Using genomics to understand the mechanisms of virulence and drug resistance in fungal pathogens

Fungal pathogens pose an increasingly worrying threat to human health, food security and ecosystem diversity. To tackle fungal infections and improve current diagnostic and therapeutic tools it is necessary to understand virulence and antifungal drug resistance mechanisms in diverse species. Recent advances in genomics approaches have provided a suitable framework to understand these phenotypes, which ultimately depend on genetically encoded determinants. In this work, we review how the study of genome sequences has been key to ascertain the bases of virulence and drug resistance traits. We focus on the contribution of comparative genomics, population genomics and directed evolution studies. In addition, we discuss how different types of genomic mutations (small or structural variants) contribute to intraspecific differences in virulence or drug resistance. Finally, we review current challenges in the field and anticipate future directions to solve them. In summary, this work provides a short overview of how genomics can be used to understand virulence and drug resistance in fungal pathogens.TG group acknowledges support from the Spanish Ministry of Science and Innovation for grant PGC2018-099921-B-I00, cofounded by European Regional Development Fund (ERDF); from the Catalan Research Agency (AGAUR) SGR423; from the European Union's Horizon 2020 research and innovation programme (ERC-2016-724173); from the Gordon and Betty Moore Foundation (Grant GBMF9742); from the ‘La Caixa’ foundation (Grant LCF/PR/HR21/00737), and from the Instituto de Salud Carlos III (INB Grant PT17/0009/0023 and CIBERINFEC CB21/13/00061- ISCIII-SGEFI/ERDF). MAST received a predoctoral fellowship from the ‘Caixa' Foundation (LCF/BQ/DR19/11740023).Peer ReviewedPostprint (published version

A hybrid approach to assess the structural impact of long noncoding RNA mutations uncovers key NEAT1 interactions in colorectal cancer

Long noncoding RNAs (lncRNAs) are emerging players in cancer and they entail potential as prognostic biomarkers or therapeutic targets. Earlier studies have identified somatic mutations in lncRNAs that are associated with tumor relapse after therapy, but the underlying mechanisms behind these associations remain unknown. Given the relevance of secondary structure for the function of some lncRNAs, some of these mutations may have a functional impact through structural disturbance. Here, we examined the potential structural and functional impact of a novel A > G point mutation in NEAT1 that has been recurrently observed in tumors of colorectal cancer patients experiencing relapse after treatment. Here, we used the nextPARS structural probing approach to provide first empirical evidence that this mutation alters NEAT1 structure. We further evaluated the potential effects of this structural alteration using computational tools and found that this mutation likely alters the binding propensities of several NEAT1-interacting miRNAs. Differential expression analysis on these miRNA networks shows upregulation of Vimentin, consistent with previous findings. We propose a hybrid pipeline that can be used to explore the potential functional effects of lncRNA somatic mutations.Funding information H2020 European Research Council, Grant/Award Number: 724173Peer ReviewedPostprint (published version

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.The authors thank Ester Saus, Jesse Willis, and Cinta Pegueroles for their help and technical assistance with some of the analyses. M.A.S.-T. received a predoctoral fellowship from the ‘‘Caixa’’ Foundation (LCF/BQ/DR19/11740023). The T.G. group acknowledges support from the Spanish Ministry of Science and Innovation grant no. PGC2018-099921-B-I00, cofounded by the European Regional Development Fund (ERDF); from the CERCA Programme/Generalitat de Catalunya; from the Catalan Research Agency (AGAUR) SGR423; the European Union’s Horizon 2020 research and innovation program under grant agreement no. ERC-2016-724173; and the Marie Sk1odowska-Curie grant agreement no. H2020-MSCA-IF-2017-793699. The group also receives support from an INB grant (PT17/0009/0023-ISCIII-SGEFI/ERDF). The Bioactive Microbial Metabolites research platform (BiMM) is supported by grants K3- G-2/026-2013 and COMBIS/ LS16005, both funded by the Lower Austria Science and Education Fund (NfB).Peer ReviewedPostprint (published version

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.This work was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. H2020-MSCA-ITN-2014-642095. The TG group also acknowledges the support from the Spanish Ministry of Economy, Industry, and Competitiveness (MEIC) for the EMBL partnership and grants “Centro de Excelencia Severo Ochoa 2013-2017” SEV-2012-0208 and BFU2015-67107 co-founded by the 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 program under the grant agreement ERC-2016-724173. TG also receives support from an INB Grant (PT17/0009/0023—ISCIII-SGEFI/ERDF). The authors thank Dr. Powel Golik’s guidance in the identification of PPR proteins, Simone Mozzachiodi and Dr. Gianni Liti for the helpful discussions on the analysis of meiotic patterns, and all Gabaldón lab members for the helpful discussions and comments on this work, especially Marina Marcet-Houben.Peer ReviewedPostprint (author's final draft

The Quest for Orthologs orthology benchmark service in 2022

The Orthology Benchmark Service (https://orthology.benchmarkservice.org) is the gold standard for orthology inference evaluation, supported and maintained by the Quest for Orthologs consortium. It is an essential resource to compare existing and new methods of orthology inference (the bedrock for many comparative genomics and phylogenetic analysis) over a standard dataset and through common procedures. The Quest for Orthologs Consortium is dedicated to maintaining the resource up to date, through regular updates of the Reference Proteomes and increasingly accessible data through the OpenEBench platform. For this update, we have added a new benchmark based on curated orthology assertion from the Vertebrate Gene Nomenclature Committee, and provided an example meta-analysis of the public predictions present on the platform.European Molecular Biology Laboratory (EMBL) (core funds to D.J. and M.J.M.); National Institutes of Health [U24HG007822 to D.J. and M.J.M., 75N93019C00077 to D.S.R.]; National Human Genome Research Institute (NHGRI) [U24HG003345 to T.E.M.J, B.Y., E.A.B.]; JSPS KAKENHI [16H06279, 19H05688 to W.I.]; JST CREST [JPMJCR19S2 to W.I.]; MEXT [JPMXD1521474594 to W.I.]; Horizon 2020 [676559 to S.C.-G., 637765] (to D.M.E.), ELIXIR (to S.C.-G.); Wellcome Grant [208349/Z/17/Z to E.A.B.]; National Science Foundation (USA) [1917302 to P.D.T.]; Wellcome Trust [WT-218288, WT-212929 to D.S.R.]; Service and Infrastructure grant from the Swiss Institute of Bioinformatics, Swiss National Science Foundation [186397, 205085 to C.D.]. Funding for open access charge: Swiss National Science Foundation [205085].Peer Reviewed"Article signat per 31 autors/es: Yannis Nevers, Tamsin E M Jones, Dushyanth Jyothi, Bethan Yates, Meritxell Ferret, Laura Portell-Silva, Laia Codo, Salvatore Cosentino, Marina Marcet-Houben, Anna Vlasova, Laetitia Poidevin, Arnaud Kress, Mark Hickman, Emma Persson, Ivana Piližota, Cristina Guijarro-Clarke, the OpenEBench team the Quest for Orthologs Consortium , Wataru Iwasaki, Odile Lecompte, Erik Sonnhammer, David S Roos, Toni Gabaldón, David Thybert, Paul D Thomas, Yanhui Hu, David M Emms, Elspeth Bruford, Salvador Capella-Gutierrez, Maria J Martin, Christophe Dessimoz, Adrian Altenhoff"Postprint (published version

The Genome and Development-Dependent Transcriptomes of Pyronema confluens: A Window into Fungal Evolution

Fungi are a large group of eukaryotes found in nearly all ecosystems. More than 250 fungal genomes have already been sequenced, greatly improving our understanding of fungal evolution, physiology, and development. However, for the Pezizomycetes, an early-diverging lineage of filamentous ascomycetes, there is so far only one genome available, namely that of the black truffle, Tuber melanosporum, a mycorrhizal species with unusual subterranean fruiting bodies. To help close the sequence gap among basal filamentous ascomycetes, and to allow conclusions about the evolution of fungal development, we sequenced the genome and assayed transcriptomes during development of Pyronema confluens, a saprobic Pezizomycete with a typical apothecium as fruiting body. With a size of 50 Mb and similar to 13,400 protein-coding genes, the genome is more characteristic of higher filamentous ascomycetes than the large, repeat-rich truffle genome; however, some typical features are different in the P. confluens lineage, e.g. the genomic environment of the mating type genes that is conserved in higher filamentous ascomycetes, but only partly conserved in P. confluens. On the other hand, P. confluens has a full complement of fungal photoreceptors, and expression studies indicate that light perception might be similar to distantly related ascomycetes and, thus, represent a basic feature of filamentous ascomycetes. Analysis of spliced RNA-seq sequence reads allowed the detection of natural antisense transcripts for 281 genes. The P. confluens genome contains an unusually high number of predicted orphan genes, many of which are upregulated during sexual development, consistent with the idea of rapid evolution of sex-associated genes. Comparative transcriptomics identified the transcription factor gene pro44 that is upregulated during development in P. confluens and the Sordariomycete Sordaria macrospora. The P. confluens pro44 gene (PCON_06721) was used to complement the S. macrospora pro44 deletion mutant, showing functional conservation of this developmental regulator

PeroxisomeDB 2.0: an integrative view of the global peroxisomal metabolome

Peroxisomes are essential organelles that play a key role in redox signalling and lipid homeostasis. They contain a highly diverse enzymatic network among different species, mirroring the varied metabolic needs of the organisms. The previous PeroxisomeDB version organized the peroxisomal proteome of humans and Saccharomyces cerevisiae based on genetic and functional information into metabolic categories with a special focus on peroxisomal disease. The new release (http://www.peroxisomeDB.org) adds peroxisomal proteins from 35 newly sequenced eukaryotic genomes including fungi, yeasts, plants and lower eukaryotes. We searched these genomes for a core ensemble of 139 peroxisomal protein families and identified 2706 putative peroxisomal protein homologs. Approximately 37% of the identified homologs contained putative peroxisome targeting signals (PTS). To help develop understanding of the evolutionary relationships among peroxisomal proteins, the new database includes phylogenetic trees for 2386 of the peroxisomal proteins. Additional new features are provided, such as a tool to capture kinetic information from Brenda, CheBI and Sabio-RK databases and more than 1400 selected bibliographic references. PeroxisomeDB 2.0 is a freely available, highly interactive functional genomics platform that offers an extensive view on the peroxisomal metabolome across lineages, thus facilitating comparative genomics and systems analysis of the organelle

Impact of the Mitochondrial Genetic Background in Complex III Deficiency

BACKGROUND: In recent years clinical evidence has emphasized the importance of the mtDNA genetic background that hosts a primary pathogenic mutation in the clinical expression of mitochondrial disorders, but little experimental confirmation has been provided. We have analyzed the pathogenic role of a novel homoplasmic mutation (m.15533 A>G) in the cytochrome b (MT-CYB) gene in a patient presenting with lactic acidosis, seizures, mild mental delay, and behaviour abnormalities. METHODOLOGY: Spectrophotometric analyses of the respiratory chain enzyme activities were performed in different tissues, the whole muscle mitochondrial DNA of the patient was sequenced, and the novel mutation was confirmed by PCR-RFLP. Transmitochondrial cybrids were constructed to confirm the pathogenicity of the mutation, and assembly/stability studies were carried out in fibroblasts and cybrids by means of mitochondrial translation inhibition in combination with blue native gel electrophoresis. PRINCIPAL FINDINGS: Biochemical analyses revealed a decrease in respiratory chain complex III activity in patient's skeletal muscle, and a combined enzyme defect of complexes III and IV in fibroblasts. Mutant transmitochondrial cybrids restored normal enzyme activities and steady-state protein levels, the mutation was mildly conserved along evolution, and the proband's mother and maternal aunt, both clinically unaffected, also harboured the homoplasmic mutation. These data suggested a nuclear genetic origin of the disease. However, by forcing the de novo functioning of the OXPHOS system, a severe delay in the biogenesis of the respiratory chain complexes was observed in the mutants, which demonstrated a direct functional effect of the mitochondrial genetic background. CONCLUSIONS: Our results point to possible pitfalls in the detection of pathogenic mitochondrial mutations, and highlight the role of the genetic mtDNA background in the development of mitochondrial disorders