13
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Genomes of Leishmania parasites directly sequenced from patients with visceral leishmaniasis in the Indian subcontinent

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Whole genome sequencing (WGS) is increasingly used for molecular diagnosis and epidemiology of infectious diseases. Current Leishmania genomic studies rely on DNA extracted from cultured parasites, which might introduce sampling and biological biases into the subsequent analyses. Up to now, direct analysis of Leishmania genome in clinical samples is hampered by high levels of human DNA and large variation in parasite load in clinical samples. Here, we present a method, based on target enrichment of Leishmania donovani DNA with Agilent SureSelect technology, that allows the analysis of Leishmania genomes directly in clinical samples. We validated our protocol with a set of artificially mixed samples, followed by the analysis of 63 clinical samples (bone marrow or spleen aspirates) from visceral leishmaniasis patients in Nepal. We were able to identify genotypes using a set of diagnostic SNPs in almost all of these samples (97%) and access comprehensive genome-wide information in most (83%). This allowed us to perform phylogenomic analysis, assess chromosome copy number and identify large copy number variants (CNVs). Pairwise comparisons between the parasite genomes in clinical samples and derived in vitro cultured promastigotes showed a lower aneuploidy in amastigotes as well as genomic differences, suggesting polyclonal infections in patients. Altogether our results underline the need for sequencing parasite genomes directly in the host samples

          Author summary

          Visceral leishmaniasis (VL) is caused by parasitic protozoa of the Leishmania donovani complex and is lethal in the absence of treatment. Whole Genome Sequencing (WGS) of L. donovani clinical isolates revealed hitherto cryptic population structure in the Indian Sub-Continent and provided insights into the epidemiology and potential mechanisms of drug resistance. However, several biases are likely introduced during the culture step. We report here the development of a method that allows determination of parasite genomes directly in clinical samples, and validate it on bone marrow and splenic aspirates of VL patients in Nepal. Our study sheds a new light on the biology of Leishmania in the human host: we found that intracellular parasites of the patients had very low levels of aneuploidy, in sharp contrast to the situation in cultivated isolates. Moreover, the observed differences in genomes between intracellular amastigotes of the patient and the derived cultured parasites suggests polyclonality of infections, with different clones dominating in clinical samples and in culture, likely due to fitness differences. We believe this method is most suitable for clinical studies and for molecular tracking in the context of elimination programs.

          Related collections

          Most cited references30

          • Record: found
          • Abstract: found
          • Article: not found

          Chromosome and gene copy number variation allow major structural change between species and strains of Leishmania.

          Leishmania parasites cause a spectrum of clinical pathology in humans ranging from disfiguring cutaneous lesions to fatal visceral leishmaniasis. We have generated a reference genome for Leishmania mexicana and refined the reference genomes for Leishmania major, Leishmania infantum, and Leishmania braziliensis. This has allowed the identification of a remarkably low number of genes or paralog groups (2, 14, 19, and 67, respectively) unique to one species. These were found to be conserved in additional isolates of the same species. We have predicted allelic variation and find that in these isolates, L. major and L. infantum have a surprisingly low number of predicted heterozygous SNPs compared with L. braziliensis and L. mexicana. We used short read coverage to infer ploidy and gene copy numbers, identifying large copy number variations between species, with 200 tandem gene arrays in L. major and 132 in L. mexicana. Chromosome copy number also varied significantly between species, with nine supernumerary chromosomes in L. infantum, four in L. mexicana, two in L. braziliensis, and one in L. major. A significant bias against gene arrays on supernumerary chromosomes was shown to exist, indicating that duplication events occur more frequently on disomic chromosomes. Taken together, our data demonstrate that there is little variation in unique gene content across Leishmania species, but large-scale genetic heterogeneity can result through gene amplification on disomic chromosomes and variation in chromosome number. Increased gene copy number due to chromosome amplification may contribute to alterations in gene expression in response to environmental conditions in the host, providing a genetic basis for disease tropism.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: found
            Is Open Access

            Single Cell Genomics: Advances and Future Perspectives

            Advances in whole-genome and whole-transcriptome amplification have permitted the sequencing of the minute amounts of DNA and RNA present in a single cell, offering a window into the extent and nature of genomic and transcriptomic heterogeneity which occurs in both normal development and disease. Single-cell approaches stand poised to revolutionise our capacity to understand the scale of genomic, epigenomic, and transcriptomic diversity that occurs during the lifetime of an individual organism. Here, we review the major technological and biological breakthroughs achieved, describe the remaining challenges to overcome, and provide a glimpse into the promise of recent and future developments.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: found
              Is Open Access

              Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression

              ABSTRACT Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania, a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro-cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro, aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness.
                Bookmark

                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: SoftwareRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: SoftwareRole: VisualizationRole: Writing – review & editing
                Role: InvestigationRole: ResourcesRole: Writing – review & editing
                Role: InvestigationRole: ResourcesRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: ResourcesRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: ResourcesRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: SoftwareRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                12 December 2019
                December 2019
                : 13
                : 12
                : e0007900
                Affiliations
                [1 ] Institute of Tropical Medicine Antwerp, Molecular Parasitology Unit, Antwerp, Belgium
                [2 ] Wellcome Sanger Institute, Hinxton, United Kingdom
                [3 ] BP Koirala Institute of Health Sciences, Dharan, Nepal
                [4 ] University of Antwerp, Department of Biomedical Sciences, Antwerp, Belgium
                University of Pittsburgh, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                [¤a]

                Current address: Columbia University, Department of Microbiology and Immunology, New York, United States of America

                [¤b]

                Current address: University Hospital of Ghent, Center for Medical Genetics, Ghent, Belgium

                Author information
                http://orcid.org/0000-0003-0839-0325
                http://orcid.org/0000-0002-3282-1319
                http://orcid.org/0000-0002-9141-2347
                http://orcid.org/0000-0001-5475-3583
                http://orcid.org/0000-0002-9217-5240
                Article
                PNTD-D-19-01135
                10.1371/journal.pntd.0007900
                6932831
                31830038
                a282a82d-0df9-4ec0-92ca-6c58462ae759
                © 2019 Domagalska et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 10 July 2019
                : 1 November 2019
                Page count
                Figures: 3, Tables: 1, Pages: 22
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100000780, European Commission;
                Award ID: EC-FP-222895
                Award Recipient :
                Funded by: Belgian Science Policy Office
                Award ID: TROT, P7/41
                Award Recipient :
                Funded by: Flemish Fund for Scientific Research
                Award ID: G0B8112
                Award Recipient :
                Funded by: Department of Economy, Science and Innovation in Flanders
                Award ID: SINGLE project
                Award Recipient :
                Funded by: Welcome
                Award ID: 206194
                Award Recipient :
                This study was supported by the European Commission (EC-FP-222895), Belgian Science Policy Office (TRIT, P7/41), Flemish Fund for Scientific Research (G.0.B81.12), and Department of Economy, Science and Innovation in Flanders ITM-SOFIB (SINGLE project, to J.C.D., M.V.A. and M.A.D.). MB, MJS and JAC are supported by Wellcome via their core support for the Wellcome Sanger Institute (grant 206194). The funders had no role in study design, data collection and analysis, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Eukaryota
                Protozoans
                Parasitic Protozoans
                Leishmania
                Biology and Life Sciences
                Computational Biology
                Genome Analysis
                Genomic Libraries
                Biology and Life Sciences
                Genetics
                Genomics
                Genome Analysis
                Genomic Libraries
                Biology and Life Sciences
                Genetics
                Heredity
                Genetic Mapping
                Variant Genotypes
                Biology and Life Sciences
                Physiology
                Immune Physiology
                Bone Marrow
                Medicine and Health Sciences
                Physiology
                Immune Physiology
                Bone Marrow
                Biology and Life Sciences
                Immunology
                Immune System
                Bone Marrow
                Medicine and Health Sciences
                Immunology
                Immune System
                Bone Marrow
                Biology and Life Sciences
                Organisms
                Eukaryota
                Protozoans
                Parasitic Protozoans
                Leishmania
                Leishmania Donovani
                Biology and Life Sciences
                Genetics
                Molecular Genetics
                Biology and Life Sciences
                Molecular Biology
                Molecular Genetics
                Biology and Life Sciences
                Developmental Biology
                Life Cycles
                Protozoan Life Cycles
                Promastigotes
                Biology and Life Sciences
                Microbiology
                Protozoology
                Protozoan Life Cycles
                Promastigotes
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Genome Sequencing
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Genome Sequencing
                Custom metadata
                vor-update-to-uncorrected-proof
                2019-12-26
                Raw sequencing data was deposited in the European Nucleotide Archive (ENA) with the accession number ERP110990. Not all of the data can be shared publicly due to patient confidentiality concerns, but researchers can make reasonable requests to access data from ITMresearchdataaccess@ 123456itg.be . The rest of the data can be found in the manuscript and supporting information files.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

                Comments

                Comment on this article