Origin of fungal hybrids with pathogenic potential from warm seawater environments.

Hybridisation is a common event in yeasts often leading to genomic variability and adaptation. The yeast Candida orthopsilosis is a human-associated opportunistic pathogen belonging to the Candida parapsilosis species complex. Most C. orthopsilosis clinical isolates are hybrids resulting from at least four independent crosses between two parental lineages, of which only one has been identified. The rare presence or total absence of parentals amongst clinical isolates is hypothesised to be a consequence of a reduced pathogenicity with respect to their hybrids. Here, we sequence and analyse the genomes of environmental C. orthopsilosis strains isolated from warm marine ecosystems. We find that a majority of environmental isolates are hybrids, phylogenetically closely related to hybrid clinical isolates. Furthermore, we identify the missing parental lineage, thus providing a more complete overview of the genomic evolution of this species. Additionally, we discover phenotypic differences between the two parental lineages, as well as between parents and hybrids, under conditions relevant for pathogenesis. Our results suggest a marine origin of C. orthopsilosis hybrids, with intrinsic pathogenic potential, and pave the way to identify pre-existing environmental adaptations that rendered hybrids more prone than parental lineages to colonise and infect the mammalian host.

Evolution of loss of heterozygosity patterns in hybrid genomes of Candida yeast pathogens.

BACKGROUND: Hybrids are chimeric organisms with highly plastic heterozygous genomes that may confer unique traits enabling the adaptation to new environments. However, most evolutionary theory frameworks predict that the high levels of genetic heterozygosity present in hybrids from divergent parents are likely to result in numerous deleterious epistatic interactions. Under this scenario, selection is expected to favor recombination events resulting in loss of heterozygosity (LOH) affecting genes involved in such negative interactions. Nevertheless, it is so far unknown whether this phenomenon actually drives genomic evolution in natural populations of hybrids. To determine the balance between selection and drift in the evolution of LOH patterns in natural yeast hybrids, we analyzed the genomic sequences from fifty-five hybrid strains of the pathogenic yeasts Candida orthopsilosis and Candida metapsilosis, which derived from at least six distinct natural hybridization events. RESULTS: We found that, although LOH patterns in independent hybrid clades share some level of convergence that would not be expected from random occurrence, there is an apparent lack of strong functional selection. Moreover, while mitosis is associated with a limited number of inter-homeologous chromosome recombinations in these genomes, induced DNA breaks seem to increase the LOH rate. We also found that LOH does not accumulate linearly with time in these hybrids. Furthermore, some C. orthopsilosis hybrids present LOH patterns compatible with footprints of meiotic recombination. These meiotic-like patterns are at odds with a lack of evidence of sexual recombination and with our inability to experimentally induce sporulation in these hybrids. CONCLUSIONS: Our results suggest that genetic drift is the prevailing force shaping LOH patterns in these hybrid genomes. Moreover, the observed LOH patterns suggest that these are likely not the result of continuous accumulation of sporadic events-as expected by mitotic repair of rare chromosomal breaks-but rather of acute episodes involving many LOH events in a short period of time.

Chromosome-level assemblies from diverse clades reveal limited structural and gene content variation in the genome of Candida glabrata.

BACKGROUND: Candida glabrata is an opportunistic yeast pathogen thought to have a large genetic and phenotypic diversity and a highly plastic genome. However, the lack of chromosome-level genome assemblies representing this diversity limits our ability to accurately establish how chromosomal structure and gene content vary across strains. RESULTS: Here, we expanded publicly available assemblies by using long-read sequencing technologies in twelve diverse strains, obtaining a final set of twenty-one chromosome-level genomes spanning the known C. glabrata diversity. Using comparative approaches, we inferred variation in chromosome structure and determined the pan-genome, including an analysis of the adhesin gene repertoire. Our analysis uncovered four new adhesin orthogroups and inferred a rich ancestral adhesion repertoire, which was subsequently shaped through a still ongoing process of gene loss, gene duplication, and gene conversion. CONCLUSIONS: C. glabrata has a largely stable pan-genome except for a highly variable subset of genes encoding cell wall-associated functions. Adhesin repertoire was established for each strain and showed variability among clades.

Citizen-science reveals changes in the oral microbiome in Spain through age and lifestyle factors.

The relevance of the human oral microbiome to our understanding of human health has grown in recent years as microbiome studies continue to develop. Given the links of the oral cavity with the digestive, respiratory and circulatory systems, the composition of the oral microbiome is relevant beyond just oral health, impacting systemic processes across the body. However, we still have a very limited understanding about intrinsic and extrinsic factors that shape the composition of the healthy oral microbiome. Here, we followed a citizen-science approach to assess the relative impact on the oral microbiome of selected biological, social, and lifestyle factors in 1648 Spanish individuals. We found that the oral microbiome changes across age, with middle ages showing a more homogeneous composition, and older ages showing more diverse microbiomes with increased representation of typically low abundance taxa. By measuring differences within and between groups of individuals sharing a given parameter, we were able to assess the relative impact of different factors in driving specific microbial compositions. Chronic health disorders present in the analyzed population were the most impactful factors, followed by smoking and the presence of yeasts in the oral cavity. Finally, we corroborate findings in the literature that relatives tend to have more similar oral microbiomes, and show for the first time a similar effect for classmates. Multiple intrinsic and extrinsic factors jointly shape the oral microbiome. Comparative analysis of metabarcoding data from a large sample set allows us to disentangle the individual effects.

Benzylic Dehydroxylation of Echinocandin Antifungal Drugs Restores Efficacy against Resistance Conferred by Mutated Glucan Synthase.

Each year, infections caused by fungal pathogens claim the lives of about 1.6 million people and affect the health of over a billion people worldwide. Among the most recently developed antifungal drugs are the echinocandins, which noncompetitively inhibit ß-glucan synthase, a membrane-bound protein complex that catalyzes the formation of the main polysaccharide component of the fungal cell wall. Resistance to echinocandins is conferred by mutations in FKS genes, which encode the catalytic subunit of the ß-glucan synthase complex. Here, we report that selective removal of the benzylic alcohol of the nonproteinogenic amino acid 3S,4S-dihydroxy-l-homotyrosine of the echinocandins anidulafungin and rezafungin, restored their efficacy against a large panel of echinocandin-resistant Candida strains. The dehydroxylated compounds did not significantly affect the viability of human-derived cell culture lines. An analysis of the efficacy of the dehydroxylated echinocandins against resistant Candida strains, which contain mutations in the FKS1 and/or FKS2 genes of the parental strains, identified amino acids of the Fks proteins that are likely to reside in proximity to the l-homotyrosine residue of the bound drug. This study describes the first example of a chemical modification strategy to restore the efficacy of echinocandin drugs, which have a critical place in the arsenal of antifungal drugs, against resistant fungal pathogens.

Narrow mutational signatures drive acquisition of multidrug resistance in the fungal pathogen Candida glabrata.

Fungal infections are a growing medical concern, in part due to increased resistance to one or multiple antifungal drugs. However, the evolutionary processes underpinning the acquisition of antifungal drug resistance are poorly understood. Here, we used experimental microevolution to study the adaptation of the yeast pathogen Candida glabrata to fluconazole and anidulafungin, two widely used antifungal drugs with different modes of action. Our results show widespread ability of rapid adaptation to one or both drugs. Resistance, including multidrug resistance, is often acquired at moderate fitness costs and mediated by mutations in a limited set of genes that are recurrently and specifically mutated in strains adapted to each of the drugs. Importantly, we uncover a dual role of ERG3 mutations in resistance to anidulafungin and cross-resistance to fluconazole in a subset of anidulafungin-adapted strains. Our results shed light on the mutational paths leading to resistance and cross-resistance to antifungal drugs.

Citizen-science based study of the oral microbiome in Cystic fibrosis and matched controls reveals major differences in diversity and abundance of bacterial and fungal species.

Introduction: Cystic fibrosis (CF) is an autosomal genetic disease, associated with the production of excessively thick mucosa and with life-threatening chronic lung infections. The microbiota of the oral cavity can act as a reservoir or as a barrier for infectious microorganisms that can colonize the lungs. However, the specific composition of the oral microbiome in CF is poorly understood.Methods: In collaboration with CF associations in Spain, we collected oral rinse samples from 31 CF persons (age range 7-47) and matched controls, and then performed 16S rRNA metabarcoding and high-throughput sequencing, combined with culture and proteomics-based identification of fungi to survey the bacterial and fungal oral microbiome.Results: We found that CF is associated with less diverse oral microbiomes, which were characterized by higher prevalence of Candida albicans and differential abundances of a number of bacterial taxa that have implications in both the connection to lung infections in CF, as well as potential oral health concerns, particularly periodontitis and dental caries.Conclusion: Overall, our study provides a first global snapshot of the oral microbiome in CF. Future studies are required to establish the relationships between the composition of the oral and lung microbiomes in CF.

Elevated Vacuolar Uptake of Fluorescently Labeled Antifungal Drug Caspofungin Predicts Echinocandin Resistance in Pathogenic Yeast.

Echinocandins are the newest class of antifungal drugs in clinical use. These agents inhibit ß-glucan synthase, which catalyzes the synthesis of ß-glucan, an essential component of the fungal cell wall, and have a high clinical efficacy and low toxicity. Echinocandin resistance is largely due to mutations in the gene encoding ß-glucan synthase, but the mode of action is not fully understood. We developed fluorescent probes based on caspofungin, the first clinically approved echinocandin, and studied their cellular biology in Candida species, the most common cause of human fungal infections worldwide. Fluorescently labeled caspofungin probes, like the unlabeled drug, were most effective against metabolically active cells. The probes rapidly accumulated in Candida vacuoles, as shown by colocalization with vacuolar proteins and vacuole-specific stains. The uptake of fluorescent caspofungin is facilitated by endocytosis: The labeled drug formed vesicles similar to fluorescently labeled endocytic vesicles, the vacuolar accumulation of fluorescent caspofungin was energy-dependent, and inhibitors of endocytosis reduced its uptake. In a panel comprised of isogenic Candida strains carrying different ß-glucan synthase mutations as well as clinical isolates, resistance correlated with increased fluorescent drug uptake into vacuoles. Fluorescent drug uptake also associated with elevated levels of chitin, a sugar polymer that increases cell-wall rigidity. Monitoring the intracellular uptake of fluorescent caspofungin provides a rapid and simple assay that can enable the prediction of echinocandin resistance, which is useful for research applications as well as for selecting the appropriate drugs for treatments of invasive fungal infections.

The Transcriptional Aftermath in Two Independently Formed Hybrids of the Opportunistic Pathogen Candida orthopsilosis.

Interspecific hybridization can drive evolutionary adaptation to novel environments. The Saccharomycotina clade of budding yeasts includes many hybrid lineages, and hybridization has been proposed as a source for new pathogenic species. Candida orthopsilosis is an emerging opportunistic pathogen for which most clinical isolates are hybrids, each derived from one of at least four independent crosses between the same two parental lineages. To gain insight into the transcriptomic aftermath of hybridization in these pathogens, we analyzed allele-specific gene expression in two independently formed hybrid strains and in a homozygous strain representative of one parental lineage. Our results show that the effect of hybridization on overall gene expression is rather limited, affecting ∼4% of the genes studied. However, we identified a larger effect in terms of imbalanced allelic expression, affecting ∼9.5% of the heterozygous genes in the hybrids. This effect was larger in the hybrid with more extensive loss of heterozygosity, which may indicate a tendency to avoid loss of heterozygosity in these genes. Consistently, the number of shared genes with allele-specific expression in the two independently formed hybrids was higher than random expectation, suggesting selective retention. Some of the imbalanced genes have functions related to pathogenicity, including zinc transport and superoxide dismutase activities. While it remains unclear whether the observed imbalanced genes play a role in virulence, our results suggest that differences in allele-specific expression may add an additional layer of phenotypic plasticity to traits related to virulence in C. orthopsilosis hybrids.IMPORTANCE How new pathogens emerge is an important question that remains largely unanswered. Some emerging yeast pathogens are hybrids originated through the crossing of two different species, but how hybridization contributes to higher virulence is unclear. Here, we show that hybrids selectively retain gene regulation plasticity inherited from the two parents and that this plasticity affects genes involved in virulence.

Integrative Omics Analysis Reveals a Limited Transcriptional Shock After Yeast Interspecies Hybridization.

The formation of interspecific hybrids results in the coexistence of two diverged genomes within the same nucleus. It has been hypothesized that negative epistatic interactions and regulatory interferences between the two sub-genomes may elicit a so-called genomic shock involving, among other alterations, broad transcriptional changes. To assess the magnitude of this shock in hybrid yeasts, we investigated the transcriptomic differences between a newly formed Saccharomyces cerevisiae × Saccharomyces uvarum diploid hybrid and its diploid parentals, which diverged ∼20 mya. RNA sequencing (RNA-Seq) based allele-specific expression (ASE) analysis indicated that gene expression changes in the hybrid genome are limited, with only ∼1-2% of genes significantly altering their expression with respect to a non-hybrid context. In comparison, a thermal shock altered six times more genes. Furthermore, differences in the expression between orthologous genes in the two parental species tended to be diminished for the corresponding homeologous genes in the hybrid. Finally, and consistent with the RNA-Seq results, we show a limited impact of hybridization on chromatin accessibility patterns, as assessed with assay for transposase-accessible chromatin using sequencing (ATAC-Seq). Overall, our results suggest a limited genomic shock in a newly formed yeast hybrid, which may explain the high frequency of successful hybridization in these organisms.

Oral microbiome in down syndrome and its implications on oral health.

Introduction: The oral cavity harbors an abundant and diverse microbial community (i.e. the microbiome), whose composition and roles in health and disease have been the focus of intense research. Down syndrome (DS) is associated with particular characteristics in the oral cavity, and with a lower incidence of caries and higher incidence of periodontitis and gingivitis compared to control populations. However, the overall composition of the oral microbiome in DS and how it varies with diverse factors like host age or the pH within the mouth are still poorly understood. Methods: Using a Citizen-Science approach in collaboration with DS associations in Spain, we performed 16S rRNA metabarcoding and high-throughput sequencing, combined with culture and proteomics-based identification of fungi to survey the bacterial and fungal oral microbiome in 27 DS persons (age range 7-55) and control samples matched by geographical distribution, age range, and gender. Results: We found that DS is associated with low salivary pH and less diverse oral microbiomes, which were characterized by lower levels of Alloprevotella, Atopobium, Candidatus Saccharimonas, and higher amounts of Kingella, Staphylococcus, Gemella, Cardiobacterium, Rothia, Actinobacillus, and greater prevalence of Candida. Conclusion: Altogether, our study provides a first global snapshot of the oral microbiome in DS. Future studies are required to establish whether the observed differences are related to differential pathology in the oral cavity in DS.

Genome Comparisons of Candida glabrata Serial Clinical Isolates Reveal Patterns of Genetic Variation in Infecting Clonal Populations.

Candida glabrata is an opportunistic fungal pathogen that currently ranks as the second most common cause of candidiasis. Although the mechanisms underlying virulence and drug resistance in C. glabrata are now starting to be elucidated, we still lack a good understanding of how this yeast adapts during the course of an infection. Outstanding questions are whether the observed genomic plasticity of C. glabrata plays a role during infection, or what levels of genetic variation exist within an infecting clonal population. To shed light onto the genomic variation within infecting C. glabrata populations, we compared the genomes of 11 pairs and one trio of serial clinical isolates, each obtained from a single patient. Our results provide a catalog of genetic variations existing within clonal infecting isolates, and reveal an enrichment of non-synonymous changes in genes encoding cell-wall proteins. Genetic variation and the presence of non-synonymous mutations and copy number variations accumulated within the host, suggest that clonal populations entail a non-negligible level of genetic variation that may reflect selection processes that occur within the human body. As we show here, these genomic changes can underlie phenotypic differences in traits that are relevant for infection.

Transcriptomic analyses reveal groups of co-expressed, syntenic lncRNAs in four species of the genus Caenorhabditis.

Long non-coding RNAs (lncRNAs) are a heterogeneous class of genes that do not code for proteins. Since lncRNAs (or a fraction thereof) are expected to be functional, many efforts have been dedicated to catalog lncRNAs in numerous organisms, but our knowledge of lncRNAs in non vertebrate species remains very limited. Here, we annotated lncRNAs using transcriptomic data from the same larval stage of four Caenorhabditis species. The number of annotated lncRNAs in self-fertile nematodes was lower than in out-crossing species. We used a combination of approaches to identify putatively homologous lncRNAs: synteny, sequence conservation, and structural conservation. We classified a total of 1,532 out of 7,635 genes from the four species into families of lncRNAs with conserved synteny and expression at the larval stage, suggesting that a large fraction of the predicted lncRNAs may be species specific. Despite both sequence and local secondary structure seem to be poorly conserved, sequences within families frequently shared BLASTn hits and short sequence motifs, which were more likely to be unpaired in the predicted structures. We provide the first multi-species catalog of lncRNAs in nematodes and identify groups of lncRNAs with conserved synteny and expression, that share exposed motifs.

Citizen science charts two major "stomatotypes" in the oral microbiome of adolescents and reveals links with habits and drinking water composition.

BACKGROUND: The oral cavity comprises a rich and diverse microbiome, which plays important roles in health and disease. Previous studies have mostly focused on adult populations or in very young children, whereas the adolescent oral microbiome remains poorly studied. Here, we used a citizen science approach and 16S profiling to assess the oral microbiome of 1500 adolescents around Spain and its relationships with lifestyle, diet, hygiene, and socioeconomic and environmental parameters. RESULTS: Our results provide a detailed snapshot of the adolescent oral microbiome and how it varies with lifestyle and other factors. In addition to hygiene and dietary habits, we found that the composition of tap water was related to important changes in the abundance of several bacterial genera. This points to an important role of drinking water in shaping the oral microbiota, which has been so far poorly explored. Overall, the microbiome samples of our study can be clustered into two broad compositional patterns (stomatotypes), driven mostly by Neisseria and Prevotella, respectively. These patterns show striking similarities with those found in unrelated populations. CONCLUSIONS: We hypothesize that these stomatotypes represent two possible global optimal equilibria in the oral microbiome that reflect underlying constraints of the human oral niche. As such, they should be found across a variety of geographical regions, lifestyles, and ages.

Echinocandin-Induced Microevolution of Candida parapsilosis Influences Virulence and Abiotic Stress Tolerance.

Candida species are a major cause of life-threatening bloodstream infections worldwide. Although Candida albicans is responsible for the vast majority of infections, the clinical relevance of other Candida species has also emerged over the last twenty years. This shift might be due in part to changes in clinical guidelines, as echinocandins became the first line of therapeutics for the treatment. Candida parapsilosis is an emerging non-albicans Candida species that exhibits lower susceptibility levels to these drugs. Candida species frequently display resistance to echinocandins, and the mechanism for this is well-known in C. albicans and Candida glabrata, where it is mediated by amino acid substitutions at defined locations of the ß-1,3-glucan synthase, Fks1p. In C. parapsilosis isolates, Fks1p harbors an intrinsic amino acid change at position 660 of the hot spot 1 (HS1) region, which is thought to be responsible for the high MIC values. Less is known about acquired substitutions in this species. In this study, we used directed evolution experiments to generate C. parapsilosis strains with acquired resistance to caspofungin, anidulafungin, and micafungin. We showed that cross-resistance was dependent on the type of echinocandin used to generate the evolved strains. During their characterization, all mutant strains showed attenuated virulence in vivo and also displayed alterations in the exposure of inner cell wall components. The evolved strains harbored 251 amino acid changes, including three in the HS1, HS2, and HS3 regions of Fks1p. Altogether, our results demonstrate a direct connection between acquired antifungal resistance and virulence of C. parapsilosisIMPORTANCECandida parapsilosis is an opportunistic fungal pathogen with the ability to cause infections in immunocompromised patients. Echinocandins are the currently recommended first line of treatment for all Candida species. Resistance of Candida albicans to this drug type is well characterized. C. parapsilosis strains have the lowest in vitro susceptibility to echinocandins; however, patients with such infections typically respond well to echinocandin therapy. There is little knowledge of acquired resistance in C. parapsilosis and its consequences on other characteristics such as virulence properties. In this study, we aimed to dissect how acquired echinocandin resistance influences the pathogenicity of C. parapsilosis and to develop explanations for why echinocandins are clinically effective in the setting of acquired resistance.

Evolutionary Emergence of Drug Resistance in Candida Opportunistic Pathogens.

Fungal infections, such as candidiasis caused by Candida, pose a problem of growing medical concern. In developed countries, the incidence of Candida infections is increasing due to the higher survival of susceptible populations, such as immunocompromised patients or the elderly. Existing treatment options are limited to few antifungal drug families with efficacies that vary depending on the infecting species. In this context, the emergence and spread of resistant Candida isolates are being increasingly reported. Understanding how resistance can evolve within naturally susceptible species is key to developing novel, more effective treatment strategies. However, in contrast to the situation of antibiotic resistance in bacteria, few studies have focused on the evolutionary mechanisms leading to drug resistance in fungal species. In this review, we will survey and discuss current knowledge on the genetic bases of resistance to antifungal drugs in Candida opportunistic pathogens. We will do so from an evolutionary genomics perspective, focusing on the possible evolutionary paths that may lead to the emergence and selection of the resistant phenotype. Finally, we will discuss the potential of future studies enabled by current developments in sequencing technologies, in vitro evolution approaches, and the analysis of serial clinical isolates.

Patterns of Genomic Variation in the Opportunistic Pathogen Candida glabrata Suggest the Existence of Mating and a Secondary Association with Humans.

Candida glabrata is an opportunistic fungal pathogen that ranks as the second most common cause of systemic candidiasis. Despite its genus name, this yeast is more closely related to the model yeast Saccharomyces cerevisiae than to other Candida pathogens, and hence its ability to infect humans is thought to have emerged independently. Moreover, C. glabrata has all the necessary genes to undergo a sexual cycle but is considered an asexual organism due to the lack of direct evidence of sexual reproduction. To reconstruct the recent evolution of this pathogen and find footprints of sexual reproduction, we assessed genomic and phenotypic variation across 33 globally distributed C. glabrata isolates. We cataloged extensive copy-number variation, which particularly affects genes encoding cell-wall-associated proteins, including adhesins. The observed level of genetic variation in C. glabrata is significantly higher than that found in Candida albicans. This variation is structured into seven deeply divergent clades, which show recent geographical dispersion and large within-clade genomic and phenotypic differences. We show compelling evidence of recent admixture between differentiated lineages and of purifying selection on mating genes, which provides the first evidence for the existence of an active sexual cycle in this yeast. Altogether, our data point to a recent global spread of previously genetically isolated populations and suggest that humans are only a secondary niche for this yeast.

Correction: The Genomic Aftermath of Hybridization in the Opportunistic Pathogen Candida metapsilosis.

[This corrects the article DOI: 10.1371/journal.pgen.1005626.].

The Genomic Aftermath of Hybridization in the Opportunistic Pathogen Candida metapsilosis.

Candida metapsilosis is a rarely-isolated, opportunistic pathogen that belongs to a clade of pathogenic yeasts known as the C. parapsilosis sensu lato species complex. To gain insight into the recent evolution of C. metapsilosis and the genetic basis of its virulence, we sequenced the genome of 11 clinical isolates from various locations, which we compared to each other and to the available genomes of the two remaining members of the complex: C. orthopsilosis and C. parapsilosis. Unexpectedly, we found compelling genomic evidence that C. metapsilosis is a highly heterozygous hybrid species, with all sequenced clinical strains resulting from the same past hybridization event involving two parental lineages that were approximately 4.5% divergent in sequence. This result indicates that the parental species are non-pathogenic, but that hybridization between them formed a new opportunistic pathogen, C. metapsilosis, that has achieved a worldwide distribution. We show that these hybrids are diploid and we identified strains carrying loci for both alternative mating types, which supports mating as the initial mechanism for hybrid formation. We trace the aftermath of this hybridization at the genomic level, and reconstruct the evolutionary relationships among the different strains. Recombination and introgression -resulting in loss of heterozygosis- between the two subgenomes have been rampant, and includes the partial overwriting of the MTLa mating locus in all strains. Collectively, our results shed light on the recent genomic evolution within the C. parapsilosis sensu lato complex, and argue for a re-definition of species within this clade, with at least five distinct homozygous lineages, some of which having the ability to form hybrids.