlling growth, antifungal drug and stress responses, sexual development and virulence. Our findings define the essential transcriptional regulator landscape of C. neoformans and provide a framework for prioritising divergent essential regulators, particularly Ezt1, for antifungal target discovery.

microbiology↗

bioRxiv2026

Fourier transform infrared spectroscopy reveals high intraspecies diversity of Malassezia pachydermatis in dogs with atopic dermatitis

1Canine atopic dermatitis (CAD) is a chronic inflammatory skin condition sometimes associated with microbial dysbiosis, including alterations in colonization by the lipophilic yeast Malassezia pachydermatis. This study investigated the population diversity of M. pachydermatis in the ear canals of healthy and CAD-affected dogs using Fourier-transform infrared (FTIR) spectroscopy and whole genome sequencing (WGS). Among 60 dogs, M. pachydermatis prevalence was significantly higher in CAD cases than in healthy controls. FTIR spectroscopy revealed greater strain heterogeneity in CAD-affected dogs, often with distinct genotypes in each ear, while healthy dogs exhibited more homogeneous populations. Using a previously developed FTIR-based artificial neural network classifier, we assigned strains to three phylogroups. Strains from phylogroups I and III were significantly enriched in CAD-affected dogs, while phylogroup II was most prevalent overall and the dominant phylogroup in healthy controls. This suggests that CAD-associated inflammation may favor specific M. pachydermatis phylogroups and sub-clusters within phylogroups, shaping colonization dynamics. FTIR-based typing showed full concordance with WGS across 35 sequenced isolates, recapitulating relationships among phylogenetically related isolates and their similar phenotypic profiles. Overall, our findings reveal strain-level shifts in M. pachydermatis populations associated with CAD and establish FTIR spectroscopy as a rapid, cost-effective tool for large-scale epidemiological studies.

microbiology↗

bioRxiv2026

Fluorescent organelle markers in Cryptococcus neoformans: a versatile toolkit for live-cell subcellular localization

Understanding how intracellular organelles are organized and dynamically remodeled is essential for elucidating fungal growth, differentiation, and pathogenicity. The human fungal pathogen Cryptococcus neoformans exhibits remarkable morphological diversity, yet the contribution of intracellular organelles to adaptation across environmental and host-relevant conditions has not been systematically examined. Here, we establish a comprehensive toolkit of fluorescent organelle marker plasmids and strains expressing mCherry or GFP fusions, enabling high-resolution live-cell imaging of ten major subcellular compartments: the nucleolus, endoplasmic reticulum (ER), Golgi apparatus, mitochondria, peroxisome, endosome, autophagosome, vacuole, the plasma membrane, and processing bodies (P-bodies). With this platform, we analyzed organelle organization under host-relevant conditions, including elevated temperature, 5% CO2, and capsule- and melanin-inducing media, as well as throughout sexual development from zygote formation to basidiospore production. We further show that the toolkit supports colocalization analyses via diploid formation or dual-labeling strategies. Collectively, our results reveal condition- and stage-specific remodeling of organelle architecture during vegetative growth and mating. This imaging platform provides a robust framework for investigating how subcellular organization supports fungal adaptation, development, and virulence, and for inferring the functions of uncharacterized genes from their spatial localization.

cell biology↗

bioRxiv2026

Systems-level phosphoproteomics reveals conserved and subunit-specific STRIPAK signaling networks in Cryptococcus neoformans

The striatin-interacting phosphatase and kinase (STRIPAK) complex is a conserved PP2A-associated signaling hub that integrates kinase-phosphatase networks, yet its roles in human fungal pathogens remain poorly defined. Here, we dissected STRIPAK functions in the opportunistic pathogen Cryptococcus neoformans by combining genetic, genomic, virulence, and phosphoproteomic analyses across mutants lacking individual STRIPAK subunits. Loss of the core STRIPAK components via PPH22, FAR8, FAR9, or FAR11 mutations caused severe defects in growth, stress adaptation, cell-cycle progression, and morphogenesis, accompanied by widespread aneuploidy and genome instability. In murine infection models, far11{Delta} strains were avirulent, whereas far9{Delta} mutants caused delayed but ultimately fatal disease and underwent host-associated genome remodeling, with recovered isolates exhibiting chromosome 11 amplification despite no consistent in vitro fitness advantage. In contrast, deletion of MOB3 produced a hypervirulent phenotype. mob3{Delta} cells exhibited enhanced transmigration across an in vitro blood-brain barrier model, increased survival in macrophages, and generated small-cell morphotypes, features associated with increased dissemination. Phosphoproteomic profiling revealed extensive and overlapping phosphorylation changes among core STRIPAK mutants, affecting pathways involved in signaling, cytoskeletal and cell-cycle control, chromatin regulation, RNA metabolism, and stress responses. Conversely, mob3{Delta} mutants displayed a smaller, largely distinct phosphoproteomic signature. Network and functional enrichment analyses highlighted STRIPAK-dependent regulation of TORC2-associated signaling, MAPK/GTPase signaling, autophagy, nuclear transport, RNA processing, DNA replication, and ribosome biogenesis. Together, these findings establish STRIPAK as a coordinator of genome stability, morphological plasticity, stress adaptation, and virulence in C. neoformans, and demonstrate that individual STRIPAK subunits drive shared yet divergent signaling outputs that shape host-pathogen interactions. ImportanceFungal pathogens must rapidly adapt their growth, morphology, and stress responses to survive within the host, requiring precise coordination of cellular signaling pathways. The conserved striatin-interacting phosphatase and kinase (STRIPAK) complex controls key developmental programs in eukaryotes, but its roles in fungal pathogenesis are not fully defined. We previously showed that STRIPAK is important for genome stability, development, and virulence in the opportunistic human fungal pathogen Cryptococcus neoformans. Here, we define how individual STRIPAK subunits differentially regulate fungal morphogenesis, genome plasticity, host adaptation, and virulence, revealing both shared and subunit-specific functions within this conserved signaling complex. Core STRIPAK mutants exhibit severe growth and stress-response defects and attenuation of virulence, whereas loss of the Mob3 subunit promotes hypervirulence by enhancing dissemination and persistence within the host. Phosphoproteomic profiling reveals that individual STRIPAK components exert shared yet distinct control over phosphorylation networks that shape host-pathogen interactions, establishing STRIPAK as a central signaling hub and a potential target for antifungal intervention.

microbiology↗

bioRxiv2025

The protein phosphatase 2C domain contributes to the pathobiological function of adenylyl cyclase in Cryptococcus neoformans

Adenylyl cyclase produces cyclic adenosine monophosphate, a signalling molecule that controls fungal development and virulence. Fungal adenylyl cyclases are distinguished by the presence of a conserved protein phosphatase 2C domain, whose function remains unknown. Here we show that the protein phosphatase 2C domain of Cac1, the adenylyl cyclase of the human pathogen Cryptococcus neoformans, has an unusual structure but functions as a metal-dependent serine/threonine phosphatase. Deletion of this domain or the adenylyl cyclase catalytic domain demonstrated that the protein phosphatase 2C domain is required for full Cac1 activity, influencing melanin and capsule synthesis, sexual differentiation, titan cell formation, and cell wall integrity. Notably, loss of the protein phosphatase 2C domain induces type 2 helper T-cell-biased immunity and extensive pulmonary damage yet does not cause mortality in mice. Integrated transcriptomic and phosphoproteomic analyses further revealed shared and domain-specific signalling outputs. Together, these findings define a pivotal role for the adenylyl cyclase-linked protein phosphatase 2C domain in C. neoformans pathobiology.

microbiology↗

bioRxiv2025

Dissecting the homeodomain MAT locus and engineering novel tripolar and bipolar mating systems in Cryptococcus amylolentus

Sex in fungi is governed by the mating-type (MAT) locus, which exists as bipolar, pseudobipolar, or tetrapolar systems. The significance and impact of MAT on sexual reproduction, however, remain understudied. Furthermore, the evolution of fungal MAT loci shares features with the evolution of sex chromosomes in plants and animals. Pathogenic Cryptococcus species harbor a bipolar system with a large contiguous MAT locus, whereas closely related species, such as the non-pathogen C. amylolentus possess a tetrapolar system with unlinked P/R and HD loci. The HD locus encodes homeobox domain containing proteins that play an important and evolutionarily conserved role in sexual reproduction. Here, we explored the roles of HD genes in sexual reproduction and determined the implications of a tetrapolar to bipolar MAT transition. With a CRISPR-Cas9 system we developed for C. amylolentus, we generated gene deletion mutants and demonstrated that a single compatible Sxi1-Sxi2 pair is necessary and sufficient for mating. By relocating the HD genes to the P/R locus, we found that the artificially generated bipolar configuration led to defective sexual development, which could be partially restored through additional rounds of sexual reproduction. Transcriptomic profiling further revealed that a Sxi1-Sxi2 heterodimeric complex drives expression of genes required for DNA replication and ergosterol biosynthesis during sexual reproduction. These findings provide the first experimental demonstration of a tetrapolar-to-bipolar transition in a tetrapolar mating species, illuminating MAT locus evolution and homeodomain protein functions in Cryptococcus. ImportanceSexual reproduction is critical for fungal survival and adaptation, yet the mechanisms driving transitions between mating systems remain unclear. With Cryptococcus amylolentus, we provide the first experimental validation of a mating system transition from its original tetrapolar to an intermediate tripolar to a derived bipolar in a tetrapolar species. We show that HD heterodimers phenotypically govern dikaryotic filamentation and also transcriptionally modulate DNA replication. These findings establish a mechanistic basis for how MAT locus reorganization drives bipolar evolution from an ancestral tetrapolar state and reinforce that fertility depends on the coordinated control of MAT locus architecture and regulatory functions.

microbiology↗

bioRxiv2025

Fourier transform infrared spectroscopy enables rapid species discrimination across Malassezia and strain-level typing in M. pachydermatis

1Malassezia pachydermatis is a zoophilic yeast found on the skin and in the outer ear canal of many mammals. It normally maintains a commensal lifestyle but can cause dermatitis and otitis in predisposed hosts, particularly in atopic dogs. M. pachydermatis is genetically diverse, with strains clustering into at least three phylogroups based on molecular typing, a pattern we now confirm through whole-genome sequencing (WGS). Accurate species and strain-level identification is essential for understanding its epidemiology, pathogenic potential, and response to treatment. In this study, we established Fourier Transform Infrared (FTIR) spectroscopy as a rapid, cost-effective method for distinguishing M. pachydermatis from other Malassezia species, including M. globosa, M. furfur, M. restricta, and M. sympodialis. Within M. pachydermatis, FTIR spectroscopy resolved even closely related strains with high accuracy producing clusters congruent with WGS-based phylogeny. The incorporation of an Artificial Neural Network classifier further enhanced the discriminatory power, enabling robust and automated strain assignment. These findings demonstrate the potential of FTIR spectroscopy as a practical tool for large-scale epidemiological surveillance of M. pachydermatis and for clinical and veterinary applications where strain-level identification could inform treatment and management of Malassezia-associated diseases. ImportanceMalassezia pachydermatis is a yeast that commonly inhabits the skin and ear canals of mammals but can cause dermatitis and otitis in predisposed hosts, especially dogs with allergies. This species displays substantial genetic diversity, with strains falling into distinct phylogroups that may differ in their biology and clinical significance. Determining these differences has typically required advanced molecular or genomic methods, which can be costly and time-consuming. In this study, we demonstrate that Fourier transform infrared spectroscopy can rapidly and accurately distinguish M. pachydermatis from other Malassezia species and resolve genetic groups within the species in a way that reflects whole-genome relationships. This capability offers a practical tool for investigating the epidemiology and inter-/intraspecies diversity of M. pachydermatis and for guiding targeted management of Malassezia-associated diseases in both veterinary and, potentially, human medicine.

microbiology↗

bioRxiv2025

Whole-chromosome duplications drive antimicrobial resistance in Aspergillus fumigatus

Aneuploidy causes genome plasticity and enables adaptive responses that increase stress resistance and facilitate persistence under changing conditions in eukaryotes ranging from fungal pathogens to human cancer cells. Aspergillus fumigatus is a soil-resident mold and the most prevalent etiologic agent of invasive fungal infections globally. Invasive aspergillosis has an alarmingly high mortality rate, and fungal persistence in the infection microenvironment often renders treatments unsuccessful. Treatment failures can result from innate or acquired antifungal drug resistance and from fungal adaptation to other stressors encountered during infection including nutrient limitation, immune cell activity, and changes in pH and oxygen tension. Survival under changing environmental and host conditions therefore necessitates an ability to dynamically adapt to stress. We find that exposure of A. fumigatus to FK506, an antifungal and immunosuppressive Streptomyces natural product that inhibits the master regulatory phosphatase calcineurin, selects for unstable whole-chromosome aneuploidies that alleviate the polarized growth defects caused by calcineurin inhibition. Transcriptomic analysis revealed that Chr7 disomy leads to induction of the normally-silent neosartoricin biosynthetic gene cluster in response to FK506 exposure, and we demonstrate that constitutive genetic induction of this cluster is sufficient to largely recapitulate in a haploid background the response to FK506 in the aneuploid state. We further show that aneuploids undergo extensive metabolic rewiring but do not produce detectable neosartoricin, revealing global and cross-pathway effects resulting from both the aneuploid state and activation of nscR. We also demonstrate that the aneuploid chromosome duplications reduce susceptibility to the clinical antifungal voriconazole, underscoring the utility of aneuploidy as a flexible adaptive strategy. These findings represent a major advance in understanding how aneuploidy transiently alters stress responses and metabolism in the deadly human pathogen A. fumigatus. Highlights- A. fumigatus rapidly gains and loses supernumerary chromosomes in response to the calcineurin inhibitor FK506. - Aneuploidy leads to global transcriptional and metabolic rewiring. - Overexpression of the secondary metabolite gene cluster regulator nscR is sufficient to largely recapitulate the aneuploid response to FK506 in a haploid background. - Chr4, 6, and 7 aneuploidies reduce susceptibility to voriconazole without altering expression of the azole target gene cyp51A.

microbiology↗

bioRxiv2025

Meiosis-specific genes play roles in ploidy reduction in Cryptococcus neoformans titan cells

Cryptococcus neoformans is a fungal pathogen of humans that causes life-threatening meningoencephalitis. During infection, enlarged, polyploid titan cells are produced that promote persistence in the host, in part by resisting phagocytosis; under stress conditions, such as exposure to the antifungal drug fluconazole, titan cells can produce aneuploid or diploid, drug-resistant daughter cells. However, the mechanism underlying this ploidy reduction remains poorly understood. Interestingly, meiosis related genes have been shown to be activated during Cryptococcus infection, leading us to hypothesize that the depolyploidization of C. neoformans titan cells may occur through a process resembling the ploidy reduction during meiosis. In this study, we show that titan cells developed from diploid strains predominantly produce diploid daughter cells with haploid daughters observed infrequently. We further demonstrate that meiosis-specific genes, including DMC1 and SPO11, are critical for stable inheritance of a diploid genome in the daughter cells. Specifically, deletion of these genes in a heterozygous diploid background resulted in: 1) titan cells with a significantly reduced capacity to produce daughter cells; 2) increased phenotypic variation among daughter cells produced by the titan cells, including traits that could be relevant to cell growth and viability; and 3) daughter cells produced by the titan cells exhibiting high levels of loss of heterozygosity (LOH) and aneuploidy, suggesting elevated genome instability. Taken together, these findings demonstrate the importance of meiosis-specific genes in the ploidy reduction process of titan cells derived from a heterozygous diploid background in an important human fungal pathogen. SIGNIFICANCEPolyploid titan cells are central to Cryptococcus neoformans persistence during infection, yet how they reduce ploidy to generate daughter cells was unclear. In most eukaryotes, the meiosis-related proteins Spo11 and Dmc1 are best known for initiating (double-strand break formation) and executing (strand exchange) meiotic recombination, respectively. Here, we show that in C. neoformans, Dmc1 and Spo11 are critical for polyploid titan cells to produce viable daughter cells with reduced ploidy specifically in a heterozygous diploid context, ensuring genome stability without introducing detectable canonical meiotic recombination. Loss of either gene compromises daughter cell production and causes genome instability (LOH, aneuploidy) and phenotypic heterogeneity. These findings reveal a "para-meiosis" safeguard that preserves genomic configurations during titan cell depolyploidization in C. neoformans.

genetics↗

bioRxiv2025

Genomic Evidence for a-α Heterothallic and α-α Unisexual Mating and Recombination in an Environmental Cryptococcus deneoformans Population

Cryptococcus deneoformans is a human fungal pathogen capable of both a- and - mating and sexual reproduction in laboratory settings. However, the extent of a- and - sexual reproductions in natural populations remain unexplored. Here we analyzed the whole-genome sequences of 24 environmental strains of C. deneoformans from western Saudi Arabia, including one MATa and 23 MAT isolates, with 15 MAT isolates belonging to multi-locus sequence type ST160 as defined by their combined DNA sequences at seven loci. To identify signatures for a- and - reproduction, three samples were analyzed: total, MAT, and ST160. For each subpopulation, single nucleotide polymorphisms (SNPs) were identified for both the nuclear and mitochondrial genomes and subjected to four-gamete tests. In the total population and the MAT subpopulation, variable proportions of SNP pairs within as well as between the nuclear and the mitochondrial genomes showed evidence for recombination. Though no mitogenome SNPs were found among the 15 strains of ST160, the nuclear genome showed clear evidence for recombination, including among SNPs within the mating type region. In addition, the nuclear genome SNP pairs located further apart on the same chromosome showed a greater frequency of recombination in all three sample types. In contrast, mitogenome recombination breakpoints were mainly located in two genomic regions. Together, these results provide robust evidence for both a- and - sexual reproduction within this environmental population of C. deneoformans. Article SummaryThis study compares conclusions drawn from multilocus sequence data and whole-genome SNP data regarding genetic diversity and reproduction in an environmental population of Cryptococcus deneoformans. By analyzing bi-allelic SNPs across 24 strains isolated from western Saudi Arabian soils, evidence of both a- and - recombination was found, suggesting the occurrence of both a- and - sexual reproduction in nature. This provides strong support for these reproductive modes and indicates diverse mechanisms of genetic exchange in fungal pathogens in nature. Additionally, our results suggest that multi-locus sequence typing underestimates genetic diversity, potentially confounding interpretations of population structure and evolutionary history.

microbiology↗