Overlooked Candida glabrata petites are echinocandin tolerant, induce host inflammatory responses, and display poor in vivo fitness.

IMPORTANCE: Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called "petite." This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omics technologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and, therefore, are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, which can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

Overlooked Candida glabrata petites are echinocandin tolerant, induce host inflammatory responses, and display poor in vivo fitness.

Small colony variants (SCVs) are relatively common among some bacterial species and are associated with poor prognosis and recalcitrant infections. Similarly, Candida glabrata - a major intracellular fungal pathogen - produces small and slow-growing respiratory-deficient colonies, termed "petite." Despite reports of clinical petite C . glabrata strains, our understanding of petite behavior in the host remains obscure. Moreover, controversies exist regarding in-host petite fitness and its clinical relevance. Herein, we employed whole-genome sequencing (WGS), dual-RNAseq, and extensive ex vivo and in vivo studies to fill this knowledge gap. WGS identified multiple petite-specific mutations in nuclear and mitochondrially-encoded genes. Consistent with dual-RNAseq data, petite C . glabrata cells did not replicate inside host macrophages and were outcompeted by their non-petite parents in macrophages and in gut colonization and systemic infection mouse models. The intracellular petites showed hallmarks of drug tolerance and were relatively insensitive to the fungicidal activity of echinocandin drugs. Petite-infected macrophages exhibited a pro-inflammatory and type I IFN-skewed transcriptional program. Interrogation of international C . glabrata blood isolates ( n =1000) showed that petite prevalence varies by country, albeit at an overall low prevalence (0-3.5%). Collectively, our study sheds new light on the genetic basis, drug susceptibility, clinical prevalence, and host-pathogen responses of a clinically overlooked phenotype in a major fungal pathogen. Importance: Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called "petite". This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omicstechnologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and therefore are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex-vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

Multiplexed target enrichment of coding and non-coding transcriptomes enables studying Candida spp. infections from human derived samples.

The study of transcriptomic interactions between host and pathogens in in vivo conditions is challenged by the low relative amounts of the pathogen RNA. Yeast opportunistic pathogens of the genus Candida can cause life-threatening systemic infections in immunocompromised patients, and are of growing medical concern. Four phylogenetically diverse species account for over 90% of Candida infections, and their specific interactions with various human tissues are still poorly understood. To enable in vivo transcriptomic analysis in these species, we designed and validated pan-Candida target capture probes to enrich protein-coding and non-coding transcriptomes. The probe-based enrichment approach outperformed enrichment based on differential lysis of host cells, and showed similar enrichment performance as an existing capture design, yet achieving better fidelity of expression levels, enabling species multiplexing and capturing of lncRNAs. In addition, we show that our probe-based enrichment strategy allows robust genotype-based identification of the infecting strain present in the sample.

The long non-coding RNA landscape of Candida yeast pathogens.

Long non-coding RNAs (lncRNAs) constitute a poorly studied class of transcripts with emerging roles in key cellular processes. Despite efforts to characterize lncRNAs across a wide range of species, these molecules remain largely unexplored in most eukaryotic microbes, including yeast pathogens of the Candida clade. Here, we analyze thousands of publicly available sequencing datasets to infer and characterize the lncRNA repertoires of five major Candida pathogens: Candida albicans, Candida tropicalis, Candida parapsilosis, Candida auris and Candida glabrata. Our results indicate that genomes of these species encode hundreds of lncRNAs that show levels of evolutionary constraint intermediate between those of intergenic genomic regions and protein-coding genes. Despite their low sequence conservation across the studied species, some lncRNAs are syntenic and are enriched in shared sequence motifs. We find co-expression of lncRNAs with certain protein-coding transcripts, hinting at potential functional associations. Finally, we identify lncRNAs that are differentially expressed during infection of human epithelial cells for four of the studied species. Our comprehensive bioinformatic analyses of Candida lncRNAs pave the way for future functional characterization of these transcripts.

Human albumin enhances the pathogenic potential of Candida glabrata on vaginal epithelial cells.

The opportunistic pathogen Candida glabrata is the second most frequent causative agent of vulvovaginal candidiasis (VVC), a disease that affects 70-75% of women at least once during their life. However, C. glabrata is almost avirulent in mice and normally incapable of inflicting damage to vaginal epithelial cells in vitro. We thus proposed that host factors present in vivo may influence C. glabrata pathogenicity. We, therefore, analyzed the impact of albumin, one of the most abundant proteins of the vaginal fluid. The presence of human, but not murine, albumin dramatically increased the potential of C. glabrata to damage vaginal epithelial cells. This effect depended on macropinocytosis-mediated epithelial uptake of albumin and subsequent proteolytic processing. The enhanced pathogenicity of C. glabrata can be explained by a combination of beneficial effects for the fungus, which includes an increased access to iron, accelerated growth, and increased adhesion. Screening of C. glabrata deletion mutants revealed that Hap5, a key regulator of iron homeostasis, is essential for the albumin-augmented damage potential. The albumin-augmented pathogenicity was reversed by the addition of iron chelators and a similar increase in pathogenicity was shown by increasing the iron availability, confirming a key role of iron. Accelerated growth not only led to higher cell numbers, but also to increased fungal metabolic activity and oxidative stress resistance. Finally, the albumin-driven enhanced damage potential was associated with the expression of distinct C. glabrata virulence genes. Transcriptional responses of the epithelial cells suggested an unfolded protein response (UPR) and ER-stress responses combined with glucose starvation induced by fast growing C. glabrata cells as potential mechanisms by which cytotoxicity is mediated.Collectively, we demonstrate that albumin augments the pathogenic potential of C. glabrata during interaction with vaginal epithelial cells. This suggests a role for albumin as a key player in the pathogenesis of VVC.

Candida pathogens induce protective mitochondria-associated type I interferon signalling and a damage-driven response in vaginal epithelial cells.

Vaginal candidiasis is an extremely common disease predominantly caused by four phylogenetically diverse species: Candida albicans; Candida glabrata; Candida parapsilosis; and Candida tropicalis. Using a time course infection model of vaginal epithelial cells and dual RNA sequencing, we show that these species exhibit distinct pathogenicity patterns, which are defined by highly species-specific transcriptional profiles during infection of vaginal epithelial cells. In contrast, host cells exhibit a homogeneous response to all species at the early stages of infection, which is characterized by sublethal mitochondrial signalling inducing a protective type I interferon response. At the later stages, the transcriptional response of the host diverges in a species-dependent manner. This divergence is primarily driven by the extent of epithelial damage elicited by species-specific mechanisms, such as secretion of the toxin candidalysin by C. albicans. Our results uncover a dynamic, biphasic response of vaginal epithelial cells to Candida species, which is characterized by protective mitochondria-associated type I interferon signalling and a species-specific damage-driven response.

Target Enrichment Enables the Discovery of lncRNAs with Somatic Mutations or Altered Expression in Paraffin-Embedded Colorectal Cancer Samples.

Long non-coding RNAs (lncRNAs) play important roles in cancer and are potential new biomarkers or targets for therapy. However, given the low and tissue-specific expression of lncRNAs, linking these molecules to particular cancer types and processes through transcriptional profiling is challenging. Formalin-fixed, paraffin-embedded (FFPE) tissues are abundant resources for research but are prone to nucleic acid degradation, thereby complicating the study of lncRNAs. Here, we designed and validated a probe-based enrichment strategy to efficiently profile lncRNA expression in FFPE samples, and we applied it for the detection of lncRNAs associated with colorectal cancer (CRC). Our approach efficiently enriched targeted lncRNAs from FFPE samples, while preserving their relative abundance, and enabled the detection of tumor-specific mutations. We identified 379 lncRNAs differentially expressed between CRC tumors and matched healthy tissues and found tumor-specific lncRNA variants. Our results show that numerous lncRNAs are differentially expressed and/or accumulate variants in CRC tumors, thereby suggesting a role in CRC progression. More generally, our approach unlocks the study of lncRNAs in FFPE samples, thus enabling the retrospective use of abundant, well documented material available in hospital biobanks.

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.

CROSSMAPPER: estimating cross-mapping rates and optimizing experimental design in multi-species sequencing studies.

MOTIVATION: Numerous sequencing studies, including transcriptomics of host-pathogen systems, sequencing of hybrid genomes, xenografts, mixed species systems, metagenomics and meta-transcriptomics, involve samples containing genetic material from divergent organisms. A crucial step in these studies is identifying from which organism each sequencing read originated, and the experimental design should be directed to minimize biases caused by cross-mapping of reads to incorrect source genomes. Additionally, pooling of sufficiently different genetic material into a single sequencing library could significantly reduce experimental costs but requires careful planning and assessment of the impact of cross-mapping. Having these applications in mind we designed Crossmapper, the first to our knowledge tool able to assess cross-mapping prior to sequencing, therefore allowing optimization of experimental design. RESULTS: Using any combination of reference genomes, Crossmapper performs read simulation and back-mapping of those reads to the pool of references, quantifies and reports the cross-mapping rates for each organism. Crossmapper performs these analyses with numerous user-specified parameters, including, among others, read length, read layout, coverage, mapping parameters, genomic or transcriptomic data. Additionally, it outputs the results in highly interactive and publication-ready reports. This allows the user to perform multiple comparisons at once and choose the experimental setup minimizing cross-mapping rates. Moreover, Crossmapper can be used for resource optimization in sequencing facilities by pooling different samples into one sequencing library. AVAILABILITY AND IMPLEMENTATION: Crossmapper is a command line tool implemented in Python 3.6 and available as a conda package, allowing effortless installation. The source code, detailed information and a step-by-step tutorial is available at our GitHub page https://github.com/Gabaldonlab/crossmapper. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Transcriptome Sequencing Approaches to Elucidate Host-Microbe Interactions in Opportunistic Human Fungal Pathogens.

Infections caused by opportunistic human fungal pathogens are a source of increasing medical concern, due to their growing incidence, the emergence of novel pathogenic species, and the lack of effective diagnostics tools. Fungal pathogens are phylogenetically diverse, and their virulence mechanisms can differ widely across species. Despite extensive efforts, the molecular bases of virulence in pathogenic fungi and their interactions with the human host remain poorly understood for most species. In this context, next-generation sequencing approaches hold the promise of helping to close this knowledge gap. In particular, high-throughput transcriptome sequencing (RNA-Seq) enables monitoring the transcriptional profile of both host and microbes to elucidate their interactions and discover molecular mechanisms of virulence and host defense. Here, we provide an overview of transcriptome sequencing techniques and approaches, and survey their application in studying the interplay between humans and fungal pathogens. Finally, we discuss novel RNA-Seq approaches in studying host-pathogen interactions and their potential role in advancing the clinical diagnostics of fungal infections.

Eight Millennia of Matrilineal Genetic Continuity in the South Caucasus.

The South Caucasus, situated between the Black and Caspian Seas, geographically links Europe with the Near East and has served as a crossroad for human migrations for many millennia [1-7]. Despite a vast archaeological record showing distinct cultural turnovers, the demographic events that shaped the human populations of this region is not known [8, 9]. To shed light on the maternal genetic history of the region, we analyzed the complete mitochondrial genomes of 52 ancient skeletons from present-day Armenia and Artsakh spanning 7,800 years and combined this dataset with 206 mitochondrial genomes of modern Armenians. We also included previously published data of seven neighboring populations (n = 482). Coalescence-based analyses suggest that the population size in this region rapidly increased after the Last Glacial Maximum ca. 18 kya. We find that the lowest genetic distance in this dataset is between modern Armenians and the ancient individuals, as also reflected in both network analyses and discriminant analysis of principal components. We used approximate Bayesian computation to test five different demographic scenarios explaining the formation of the modern Armenian gene pool. Despite well documented cultural shifts in the South Caucasus across this time period, our results strongly favor a genetic continuity model in the maternal gene pool. This has implications for interpreting prehistoric migration dynamics and cultural shifts in this part of the world.

Origin and spread of human mitochondrial DNA haplogroup U7.

Human mitochondrial DNA haplogroup U is among the initial maternal founders in Southwest Asia and Europe and one that best indicates matrilineal genetic continuity between late Pleistocene hunter-gatherer groups and present-day populations of Europe. While most haplogroup U subclades are older than 30 thousand years, the comparatively recent coalescence time of the extant variation of haplogroup U7 (~16-19 thousand years ago) suggests that its current distribution is the consequence of more recent dispersal events, despite its wide geographical range across Europe, the Near East and South Asia. Here we report 267 new U7 mitogenomes that - analysed alongside 100 published ones - enable us to discern at least two distinct temporal phases of dispersal, both of which most likely emanated from the Near East. The earlier one began prior to the Holocene (~11.5 thousand years ago) towards South Asia, while the later dispersal took place more recently towards Mediterranean Europe during the Neolithic (~8 thousand years ago). These findings imply that the carriers of haplogroup U7 spread to South Asia and Europe before the suggested Bronze Age expansion of Indo-European languages from the Pontic-Caspian Steppe region.