Biology subjects

Rokas, A.

Publications and source records attributed to Rokas, A..

At least 91 records · Page 5Linked to original sources

bioRxiv2020

Biosynthetic gene clusters, secondary metabolite profiles, and cards of virulence in the closest nonpathogenic relatives of Aspergillus fumigatus

Aspergillus fumigatus is a major human pathogen that causes hundreds of thousands of infections yearly with high mortality rates. In contrast, Aspergillus fischeri and the recently described Aspergillus oerlinghausenensis, the two species most closely related to A. fumigatus, are not known to be pathogenic. Some of the "cards of virulence" that A. fumigatus possesses are secondary metabolites that impair the host immune system, protect from host immune cell attacks, or acquire key nutrients. Secondary metabolites and the biosynthetic gene clusters (BGCs) that typically encode them often vary within and between fungal species. To gain insight into whether secondary metabolism-associated cards of virulence vary between A. fumigatus, A. oerlinghausenensis, and A. fischeri, we conducted extensive genomic and secondary metabolite profiling analyses. By analyzing multiple A. fumigatus, one A. oerlinghausenensis, and multiple A. fischeri strains, we identified both conserved and diverged secondary metabolism-associated cards of virulence. For example, we found that all species and strains examined biosynthesized the major virulence factor gliotoxin, consistent with the conservation of the gliotoxin BGC across genomes. However, species differed in their biosynthesis of fumagillin and pseurotin, both contributors to host tissue damage during invasive aspergillosis; these differences were reflected in sequence divergence of the intertwined fumagillin/pseurotin BGCs across genomes. These results delineate the similarities and differences in secondary metabolism-associated cards of virulence between a major fungal pathogen and its nonpathogenic closest relatives, shedding light into the genetic and phenotypic changes associated with the evolution of fungal pathogenicity. ImportanceThe major fungal pathogen Aspergillus fumigatus kills tens of thousands each year. In contrast, the two closest relatives of A. fumigatus, namely Aspergillus fischeri and Aspergillus oerlinghausenensis, are not considered pathogenic. A. fumigatus virulence stems, partly, from its ability to produce small molecules called secondary metabolites that have potent activities during infection. In this study, we examined whether A. fumigatus secondary metabolites and the metabolic pathways involved in their production are conserved in A. oerlinghausenensis and A. fischeri. We found that the nonpathogenic close relatives of A. fumigatus produce some, but not all, secondary metabolites thought to contribute to the success of A. fumigatus in causing human disease and that these similarities and differences were reflected in the underlying metabolic pathways involved in their biosynthesis. Compared to its nonpathogenic close relatives, A. fumigatus produces a distinct cocktail of secondary metabolites, which likely contributes to these organisms vastly different potentials to cause human disease. More broadly, the study of nonpathogenic organisms that have virulence-related traits, but are not currently considered agents of human disease, may facilitate the prediction of species capable of posing future threats to human health.

evolutionary biology↗

bioRxiv2020

Genomic and phenotypic heterogeneity of clinical isolates of the human pathogens Aspergillus fumigatus, Aspergillus lentulus and Aspergillus fumigatiaffinis

Fungal pathogens are a global threat to human health. For example, fungi from the genus Aspergillus cause a spectrum of diseases collectively known as aspergillosis. Most of the >200,000 life-threatening aspergillosis infections per year worldwide are caused by Aspergillus fumigatus. Recently, molecular typing techniques have revealed that aspergillosis can also be caused by organisms that are phenotypically similar to A. fumigatus but genetically distinct, such as Aspergillus lentulus and Aspergillus fumigatiaffinis. Importantly, some of these so-called cryptic species exhibit different virulence and drug susceptibility profiles than A. fumigatus, however, our understanding of their biology and pathogenic potential has been stymied by the lack of genome sequences and phenotypic profiling. To fill this gap, we phenotypically characterized the virulence and drug susceptibility of 15 clinical strains of A. fumigatus, A. lentulus, and A. fumigatiaffinis from Spain and sequenced their genomes. We found heterogeneity in virulence and drug susceptibility across species and strains. Genes known to influence drug susceptibility (cyp51A and fks1) vary in paralog number and sequence among these species and strains and correlate with differences in drug susceptibility. Similarly, genes known to be important for virulence in A. fumigatus showed variability in number of paralogs across strains and across species. Characterization of the genomic similarities and differences of clinical strains of A. lentulus, A. fumigatiaffinis, and A. fumigatus that vary in disease-relevant traits will advance our understanding of the variance in pathogenicity between Aspergillus species and strains that are collectively responsible for the vast majority of aspergillosis infections in humans.

microbiology↗

bioRxiv2020

Functional characterization of clinical isolates of the opportunistic fungal pathogen Aspergillus nidulans

Aspergillus nidulans is an opportunistic fungal pathogen in patients with immunodeficiency and virulence of A. nidulans isolates has mainly been studied in the context of the chronic granulomatous disease (CGD), with characterization of clinical isolates obtained from non-CGD patients remaining elusive. This study therefore carried out a detailed biological characterization of two A. nidulans clinical isolates (CIs), obtained from a patient with breast carcinoma and pneumonia and from a patient with cystic fibrosis that underwent lung transplantation, and compared them to the reference, non-clinical A4 strain. Both CIs presented increased growth in comparison to the reference strain in the presence of physiologically-relevant carbon sources. Metabolomic analyses showed that the three strains are metabolically very different from each other in these carbon sources. Furthermore, the CIs were highly susceptible to cell wall perturbing agents but not to other physiologically-relevant stresses. Genome analyses identified several frame-shift variants in genes encoding cell wall integrity (CWI) signalling components. Significant differences in CWI signalling were confirmed by western blotting among the three strains. In vivo virulence studies using several different models revealed that strain MO80069 had significantly higher virulence in hosts with impaired neutrophil function when compared to the other strains. In summary, this study presents detailed biological characterization of two A. nidulans sensu stricto clinical isolates. Just like in A. fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits. Further studies are required to fully characterize A. nidulans strain-specific virulence traits and pathogenicity. ImportanceImmunocompromised patients are susceptible to infections with opportunistic filamentous fungi from the genus Aspergillus. Although A. fumigatus is the main etiological agent of Aspergillus spp.-related infections, other species, such as A. nidulans are prevalent in a condition-specific manner. A. nidulans is a predominant infective agent in patients suffering from chronic granulomatous disease (CGD). A. nidulans isolates have mainly been studied in the context of CGD, although infection with A. nidulans also occurs in non-CGD patients. This study carried out a detailed biological characterization of two non-CGD A. nidulans clinical isolates and compared it to a reference strain. Phenotypic, metabolomic and genomic analyses highlight fundamental differences in carbon source utilization, stress responses and maintenance of cell wall integrity among the strains. One clinical strain had increased virulence in models with impaired neutrophil function. Just as in A. fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits.

microbiology↗

bioRxiv2019

Gliotoxin, a known virulence factor in the major human pathogen Aspergillus fumigatus, is also biosynthesized by the non-pathogenic relative A. fischeri

Aspergillus fumigatus is a major opportunistic human pathogen. Multiple traits contribute to A. fumigatus pathogenicity, including its ability to produce specific secondary metabolites, such as gliotoxin. Gliotoxin is known to inhibit the host immune response, and genetic mutants that inactivate gliotoxin biosynthesis (or secondary metabolism in general) attenuate A. fumigatus virulence. The genome of A. fischeri, a very close non-pathogenic relative of A. fumigatus, contains a biosynthetic gene cluster that exhibits high sequence similarity to the A. fumigatus gliotoxin cluster. However, A. fischeri is not known to produce gliotoxin. To gain further insight into the similarities and differences between the major pathogen A. fumigatus and the non-pathogen A. fischeri, we examined whether A. fischeri strain NRRL 181 biosynthesizes gliotoxin and whether its production, and of secondary metabolites more generally, influence its virulence profile. We found that A. fischeri biosynthesizes gliotoxin in the same conditions as A. fumigatus. However, whereas loss of laeA, a master regulator of secondary metabolite production, has been previously shown to reduce the virulence of A. fumigatus, we found that laeA loss (and loss of secondary metabolite production, including gliotoxin) in A. fischeri does not influence its virulence. These results suggest that gliotoxin and secondary metabolite production are virulence factors in the genomic and phenotypic background of the major pathogen A. fumigatus but are much less important in the background of the non-pathogen A. fischeri. We submit that understanding the observed spectrum of pathogenicity across closely related pathogenic and non-pathogenic Aspergillus species will require detailed characterization of their biological, chemical, and genomic similarities and differences. IMPORTANCEAspergillus fumigatus is a major opportunistic fungal pathogen of humans but most of its close relatives are non-pathogenic. Why is that so? This important, yet largely unanswered, question can be addressed by examining how A. fumigatus and its non-pathogenic close relatives are similar or different with respect to virulence-associated traits. We investigated whether Aspergillus fischeri, a non-pathogenic close relative of A. fumigatus, can produce gliotoxin, a mycotoxin known to contribute to A. fumigatus virulence. We discovered that the non-pathogenic A. fischeri produces gliotoxin under the same conditions as the major pathogen A. fumigatus. However, we also discovered that, in contrast to what has been previously observed in A. fumigatus, loss of secondary metabolite, including gliotoxin, production in A. fischeri does not alter its virulence. Our results are consistent with the "cards of virulence" model of opportunistic fungal disease, where the ability to cause disease stems from the combination ("hand") of individual virulence factors ("cards"), but not from individual factors per se.

microbiology↗

bioRxiv2019

Recurrent loss of abaA, a master regulator of asexual development in filamentous fungi, correlates with changes in genomic and morphological traits

Gene regulatory networks (GRNs) drive developmental and cellular differentiation, and variation in their architectures gives rise to morphological diversity. Pioneering studies in Aspergillus fungi, coupled with subsequent work in other filamentous fungi, have shown that the GRN governed by the BrlA, AbaA, and WetA proteins controls the development of the asexual fruiting body or conidiophore. A specific aspect of conidiophore development is the production of phialides, conidiophore structures that are under the developmental control of AbaA and function to repetitively generate spores. Fungal genome sequencing has revealed that some filamentous fungi lack abaA, and also produce asexual structures that lack phialides, raising the hypothesis that abaA loss is functionally linked to diversity in asexual fruiting body morphology. To examine this hypothesis, we carried out an extensive search for the abaA gene across 241 genomes of species from the fungal subphylum Pezizomycotina. We found that abaA was independently lost in four lineages of Eurotiomycetes, including from all sequenced species within the order Onygenales, and that all four lineages that have lost abaA also lack the ability to form phialides. Genetic restoration of abaA from Aspergillus nidulans into Histoplasma capsulatum, a pathogenic species from the order Onygenales that lacks an endogenous copy of abaA, did not alter Histoplasma conidiation morphology but resulted in a marked increase in spore viability. We also discovered that species lacking abaA contain fewer AbaA binding motifs in the regulatory regions of orthologs of some AbaA target genes, suggesting that the asexual fruiting body GRN of organisms that have lost abaA has been rewired. Our results provide an illustration of how repeated losses of a key regulatory transcription factor and concomitant changes in non-coding regulatory regions of the genome have contributed to the diversity of an iconic fungal morphological trait.\n\nAuthor SummaryFungi exhibit tremendous variation in their asexual fruiting bodies. For example, whereas some fungi form complex fruiting bodies whose tips repeatedly generate and release spores, others produce single spores in the absence of a specialized structure. To gain insights into the molecular differences that underpin fungal asexual fruiting body diversity, we examined the genomes of hundreds of filamentous fungi for the presence of abaA, a master regulatory gene previously shown to control the development of fungal asexual fruiting bodies. We found that abaA was repeatedly lost during fungal evolution, including in a lineage of human pathogenic fungi, and that the loss of the gene was always associated with the loss of specialized structures in fungal asexual fruiting bodies. Reintroduction of abaA into the human pathogenic fungus Histoplasma capsulatum, which normally lacks the regulator, did not result in a change in the spore-producing structure but did increase spore outgrowth. Based on these results, we hypothesize that the loss of the master regulatory gene abaA has contributed to the observed diversity of fungal fruiting body morphology. This work advances our understanding of how fungal developmental networks evolve over time and advances our understanding of how infectious spores form in pathogenic fungi.

evolutionary biology↗

bioRxiv2019

Accounting for diverse evolutionary forces reveals the mosaic nature of selection on genomic regions associated with human preterm birth

Human pregnancy requires the coordinated function of multiple tissues in both mother and fetus and has evolved in concert with major human adaptations. As a result, pregnancy-associated phenotypes and related disorders are genetically complex and have likely been sculpted by diverse evolutionary forces. However, there is no framework to comprehensively evaluate how these traits evolved or to explore the relationship of evolutionary signatures on trait-associated genetic variants to molecular function. Here we develop an approach to test for signatures of diverse evolutionary forces, including multiple types of selection, and apply it to genomic regions associated with spontaneous preterm birth (sPTB), a complex disorder of global health concern. We find that sPTB-associated regions harbor diverse evolutionary signatures including evolutionary sequence conservation (consistent with the action of negative selection), excess population differentiation (local adaptation), accelerated evolution (positive selection), and balanced polymorphism (balancing selection). Furthermore, these genomic regions show diverse functional characteristics which enables us to use evolutionary and molecular lines of evidence to develop hypotheses about how these genomic regions contribute to sPTB risk. In summary, we introduce an approach for inferring the spectrum of evolutionary forces acting on genomic regions associated with complex disorders. When applied to sPTB-associated genomic regions, this approach both improves our understanding of the potential roles of these regions in pathology and illuminates the mosaic nature of evolutionary forces acting on genomic regions associated with sPTB.

evolutionary biology↗

bioRxiv2019

Rapid phenotypic and metabolomic domestication of wild Penicillium molds on cheese

Fermented foods provide novel ecological opportunities for natural populations of microbes to evolve through successive recolonization of resource-rich substrates. Comparative genomic data have reconstructed the evolutionary histories of microbes adapted to food environments, but experimental studies directly demonstrating the process of domestication are lacking for most fermented food microbes. Here we show that during the repeated colonization of cheese, phenotypic and metabolomic traits of wild Penicillium molds rapidly change to produce mutants with properties similar to industrial cultures used to make Camembert and other bloomy rind cheeses. Over a period of just a few weeks, populations of wild Penicillium strains serially passaged on cheese resulted in the reduction or complete loss of pigment, spore, and mycotoxin production. Mutants also had a striking change in volatile metabolite production, shifting from production of earthy or musty volatile compounds (e.g. geosmin) to fatty and cheesy volatiles (e.g. 2-nonanone, 2-undecanone). RNA-sequencing demonstrated a significant decrease in expression of 356 genes in domesticated mutants, with an enrichment of many secondary metabolite production pathways in these downregulated genes. By manipulating the presence of neighboring microbial species and overall resource availability, we demonstrate that the limited competition and high nutrient availability of the cheese environment promote rapid trait evolution of Penicillium molds.\n\nIMPORTANCEIndustrial cultures of filamentous fungi are used to add unique aesthetics and flavors to cheeses and other microbial foods. How these microbes adapted to live in food environments is generally unknown as most microbial domestication is unintentional. Our work demonstrates that wild molds closely related to the starter culture Penicillium camemberti can readily lose undesirable traits and quickly shift toward producing desirable aroma compounds. In addition to experimentally demonstrating a putative domestication pathway for P. camemberti, our work suggests that wild Penicillium isolates could be rapidly domesticated to produce new flavors and aesthetics in fermented foods.

microbiology↗

bioRxiv2019

Variation and selection on codon usage bias across an entire subphylum

Variation in synonymous codon usage is abundant across multiple levels of organization: between codons of an amino acid, between genes in a genome, and between genomes of different species. It is now well understood that variation in synonymous codon usage is influenced by mutational bias coupled with both natural selection for translational efficiency and genetic drift, but how these processes shape patterns of codon usage bias across entire lineages remains unexplored. To address this question, we used a rich genomic data set of 327 species that covers nearly one third of the known biodiversity of the budding yeast subphylum Saccharomycotina. We found that, while genome-wide relative synonymous codon usage (RSCU) for all codons was highly correlated with the GC content of the third codon position (GC3), the usage of codons for the amino acids proline, arginine, and glycine was inconsistent with the neutral expectation where mutational bias coupled with genetic drift drive codon usage. Examination between genes effective numbers of codons and their GC3 contents in individual genomes revealed that nearly a quarter of genes (381,174/1,683,203; 23%), as well as most genomes (308/327; 94%), significantly deviate from the neutral expectation. Finally, by evaluating the imprint of translational selection on codon usage, measured as the degree to which genes adaptiveness to the tRNA pool were correlated with selective pressure, we show that translational selection is widespread in budding yeast genomes (264/327; 81%). These results suggest that the contribution of translational selection and drift to patterns of synonymous codon usage across budding yeasts varies across codons, genes, and genomes; whereas drift is the primary driver of global codon usage across the subphylum, the codon bias of large numbers of genes in the majority of genomes is influenced by translational selection.\n\nLay Summary / Significance statementSynonymous mutations in genes have no effect on the encoded proteins and were once thought to be evolutionarily neutral. By examining codon usage bias across codons, genes, and genomes of 327 species in the budding yeast subphylum, we show that synonymous codon usage is shaped by both neutral processes and selection for translational efficiency. Specifically, whereas codon usage bias for most codons appears to be strongly associated with mutational bias and largely driven by genetic drift across the entire subphylum, patterns of codon usage bias in a few codons, as well as in many genes in nearly all genomes of budding yeasts, deviate from neutral expectations. Rather, the synonymous codons used within genes in most budding yeast genomes are adapted to the tRNAs present within each genome, a result most likely due to translational selection that optimizes codons to match the tRNAs. Our results suggest that patterns of codon usage bias in budding yeasts, and perhaps more broadly in fungi and other microbial eukaryotes, are shaped by both neutral and selective processes.

evolutionary biology↗

bioRxiv2019

Extensive loss of cell cycle and DNA repair genes in an ancient lineage of bipolar budding yeasts

Cell cycle checkpoints and DNA repair processes protect organisms from potentially lethal mutational damage. Compared to other budding yeasts in the subphylum Saccharomycotina, we noticed that a lineage in the genus Hanseniaspora exhibited very high evolutionary rates, low GC content, small genome sizes, and lower gene numbers. To better understand Hanseniaspora evolution, we analyzed 25 genomes, including 11 newly sequenced, representing 18 / 21 known species in the genus. Our phylogenomic analyses identify two Hanseniaspora lineages, the fast-evolving lineage (FEL), which began diversifying [~]87 million years ago (mya), and the slow-evolving lineage (SEL), which began diversifying [~]54 mya. Remarkably, both lineages lost genes associated with the cell cycle and genome integrity, but these losses were greater in the FEL. For example, all species lost the cell cycle regulator WHI5, and the FEL lost components of the spindle checkpoint pathway (e.g., MAD1, MAD2) and DNA damage checkpoint pathway (e.g., MEC3, RAD9). Similarly, both lineages lost genes involved in DNA repair pathways, including the DNA glycosylase gene MAG1, which is part of the base excision repair pathway, and the DNA photolyase gene PHR1, which is involved in pyrimidine dimer repair. Strikingly, the FEL lost 33 additional genes, including polymerases (i.e., POL4 and POL32) and telomere-associated genes (e.g., RIF1, RFA3, CDC13, PBP2). Echoing these losses, molecular evolutionary analyses reveal that, compared to the SEL, the FEL stem lineage underwent a burst of accelerated evolution, which resulted in greater mutational loads, homopolymer instabilities, and higher fractions of mutations associated with the common endogenously damaged base, 8-oxoguanine. We conclude that Hanseniaspora is an ancient lineage that has diversified and thrived, despite lacking many otherwise highly conserved cell cycle and genome integrity genes and pathways, and may represent a novel system for studying cellular life without them.

evolutionary biology↗

bioRxiv2019

Signatures of recent positive selection in enhancers across 41 human tissues

Evolutionary changes in enhancers are widely associated with variation in human traits and diseases. However, studies comprehensively quantifying levels of selection on enhancers at multiple evolutionary time points during recent human evolution and how enhancer evolution varies across human tissues are lacking. To address these questions, we integrated a dataset of 41,561 transcribed enhancers active in 41 different human tissues (FANTOM Consortium) with whole genome sequences of 1,668 individuals from the African, Asian, and European populations (1000 Genomes Project). Our analyses based on four different metrics (Tajimas D, FST, H12, nSL) showed that ~5.90% of enhancers considered showed evidence of recent positive selection and that genes associated with enhancers under positive selection are enriched for diverse immune-related functions. The distributions of these metrics for brain and testis enhancers were often statistically significantly different compared to those of other tissues; the same was true for brain and testis enhancers that are tissue-specific compared to those that are tissue-broad and for testis enhancers associated with tissue-enriched and non-tissue-enriched genes. These differences varied considerably across metrics and tissues and were generally due to changes in distributions shapes rather than shifts in their values. These results suggest that many human enhancers experienced recent positive selection throughout multiple time periods in human evolutionary history, that this selection occurred in a tissue-dependent and immune-related functional context, and that much like the evolution of their coding counterparts, the evolution of brain and testis enhancers has been markedly different from that of enhancers in other tissues.

evolutionary biology↗

bioRxiv2018

The impact of natural selection on the evolution and function of placentally expressed galectins

Immunity genes have repeatedly experienced natural selection during mammalian evolution. Galectins are carbohydrate-binding proteins that regulate diverse immune responses, including maternal-fetal immune tolerance in placental pregnancy. Seven human galectins, four conserved across vertebrates and three specific to primates, are involved in placental development. To comprehensively study the molecular evolution of these galectins both across mammals and within humans, we conducted a series of between-and within-species evolutionary analyses. By examining patterns of sequence evolution between species, we found that primate-specific galectins showed uniformly high substitution rates, whereas two of the four other galectins experienced accelerated evolution in primates. By examining human population genomic variation, we found that galectin genes and variants, including variants previously linked to immune diseases, showed signatures of recent positive selection in specific human populations. By examining one nonsynonymous variant in Galectin-8 previously associated with autoimmune diseases, we further discovered that it is tightly linked to three other nonsynonymous variants; surprisingly, the global frequency of this four-variant haplotype is [~]50%. To begin understanding the impact of this major haplotype on Galectin-8 protein structure, we modeled its 3D protein structure and found that it differed substantially from the reference protein structure. These results suggest that placentally expressed galectins experienced both ancient and more recent selection in a lineage-and population-specific manner. Furthermore, our discovery that the major Galectin-8 haplotype is structurally distinct from and more commonly found than the reference haplotype illustrates the significance of understanding the evolutionary processes that sculpted variants associated with human genetic disease.

evolutionary biology↗

bioRxiv2018

The Genomic Geography and Evolution of Clusters of Tandemly Duplicated Genes in the Human and Mammal Genomes

Clusters of duplicated genes (CTDGs) are nearly ubiquitous in lifes genomes, and are associated with several well-known gene families, such as olfactory receptors, zinc fingers, and immunity-related genes, as well as with several highly variable traits, including olfaction, body plan architecture, and pathogen resistance. However, these observations are usually anecdotal, restricted to specific cases, and lacking evolutionary context. In this study, we use a robust statistical approach to characterize the CTDG repertoire and analyze the distribution of CTDGs across 18 mammal genomes, including human. We found that, on average, 18% of the genes in each species are parts of CTDGs. Although genes in CTDGs are enriched for several biological processes, these tend to be involved in the interactions between the organism and its environment. We further found that mammalian CTDGs are not uniformly distributed across chromosomes and that orthologs of the human chromosome 19 are among the most clustered chromosomes in nearly all mammalian genomes analyzed. We also found evidence that the human chromosome 19 was formed by a fusion event that occurred before the diversification of the rodent and primate lineages and maintained its high density of CTDGs during its subsequent evolution. Finally, using chromosome-level alignments across mammalian genomes, we show how the syntenic regions of the human chromosome 19 have been shrinking, increasing their gene density and possibly increasing the compactness of its CTDGs. These results suggest that CTDGs are a major feature of mammalian genomes and provide novel insights into the origin and evolution of regions with unusually high densities of CTDGs.

evolutionary biology↗

bioRxiv2018

Characterizing the pathogenic, genomic, and chemical traits of Aspergillus fischeri, the closest sequenced relative of the major human fungal pathogen Aspergillus fumigatus

Aspergillus fischeri is closely related to Aspergillus fumigatus, the major cause of invasive mold infections. Even though A. fischeri is commonly found in diverse environments, including hospitals, it rarely causes invasive disease; why that is so is unclear. Comparison of A. fischeri and A. fumigatus for diverse pathogenic, genomic, and secondary metabolic traits revealed multiple differences for pathogenesis-related phenotypes, including that A. fischeri is less virulent than A. fumigatus in multiple animal models of disease, grows slower in low oxygen environments, and is more sensitive to oxidative stress. In contrast, the two species exhibit high genomic similarity; ~90% of the A. fumigatus proteome is conserved in A. fischeri, including 48/49 genes known to be involved in A. fumigatus virulence. However, only 10/33 A. fumigatus biosynthetic gene clusters (BGCs) likely involved in secondary metabolite production are conserved in A. fischeri and only 13/48 A. fischeri BGCs are conserved in A. fumigatus. Detailed chemical characterization of A. fischeri cultures grown on multiple substrates identified multiple secondary metabolites, including two new compounds and one never before isolated as a natural product. Interestingly, an A. fischeri deletion mutant of laeA, a master regulator of secondary metabolism, produced fewer secondary metabolites and in lower quantities, suggesting that regulation of secondary metabolism is at least partially conserved. These results suggest that the non-pathogenic A. fischeri possesses many of the genes important for A. fumigatus pathogenicity but is divergent with respect to its ability to thrive under host-relevant conditions and its secondary metabolism.\n\nImportanceAspergillus fumigatus is the primary cause of aspergillosis, a devastating ensemble of diseases associated with severe morbidity and mortality worldwide. A. fischeri is a close relative of A. fumigatus, but is not generally observed to cause human disease. To gain insights into the underlying causes of this remarkable difference in pathogenicity, we compared two representative strains (one from each species) for a range of host-relevant biological and chemical characteristics. We found that disease progression in multiple A. fischeri mouse models was slower and caused less mortality than A. fumigatus. The two species also exhibited different growth profiles when placed in a range of stress-inducing conditions encountered during infection, such as low levels of oxygen and the presence of reactive oxygen species-inducing agents. Interestingly, we also found that the vast majority of A. fumigatus genes known to be involved in virulence are conserved in A. fischeri, whereas the two species differ significantly in their secondary metabolic pathways. These similarities and differences that we identified are the first step toward understanding the evolutionary origin of a major fungal pathogen.

microbiology↗

bioRxiv2018

Eukaryotic acquisition of a bacterial operon

Operons are a hallmark of bacterial genomes, where they allow concerted expression of multiple functionally related genes as single polycistronic transcripts. They are rare in eukaryotes, where each gene usually drives expression of its own independent messenger RNAs. Here we report the horizontal operon transfer of a catecholate-class siderophore biosynthesis pathway from Enterobacteriaceae into a group of closely related yeast taxa. We further show that the co-linearly arranged secondary metabolism genes are actively expressed, exhibit mainly eukaryotic transcriptional features, and enable the sequestration and uptake of iron. After transfer to the eukaryotic host, several genetic changes occurred, including the acquisition of polyadenylation sites, structural rearrangements, integration of eukaryotic genes, and secondary loss in some lineages. We conclude that the operon genes were likely captured in the shared insect gut habitat, modified for eukaryotic gene expression, and maintained by selection to adapt to the highly-competitive, iron-limited environment.

evolutionary biology↗

bioRxiv2018

A robust phylogenomic timetree for biotechnologically and medically important fungi from Aspergillaceae (Eurotiomycetes, Ascomycota)

AbbreviationsNT, nucleotide; AA, amino acid; CI, credible interval; RCV, relative composition variability; IC, internode certainty; GSF, gene support frequencies; GLS, gene-wise log-likelihood scores; DVMC, degree of violation of a molecular clock\n\nThe filamentous fungal family Aspergillaceae contains > 1,000 known species, mostly in the genera Aspergillus and Penicillium. Several species are used in the food, biotechnology, and drug industries (e.g., Aspergillus oryzae, Penicillium camemberti), while others are dangerous human and plant pathogens (e.g., Aspergillus fumigatus, Penicillium digitatum). To infer a robust phylogeny and pinpoint poorly resolved branches and their likely underlying contributors, we used 81 genomes spanning the diversity of Aspergillus and Penicillium to construct a 1,668-gene data matrix. Phylogenies of the nucleotide and amino acid versions of this full data matrix as well as of five additional 834-gene data matrices constructed by subsampling the top 50% of genes according to different criteria associated with strong phylogenetic signal were generated using three different maximum likelihood schemes (i.e., gene-partitioned, unpartitioned, and coalescence). Examination of the topological agreement among these 36 phylogenies and measures of internode certainty identified 12 / 78 (15.4%) bipartitions that were incongruent and pinpoint the likely underlying contributing factors (incomplete lineage sorting, hybridization or introgression, and reconstruction artifacts associated with poor taxon sampling). Relaxed molecular clock analyses suggest that Aspergillaceae likely originated in the lower Cretaceous and the Aspergillus and Penicillium genera in the upper Cretaceous. Our results shed light on the ongoing debate on Aspergillus systematics and taxonomy and provide a robust evolutionary and temporal framework for comparative genomic analyses in Aspergillaceae. More broadly, our approach provides a general template for phylogenomic identification of resolved and contentious branches in densely genome-sequenced lineages across the tree of life.

evolutionary biology↗

bioRxiv2018

Conservation and Divergence in the Asexual Sporulation Gene Regulatory Network Across a Genus of Filamentous Fungi

Asexual sporulation is fundamental to the ecology and lifestyle of filamentous fungi and can facilitate both plant and human infection. In Aspergillus, the production of asexual spores is primarily governed by the BrlA[->]AbaA[->]WetA regulatory cascade. The final step in this cascade is controlled by the WetA protein and not only governs the morphological differentiation of spores but also the production and deposition of diverse metabolites into spores. While WetA is conserved across the genus Aspergillus, the structure and degree of conservation of the wetA gene regulatory network (GRN) remains largely unknown. We carried out comparative transcriptome analyses between wetA null mutant and wild type asexual spores in three representative species spanning the diversity of the genus Aspergillus: A. nidulans, A. flavus, and A. fumigatus. We discovered that WetA regulates asexual sporulation in all three species via a negative feedback loop that represses BrlA, the cascades first step. Furthermore, ChIP-seq experiments in A. nidulans asexual spores suggest that WetA is a DNA-binding protein that interacts with a novel regulatory motif. Several global regulators known to bridge spore production and the production of secondary metabolites show species-specific regulatory patterns in our data. These results suggest that the BrlA[->]AbaA[->]WetA cascades regulatory role in cellular and chemical asexual spore development is functionally conserved, but that the wetA-associated GRN has diverged during Aspergillus evolution.

microbiology↗

bioRxiv2018

integRATE: a desirability-based data integration framework for the prioritization of candidate genes across heterogeneous omics and its application to preterm birth

BackgroundThe integration of high-quality, genome-wide analyses offers a robust approach to elucidating genetic factors involved in complex human diseases. Even though several methods exist to integrate heterogeneous omics data, most biologists still manually select candidate genes by examining the intersection of lists of candidates stemming from analyses of different types of omics data that have been generated by imposing hard (strict) thresholds on quantitative variables, such as P-values and fold changes, increasing the chance of missing potentially important candidates.\n\nMethodsTo better facilitate the unbiased integration of heterogeneous omics data collected from diverse platforms and samples, we propose a desirability function framework for identifying candidate genes with strong evidence across data types as targets for follow-up functional analysis. Our approach is targeted towards disease systems with sparse, heterogeneous omics data, so we tested it on one such pathology: spontaneous preterm birth (sPTB).\n\nResultsWe developed the software integRATE, which uses desirability functions to rank genes both within and across studies, identifying well-supported candidate genes according to the cumulative weight of biological evidence rather than based on imposition of hard thresholds of key variables. Integrating 10 sPTB omics studies identified both genes in pathways previously suspected to be involved in sPTB as well as novel genes never before linked to this syndrome. integRATE is available as an R package on GitHub (https://github.com/haleyeidem/integRATE).\n\nConclusionsDesirability-based data integration is a solution most applicable in biological research areas where omics data is especially heterogeneous and sparse, allowing for the prioritization of candidate genes that can be used to inform more targeted downstream functional analyses.

genomics↗

bioRxiv2018

Genome wide association analysis identifies genetic variants associated with reproductive variation across domestic dog breeds and uncovers links to domestication

The diversity of eutherian reproductive strategies has led to variation in many traits, such as number of offspring, age of reproductive maturity, and gestation length. While reproductive trait variation has been extensively investigated and is well established in mammals, the genetic loci contributing to this variation remain largely unknown. The domestic dog, Canis lupus familiaris is a powerful model for studies of the genetics of inherited disease due to its unique history of domestication. To gain insight into the genetic basis of reproductive traits across domestic dog breeds, we collected phenotypic data for four traits - cesarean section rate (n = 97 breeds), litter size (n = 60), stillbirth rate (n = 57), and gestation length (n = 23) - from primary literature and breeders handbooks. By matching our phenotypic data to genomic data from the Cornell Veterinary Biobank, we performed genome wide association analyses for these four reproductive traits, using body mass and kinship among breeds as co-variates. We identified 14 genome-wide significant associations between these traits and genetic loci, including variants near CACNA2D3 with gestation length, MSRB3 with litter size, SMOC2 with cesarean section rate, MITF with litter size and still birth rate, KRT71 with cesarean section rate, litter size, and stillbirth rate, and HTR2C with stillbirth rate. Some of these loci, such as CACNA2D3 and MSRB3, have been previously implicated in human reproductive pathologies. Many of the variants that we identified have been previously associated with domestication-related traits, including brachycephaly (SMOC2), coat color (MITF), coat curl (KRT71), and tameness (HTR2C). These results raise the hypothesis that the artificial selection that gave rise to dog breeds also shaped the observed variation in their reproductive traits. Overall, our work establishes the domestic dog as a system for studying the genetics of reproductive biology and disease.

evolutionary biology↗