Biology subjects

Averette, A. F.

Publications and source records attributed to Averette, A. F..

7 recordsLinked to original sources

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↗

bioRxiv2024

Structure-guided design, synthesis, and characterization of C22- and C32-modified FK520 analogs with enhanced activity against human pathogenic fungi

Invasive fungal infections are a leading cause of death worldwide. Translating molecular insights into clinical benefits is challenging because fungal pathogens and their hosts share similar eukaryotic physiology. Consequently, current antifungal treatments have limited efficacy, may be poorly fungicidal in the host, can exhibit toxicity, and are increasingly compromised by emerging resistance. We have established that the phosphatase calcineurin (CaN) is required for invasive fungal disease and an attractive target for antifungal drug development. CaN is a druggable target, and there is vast clinical experience with the CaN inhibitors FK506 and cyclosporin A (CsA). However, while FK506 and its natural analog FK520 exhibit antifungal activity, they are also immunosuppressive in the host and thus not fungal-selective. We leverage our pathogenic fungal CaN-FK506-FKBP12 complex X-ray structures and biophysical data to support structure-based ligand design as well as structure-activity relationship analyses of broad-spectrum FK506/FK520 derivatives with potent antifungal activity and reduced immunosuppressive activity. Here we apply molecular docking studies to develop antifungal C22- or C32-modified FK520 derivatives with improved therapeutic index scores. Among them, the C32-modified FK520 derivative JH-FK-44 (7) demonstrates a significantly improved therapeutic index compared to JH-FK-08, our lead compound to date. NMR binding studies with C32-derivatives are consistent with our hypothesis that C32 modifications disrupt the hydrogen bonding network in the human complex while introducing favorable electrostatic and cation-{pi} interactions with the fungal FKBP12 R86 residue. These findings further reinforce calcineurin inhibition as a promising strategy for antifungal therapy. SignificanceInvasive fungal infections cause significant mortality worldwide, and current antifungal treatments are often ineffective, toxic, or face growing resistance. This research identifies calcineurin (CaN), a critical protein for fungal survival, as a potential target for developing new antifungal drugs. Although existing CaN inhibitors such as FK506 (tacrolimus) and FK520 (ascomycin) possess antifungal properties, their immunosuppressive effects limit their clinical utility. By studying the structure of human and fungal FKBP12-FK506 or FK520 complexes with CaN, we have designed and synthesized modified FK520 derivatives with strong antifungal activity and reduced immunosuppressive effects. These new derivatives are expected to have significantly improved therapeutic profiles, offering hope for more effective and safer antifungal treatments.

microbiology↗

bioRxiv2022

Obligate sexual reproduction of a homothallic fungus closely related to the Cryptococcus pathogenic species complex

Sexual reproduction is a ubiquitous and ancient trait of eukaryotic life. While sexual organisms are usually faced with the challenge of finding a compatible mating partner, species as diverse as animals, plants, and fungi have repeatedly evolved the ability to reproduce sexually without an obligate requirement for another individual. Here, we uncovered the underlying mechanism of self-compatibility (homothallism) in Cryptococcus depauperatus, a fungal species sister to the clinically relevant human fungal pathogens Cryptococcus neoformans and Cryptococcus gattii species complexes. In contrast to C. neoformans or C. gattii, which grow as a yeast in the asexual stage, and produce hyphae, basidia, and infectious spores during the sexual stage, C. depauperatus grows exclusively as hyphae decorated with basidia and abundant spores and appears to be continuously engaged in sexual reproduction. By combining the insights from comparative genomics and genetic analyses of mutants defective in key mating and meiosis genes, we demonstrate the sexual cycle of C. depauperatus involves meiosis, and reveal that self-compatibility is orchestrated by the expression, in the same cell, of an unlinked mating receptor (Ste3a) and pheromone ligand (MF) pair seemingly derived from opposite mating types of a heterothallic (self-sterile) ancestor. We identified a putative mating-type (MAT) determining region containing genes phylogenetically aligned with MATa alleles of other species, and a few MAT gene alleles scattered and unlinked throughout the genome, but no homologs of the mating-type homeodomain genes SXI1 (HD1) and SXI2 (HD2). Comparative genomic analyses suggested a dramatic remodeling of the MAT locus possibly owing to reduced selective constraints to maintain mating-type genes in tight linkage, associated with a transition to self-fertility. Our findings support C. depauperatus as an obligately sexual, homothallic fungal species and provide additional insight into the repeated transitions between modes of sexual reproduction that have occurred throughout the fungal kingdom.

microbiology↗

bioRxiv2021

Epistatic genetic interactions govern morphogenesis during sexual reproduction and infection in a global human fungal pathogen

Cellular development is orchestrated by evolutionarily conserved signaling pathways, which are often pleiotropic and involve intra- and inter-pathway epistatic interactions that form intricate, complex regulatory networks. Cryptococcus species are a group of closely-related human fungal pathogens that grow as yeasts yet transition to hyphae during sexual reproduction. Additionally, during infection they can form large, polyploid titan cells that evade immunity and develop drug resistance. Multiple known signaling pathways regulate cellular development, yet how these are coordinated and interact with genetic variation is less well understood. Here, we conducted quantitative trait locus (QTL) analyses of a mapping population generated by sexual reproduction of two parents, only one of which is unisexually fertile. We observed transgressive segregation of the unisexual phenotype among progeny, as well as a novel large-cell phenotype under mating-inducing conditions. These large-cell progeny were found to produce titan cells both in vitro and in infected animals. Two major QTLs and corresponding quantitative trait genes (QTGs) were identified: RIC8 (encoding a guanine-exchange factor) and CNC06490 (encoding a putative Rho-GTPase activator), both involved in G-protein signaling. The two QTGs interact epistatically with each other and with the mating-type locus in phenotypic determination. These findings provide insights into the complex genetics of morphogenesis during unisexual reproduction and pathogenic titan cell formation and illustrate how QTL analysis can be applied to identify epistasis between genes. This study shows that phenotypic outcomes are influenced by the genetic background upon which mutations arise, implicating dynamic, complex genotype-to-phenotype landscapes in fungal pathogens and beyond. Significance statementCellular development is regulated by a complex web of signaling pathways that respond to both intracellular and extracellular cues. Morphological transitions in pathogenic fungi, such as those observed during sexual reproduction or in response to the host environment, offer tractable models for understanding the principles that govern eukaryotic cell development and morphogenesis. Using the human fungal pathogen Cryptococcus deneoformans as a model and applying QTL analysis, we defined novel genes and gene-gene interactions involved in the yeast-hyphal transition and titanization, two morphological developments that are important for adaptation, pathogenesis, and evolution of this fungal pathogen. Our study provides new insights into the conservation and complexity of key signaling pathways in regulating cell development in fungi, as well as other eukaryotes.

genetics↗

bioRxiv2020

Pleiotropy and Epistasis Within and Between Signaling Pathways Defines the Genetic Architecture of Fungal Virulence

Cryptococcal disease is estimated to affect nearly a quarter of a million people annually. Environmental isolates of Cryptococcus deneoformans, which make up 15 to 30% of clinical infections in temperate climates such as Europe, vary in their pathogenicity, ranging from benign to hyper-virulent. Key traits that contribute to virulence, such as the production of the pigment melanin, an extracellular polysaccharide capsule, and the ability to grow at human body temperature have been identified, yet little is known about the genetic basis of variation in such traits. Here we investigate the genetic basis of melanization, capsule size, thermal tolerance, oxidative stress resistance, and antifungal drug sensitivity using quantitative trait locus (QTL) mapping in progeny derived from a cross between two divergent C. deneoformans strains. Using a "function-valued" QTL analysis framework that exploits both time-series information and growth differences across multiple environments, we identified QTL for each of these virulence traits and drug susceptibility. For three QTL we identified the underlying genes and nucleotide differences that govern variation in virulence traits. One of these genes, RIC8, which encodes a regulator of cAMP-PKA signaling, contributes to variation in four virulence traits: melanization, capsule size, thermal tolerance, and resistance to oxidative stress. Two major effect QTL for amphotericin B resistance map to the genes SSK1 and SSK2, which encode key components of the HOG pathway, a fungal-specific signal transduction network that orchestrates cellular responses to osmotic and other stresses. We also discovered complex epistatic interactions within and between genes in the HOG and cAMP-PKA pathways that regulate antifungal drug resistance and resistance to oxidative stress. Our findings advance the understanding of virulence traits among diverse lineages of Cryptococcus, and highlight the role of genetic variation in key stress-responsive signaling pathways as a major contributor to phenotypic variation. Author summaryDifferent environmental isolates (strains) of the same microbial species can vary greatly in their ability to cause disease, ranging from avirulent to hypervirulent. What makes some strains deadly pathogens, while others are relatively benign? This study describes the characterization of key genetic differences that underlie variation in traits thought to promote virulence in Cryptococcus deneoformans, a wide-spread opportunistic fungal pathogen. Using a combination of quantitative genetic and molecular genetic approaches we dissected the genetic architecture of virulence-related cellular traits (melanin production and the production of a polysaccharide capsule), physiological responses to stress (tolerance of thermal, oxidative, and osmotic stress), and sensitivity to multiple antifungal drugs. Strikingly we find that variation in most of these traits is governed by a small number of genetic differences that modify the function of two major cell signaling networks, cyclic AMP-Protein Kinase A (cAMP-PKA) signaling and a fungal specific MAP-kinase cascade called the high osmolarity glycerol (HOG) pathway. Similar to recent studies in a number of other fungal species, our findings point to an outsize role for a small number of highly pleiotropic signaling pathways in potentiating phenotypic variation both within and between fungal species.

genetics↗

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

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↗