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.TG group acknowledges support from the Spanish Ministry of Science and Innovation for grants ‘Centro de Excelencia Severo Ochoa’ and PGC2018-099921-B-I00, cofounded by European Regional Development Fund (ERDF); from the CERCA Programme/Generalitat de Catalunya; from the Catalan Research Agency (AGAUR) SGR423, and grants from the European Union’s Horizon 2020 research and innovation programme under the grant agreement ERC-2016-724173, and the Marie Sklodowska-Curie grant agreement No. 642095. The group also receives support from an INB Grant (PT17/0009/0023 - ISCIII-SGEFI/ERDF). AR was also supported by the Marie Sklodowska-Curie grant agreement No. 642095.Peer ReviewedPostprint (published version

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

Funding: M.P., H.H., T.G., and B.H. received funding from the European Union Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 642095 (OPATHY). A.K. and B. H. received support from the European Union Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 812969 (FunHoMic). S.A. and B.H. were supported by funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 847507 (HDM-FUN). B.H. is further supported by the DFG within the Collaborative Research Centre (CRC)/Transregio (TRR) 124 “FungiNet” project C1 (DFG project number 210879364) and the Balance of the Microverse Cluster (Germany´s Excellence Strategy – EXC 2051 – Project-ID 390713860). M.S.G. was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft - DFG) Emmy Noether Program (project no. 434385622 / GR 5617/1-1), and a Research Grant 2019 from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. M.P. and H.H. received salary from grant agreement No 642095 (OPATHY) (2016-2019). A.K. received salary from grant agreement No 812969 (FunHoMic) (2019-2022).Peer reviewedPublisher PD

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

Comparative transcriptomics of host-pathogen interactions and hybridization in Candida pathogens

Candida pathogenic yeasts represent a global healthcare problem. They comprise phylogenetically diverse species, including newly emerged pathogens. How human-Candida interactions vary across species, and what processes underlie the emergence of novel pathogens are poorly understood. Current thesis addresses these issues using comparative transcriptomics and bioinformatics. We established the global patterns of host-pathogen interactions between human host and the main Candida species, providing novel mechanistic insights into their interplay. We also explored lncRNAs of these pathogens, assessing their implications in infection. Further, we designed and validated a pan-Candida RNA enrichment approach, opening new possibilities for studying host-pathogen interactions in vivo. Then, we assessed the impact of hybridization on transcriptomes of hybrid yeasts, exploring the links between hybridization and virulence emergence. We also developed a new bioinformatics tool facilitating the research in the field. Altogether, results of this thesis expand our knowledge on relevant aspects of human-Candida interactions and yeast evolution.Las levaduras patógenas Candida representan un problema de salud global. Este grupo de levaduras, comprenden especies filogenéticamente diversas, e incluye patógenos emergidos recientemente. La forma en que las interacciones entre humanos y Candida varían de una especie a otra y qué procesos subyacen a la aparición de nuevos patógenos son poco conocidos. La tesis actual aborda estos problemas utilizando una aproximación de transcriptómica comparativa y bioinformática. Establecimos los patrones globales de las interacciones huésped-patógeno entre el huésped humano y las principales especies de Candida, proporcionando nuevas ideas mecanicistas sobre su interacción. También exploramos los lncRNA de estos patógenos, evaluando sus implicaciones en la infección. Además, diseñamos y validamos un enfoque de enriquecimiento de ARN pan-Candida, abriendo nuevas posibilidades para estudiar las interacciones huésped-patógeno in vivo. Luego, evaluamos el impacto de la hibridación en los transcriptomas de levaduras híbridas, explorando los vínculos entre la hibridación y la aparición de virulencia. En su conjunto, los resultados de esta tesis amplían nuestro conocimiento sobre aspectos relevantes de las interacciones humano-Candida y la evolución de las levaduras

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.This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) for the EMBL partnership and the grant ‘Centro de Excelencia Severo Ochoa’ SEV-2012-0208 cofounded by European Regional Development Fund (ERDF); from the CERCA Programme/Generalitat de Catalunya; from the Catalan Research Agency (AGAUR) SGR857 and grants from the European Union’s Horizon 2020 research and innovation programme under the grant agreement ERC-2016-724173 and the Marie Sklodowska-Curie grant agreement No H2020-MSCA-ITN-2014-642095. The group also receives support from a INB Grant (PT17/0009/0023–ISCIII-SGEFI/ERDF

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

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.TG group acknowledges support from the Spanish Ministry of Science and Innovation for grants ‘Centro de Excelencia Severo Ochoa’ and PGC2018-099921-B-I00, cofounded by European Regional Development Fund (ERDF); from the CERCA Programme/Generalitat de Catalunya; from the Catalan Research Agency (AGAUR) SGR423, and grants from the European Union’s Horizon 2020 research and innovation programme under the grant agreement ERC-2016-724173, and the Marie Sklodowska-Curie grant agreement No. 642095. The group also receives support from an INB Grant (PT17/0009/0023 - ISCIII-SGEFI/ERDF). AR was also supported by the Marie Sklodowska-Curie grant agreement No. 642095

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.This work was funded in part by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no. H2020-MSCAITN-2014-642095. TG group also acknowledges support from the Spanish Ministry of Economy, Industry, and Competitiveness (MEIC) for the EMBL partnership, and grants “Centro de Excelencia Severo Ochoa” SEV-2012-0208, and BFU2015-67107 co-founded by European Regional Development Fund (ERDF); from the CERCA Program/Generalitat de Catalunya; from the Catalan Research Agency (AGAUR) SGR857; and grants from the European Union’s Horizon 2020 Research and Innovation Program under the grant agreement ERC-2016-724173. TG also receives support from an INB grant (PT17/0009/0023—ISCIIISGEFI/ERDF

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

Alterations in long noncoding RNAs and their mutations have been increasingly recognized in tumorogenesis and cancer progression awakening especial interest as potential novel cancer biomarkers and therapeutic targets. The use of adjuvant chemotherapy in stage II colorectal cancer patients is challenging, and new biomarkers are required to identify patients with high probability of relapse. We focused on translational potential of non-coding RNAs in colorectal cancer. In this study, we aim to validate a new tool which couples target enrichment and RNAseq for transcriptomics studies of lncRNAs in formalin-fixed paraffin embedded (FFPE) tissue samples. Our results show that this new approach efficiently detects lncRNAs and differences in their expression between healthy and tumor FFPE tissues, as well as somatic mutations in expressed lncRNAs, identifying novel lncRNAs as potential candidates for colorectal cancer. This new approach could represent a promising avenue that would reduce costs and enable more efficient translational research. 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 biobank

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

Small colony variants 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 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 isolate