Comparative genomics to unravel virulence mechanisms in fungal human pathogens

Las Candidas forman uno de los grupos con mayor n´umero de hongos pat´ogenos en humanos. La relaci ´on que tienen desde un punto de vista filogen´etico demuestra que la capacidad de infectar humanos ha surgido varias veces de forma independiente en este clado. El complejo de Candida parapsilosis es ideal para investigar la aparici´on de virulencia puesto que contiene tres especies cercanas que muestran diferentes grados de virulencia y de importancia: C. parapsilosis, C. orthopsilosis y C. metapsilosis. En esta tesis presento la secuenciaci´on y posterior an´alisis de quince cepas de origen cl´ınico y medioambiental que forman parte de este clado. Cabe remarcar que los genomas de C. orthopsilosis tipo 1 y de C. metapsilosis no se hab´ıan secuenciado previamente. Nuestros resultados muestran evidencia gen´omica de la existencia de recombinaci´on, apareamiento e hibridaci´on en este clado, que previamente se habia considerado asexual. Proponemos que las cepas cl´ınicas emergieron de forma independiente en linajes de cepas encontradas en el medioambiente y una posible relaci ´on entre la hibridaci´on y la adquisici´on de caracter´ısticas relevantes para la virulencia. Finalmente, para estudiar la aparici´on de virulencia en este clado, hemos comparado, por primera vez, los genomas de las tres especies que se encuentran dentro del complejo de Candida parapsilosis. Hemos encontrado expansiones de familias g´enicas previamente implicadas en virulencia, como es el caso de las adhesinas, transportadores de membrana y enzimas extracelulares. Tambi´en hemos observado expansiones de familias de genes que hasta el momento no han estado relacionadas con virulencia. En resumen, nuestros resultados aportan una base para poder elaborar numerosas hip´ otesis sobre la aparici´on de virulencia en las especies de Candida y para que estas hip´ otesis puedan ser comprobadas posteriormente en el laboratorio.Candida species constitute one of the most prevalent groups of fungal pathogens. From their phylogenetic relationships it is clear that virulence to humans has emerged in this clade several, independent times. The Candida parapsilosis complex is particularly suitable to investigate the emergence of virulence, with three closely-related species of varying degree of pathogenicity and of growing relevance: C. parapsilosis, C. orthopsilosis and C. metapsilosis. In this thesis I present the genome sequencing and analysis of fifteen strains from this clade, sampled from clinical and environmental sources. Notably, genomes of C. orthopsilosis Type 1 and C. metapsilosis were sequenced for the first time. Our results show for the first time the genomic evidence for the existence of recombination, mating and hybridization in this clade, previously considered asexual. We propose the independent emergence of clinical isolates from environmental lineages and a possible role of hybridization in the acquisition of relevant traits for pathogenesis. Finally, in order to gain insight into the emergence of virulence in this clade, we have compared the genomes of all three species of C. parapsilosis complex. We have found expansions in gene families known to be involved in virulence, like adhesins, membrane transporters and extracellular enzymes, as well as expansions in gene families not implicated in virulence so far. Altogether, our findings provide the grounds for numerous hypotheses about the emergence of virulence in Candida spp. and for their future experimental testing.

ModPhred: an integrative toolkit for the analysis and storage of nanopore sequencing DNA and RNA modification data

Data de publicació electrònica: 22-07-2021Motivation: DNA and RNA modifications can now be identified using Nanopore sequencing. However, we currently lack a flexible software to efficiently encode, store, analyze and visualize DNA and RNA modification data. Results: Here we present ModPhred, a versatile toolkit that facilitates DNA and RNA modification analysis from nanopore sequencing reads in a user-friendly manner. ModPhred integrates probabilistic DNA and RNA modification information within the FASTQ and BAM file formats, can be used to encode multiple types of modifications simultaneously, and its output can be easily coupled to genomic track viewers, facilitating the visualization and analysis of DNA and RNA modification information in individual reads in a simple and computationally efficient manner. Availability: ModPhred is available at https://github.com/novoalab/modPhred, is implemented in Python3, and is released under an MIT license. Docker images with all dependencies preinstalled are also provided. Supplementary information: Supplementary data are available at Bioinformatics online.L.P.P. was supported by funding from the European Union’s H2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No. 754422. This work was partly supported by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) [PGC2018-098152-A-100 to E.M.N.]. They acknowledge the support of the Spanish Ministry of Science and Innovation to the EMBL partnership, Centro de Excelencia Severo Ochoa and CERCA Programme/Generalitat de Catalunya

Redundans: an assembly pipeline for highly heterozygous genomes

Many genomes display high levels of heterozygosity (i.e. presence of different alleles at the same loci in homologous chromosomes), being those of hybrid organisms an extreme such case. The assembly of highly heterozygous genomes from short sequencing reads is a challenging task because it is difficult to accurately recover the different haplotypes. When confronted with highly heterozygous genomes, the standard assembly process tends to collapse homozygous regions and reports heterozygous regions in alternative contigs. The boundaries between homozygous and heterozygous regions result in multiple assembly paths that are hard to resolve, which leads to highly fragmented assemblies with a total size larger than expected. This, in turn, causes numerous problems in downstream analyses such as fragmented gene models, wrong gene copy number, or broken synteny. To circumvent these caveats we have developed a pipeline that specifically deals with the assembly of heterozygous genomes by introducing a step to recognise and selectively remove alternative heterozygous contigs. We tested our pipeline on simulated and naturally-occurring heterozygous genomes and compared its accuracy to other existing tools. Our method is freely available at https://github.com/Gabaldonlab/redundans.Spanish Ministry of Economy and Competitiveness grants, ‘Centro de Excelencia Severo Ochoa [2013–2017’ SEV-2012-0208, BFU2015-67107 to TG group] cofounded by European Regional Development Fund (ERDF); European Union and ERC Seventh Framework Programme [FP7/2007-2013] under grant agreements [FP7-PEOPLE-2013-ITN-606786 and ERC-2012-StG-310325]; European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie [H2020-MSCA-ITN-2014-642095]; La Caixa-CRG International Fellowship Program (to L.P.P.). Funding for open access charge: ERC Seventh Framework Programme [FP7/2007-2013] under grant agreements [FP7-PEOPLE-2013-ITN-606786 and ERC-2012-StG-310325]; European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie [H2020-MSCA-ITN-2014-642095]; Spanish Ministry of Economy and Competitiveness grants, ‘Centro de Excelencia Severo Ochoa [2013–2017’ SEV-2012-0208, BFU2015-67107] cofounded by ERDF; Catalan Research Agency (AGAUR) [SGR857]

Epigenetic response of yarrowia lipolytica to stress: tracking methylation level and search for methylation patterns via whole-genome sequencing

DNA methylation is a common, but not universal, epigenetic modification that plays an important role in multiple cellular processes. While definitely settled for numerous plant, mammalian, and bacterial species, the genome methylation in different fungal species, including widely studied and industrially-relevant yeast species, Yarrowia lipolytica, is still a matter of debate. In this paper, we report a differential DNA methylation level in the genome of Y. lipolytica subjected to sequential subculturing and to heat stress conditions. To this end, we adopted repeated batch bioreactor cultivations of Y. lipolytica subjected to thermal stress in specific time intervals. To analyze the variation in DNA methylation between stressed and control cultures, we (a) quantified the global DNA methylation status using an immuno-assay, and (b) studied DNA methylation patterns through whole-genome sequencing. Primarily, we demonstrated that 5 mC modification can be detected using a commercial immuno-assay, and that the modifications are present in Y. lipolytica's genome at ~0.5% 5 mC frequency. On the other hand, we did not observe any changes in the epigenetic response of Y. lipolytica to heat shock (HS) treatment. Interestingly, we identified a general phenomenon of decreased 5 mC level in Y. lipolytica's genome in the stationary phase of growth, when compared to a late-exponential epigenome. While this study provides an insight into the subculturing stress response and adaptation to the stress at epigenetic level by Y. lipolytica, it also leaves an open question of inability to detect any genomic DNA methylation level (either in CpG context or context-less) through whole-genome sequencing. The results of ONT sequencing, suggesting that 5 mC modification is either rare or non-existent in Y. lipolytica genome, are contradicted with the results of the immunoassay.This research was financially supported by the Ministry of Science and Higher Education in Poland, project No. DI2017 001047 (in the part concerning stress-response) and partly by Poznan University of Life Sciences, project No. 506.771.09.00 B (in the part concerning methylation and genome sequencing). L.P.P. is supported by funding from the European Union’s H2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No. 75442

MetaPhOrs: orthology and paralogy predictions from multiple phylogenetic evidence using a consistency-based confidence score

Reliable prediction of orthology is central to comparative genomics. Approaches based on phylogenetic analyses closely resemble the original definition of orthology and paralogy and are known to be highly accurate. However, the large computational cost associated to these analyses is a limiting factor that often prevents its use at genomic scales. Recently, several projects have addressed the reconstruction of large collections of high-quality phylogenetic trees from which orthology and paralogy relationships can be inferred. This provides us with the opportunity to infer the evolutionary relationships of genes from multiple, independent, phylogenetic trees. Using such strategy, we combine phylogenetic information derived from different databases, to predict orthology and paralogy relationships for 4.1 million proteins in 829 fully sequenced genomes. We show that the number of independent sources from which a prediction is made, as well as the level of consistency across predictions, can be used as reliable confidence scores. A webserver has been developed to easily access these data (http://orthology.phylomedb.org), which provides users with a global repository of phylogeny-based orthology and paralogy predictions.This work was supported by La Caixa pre-doctoral fellowship to L.P.; partial support by a grant from the Spanish Ministry of Science (BFU2009-09168) for T.G.’s research. Funding for open access charge: Spanish Ministry of Science (BFU2009-09168

MetaPhOrs 2.0: integrative, phylogeny-based inference of orthology and paralogy across the tree of life

Inferring homology relationships across genes in different species is a central task in comparative genomics. Therefore, a large number of resources and methods have been developed over the years. Some public databases include phylogenetic trees of homologous gene families which can be used to further differentiate homology relationships into orthology and paralogy. MetaPhOrs is a web server that integrates phylogenetic information from different sources to provide orthology and paralogy relationships based on a common phylogeny-based predictive algorithm and associated with a consistency-based confidence score. Here we describe the latest version of the web server which includes major new implementations and provides orthology and paralogy relationships derived from ∼8.2 million gene family trees-from 13 different source repositories across ∼4000 species with sequenced genomes. MetaPhOrs server is freely available, without registration, at http://orthology.phylomedb.org/.H2020 Marie Skłodowska-Curie Actions [H2020-MSCA-IF-2017-793699 to U.C.]; Spanish Ministry of Economy, Industry, and Competitiveness (MEIC) [PGC2018-099921-B-I00]; CERCA Programme/Generalitat de Catalunya; Catalan Research Agency (AGAUR) SGR423; European Union's Horizon 2020 Research and Innovation Programme [ERC-2016-724173]; INB [PT17/0009/0023 - ISCIII-SGEFI/ERDF to T.G.]. Funding for open access charge: H2020Grant

Unexpected genomic variability in clinical and environmental strains of the pathogenic yeast Candida parapsilosis

Invasive candidiasis is the most commonly reported invasive fungal infection worldwide. Although Candida albicans remains the main cause, the incidence of emerging Candida species, such as C. parapsilosis is increasing. It has been postulated that C. parapsilosis clinical isolates result from a recent global expansion of a virulent clone. However, the availability of a single genome for this species has so far prevented testing this hypothesis at genomic scales. We present here the sequence of three additional strains from clinical and environmental samples. Our analyses reveal unexpected patterns of genomic variation, shared among distant strains, that argue against the clonal expansion hypothesis. All strains carry independent expansions involving an arsenite transporter homolog, pointing to the existence of directional selection in the environment, and independent origins of the two clinical isolates. Furthermore, we report the first evidence for the existence of recombination in this species. Altogether, our results shed new light onto the dynamics of genome evolution in C. parapsilosis.This work was supported by the La Caixa-CRG International Fellowship Program to L.P., the TAMOP 4.2.4. A/2-11-1-2012-001 “National Excellence Program” to T.N., and was supported in part by OTKA NF 84006, NN00374 (ERA-Net/nPathoGenomics Program), EMBO Installation Grant and by the Janos Bolyai Fellowship of the Hungarian Academy of Sciences to A.G. T.G. group research is funded in part by a grant from the Spanish Ministry of Economy and Competitiveness (BIO2012-37161), a grant from the Qatar National Research Fund grant (NPRP 5-298-3-086), and a grant from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC (grant agreement no. ERC-/n2012-StG-310325

Unexpected genomic variability in clinical and environmental strains of the pathogenic yeast Candida parapsilosis

Invasive candidiasis is the most commonly reported invasive fungal infection worldwide. Although Candida albicans remains the main cause, the incidence of emerging Candida species, such as C. parapsilosis is increasing. It has been postulated that C. parapsilosis clinical isolates result from a recent global expansion of a virulent clone. However, the availability of a single genome for this species has so far prevented testing this hypothesis at genomic scales. We present here the sequence of three additional strains from clinical and environmental samples. Our analyses reveal unexpected patterns of genomic variation, shared among distant strains, that argue against the clonal expansion hypothesis. All strains carry independent expansions involving an arsenite transporter homolog, pointing to the existence of directional selection in the environment, and independent origins of the two clinical isolates. Furthermore, we report the first evidence for the existence of recombination in this species. Altogether, our results shed new light onto the dynamics of genome evolution in C. parapsilosis.This work was supported by the La Caixa-CRG International Fellowship Program to L.P., the TÁMOP 4.2.4. A/2-11-1-2012-001 “National Excellence Program” to T.N., and was supported in part by OTKA NF 84006, NN00374 (ERA-Net PathoGenomics Program), EMBO Installation Grant and by the Janos Bolyai Fellowship of the Hungarian Academy of Sciences to A.G. T.G. group research is funded in part by a grant from the Spanish Ministry of Economy and Competitiveness (BIO2012-37161), a grant from the Qatar National Research Fund grant (NPRP 5-298-3-086), and a grant from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC (grant agreement no. ERC-2012-StG-310325

Genome comparison of Candida orthopsilosis clinical strains reveals the existence of hybrids between two distinct subspecies

The Candida parapsilosis species complex comprises a group of emerging human pathogens of varying virulence. This complex was recently subdivided into three different species: C. parapsilosis sensu stricto, C. metapsilosis, and C. orthopsilosis. Within the latter, at least two clearly distinct subspecies seem to be present among clinical isolates (Type 1 and Type 2). To gain insight into the genomic differences between these subspecies, we undertook the sequencing of a clinical isolate classified as Type 1 and compared it with the available sequence of a Type 2 clinical strain. Unexpectedly, the analysis of the newly sequenced strain revealed a highly heterozygous genome, which we show to be the consequence of a hybridization event between both identified subspecies. This implicitly suggests that C. orthopsilosis is able to mate, a so-far unanswered question. The resulting hybrid shows a chimeric genome that maintains a similar gene dosage from both parental lineages and displays ongoing loss of heterozygosity. Several of the differences found between the gene content in both strains relate to virulent-related families, with the hybrid strain presenting a higher copy number of genes coding for efflux pumps or secreted lipases. Remarkably, two clinical strains isolated from distant geographical locations (Texas and Singapore) are descendants of the same hybrid line, raising the intriguing possibility of a relationship between the hybridization event and the global spread of a virulent clone.This work was supported in part by a grant from the Spanish ministry of Economy and Competitiveness (BIO2012-37161), a Grant from the Qatar National Research Fund grant (NPRP 5-/n298-3-086), and a grant from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC (Grant Agreement No. ERC-2012-StG-310325) to T.G.; by La Caixa-CRG International Fellowship Program to L.P.P.; by the TA´ MOP 4.2.4. A/2-11-1-2012-001/n“National Excellence Program” to T.N.; in part by OTKA NF 84006, NN00374 (ERA-Net PathoGenomics Program), EMBO Installation Grant, and the Janos Bolyai Fellowship of the/nHungarian Academy of Sciences to A.G

PhylomeDB v4: zooming into the plurality of evolutionary histories of a genome

Phylogenetic trees representing the evolutionary relationships of homologous genes are the entry point for many evolutionary analyses. For instance, the use of a phylogenetic tree can aid in the inference of orthology and paralogy relationships, and in the detection of relevant evolutionary events such as gene family expansions and contractions, horizontal gene transfer, recombination or incomplete lineage sorting. Similarly, given the plurality of evolutionary histories among genes encoded in a given genome, there is a need for the combined analysis of genome-wide collections of phylogenetic trees (phylomes). Here, we introduce a new release of PhylomeDB (http://phylomedb.org), a public repository of phylomes. Currently, PhylomeDB hosts 120 public phylomes, comprising >1.5 million maximum likelihood trees and multiple sequence alignments. In the current release, phylogenetic trees are annotated with taxonomic, protein-domain arrangement, functional and evolutionary information. PhylomeDB is also a major source for phylogeny-based predictions of orthology and paralogy, covering >10 million proteins across 1059 sequenced species. Here we describe newly implemented PhylomeDB features, and discuss a benchmark of the orthology predictions provided by the database, the impact of proteome updates and the use of the phylome approach in the analysis of newly sequenced genomes and transcriptomes.Spanish ministry of Economy and Competitiveness [BIO2012-37161]; a Grant from the Qatar National/nResearch Fund [NPRP 5-298-3-086]; a the European Research Council under the European Union’s Seventh/nFramework Programme [FP/2007-2013/ERC and ERC-/n2012-StG-310325]; Juan de La Cierva postdoctoral program (to J.H.C.) and La Caixa-CRG International Fellowship Program (to L.P.P.). Funding for open access charge: Internal budget from the CR