Publications by authors named "Thomas D Otto"

109 Publications

Common virulence gene expression in adult first-time infected malaria patients and severe cases.

Elife 2021 Apr 28;10. Epub 2021 Apr 28.

Molecular Biology and Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.

Sequestration of ()-infected erythrocytes to host endothelium through the parasite-derived erythrocyte membrane protein 1 (EMP1) adhesion proteins is central to the development of malaria pathogenesis. EMP1 proteins have diversified and expanded to encompass many sequence variants, conferring each parasite a similar array of human endothelial receptor-binding phenotypes. Here, we analyzed RNA-seq profiles of parasites isolated from 32 infected adult travellers returning to Germany. Patients were categorized into either malaria naive (n = 15) or pre-exposed (n = 17), and into severe (n = 8) or non-severe (n = 24) cases. For differential expression analysis, EMP1-encoding gene transcripts were de novo assembled from RNA-seq data and, in parallel, expressed sequence tags were analyzed and used to predict the encoded domain composition of the transcripts. Both approaches showed in concordance that severe malaria was associated with EMP1 containing the endothelial protein C receptor (EPCR)-binding CIDRα1 domain, whereas CD36-binding EMP1 was linked to non-severe malaria outcomes. First-time infected adults were more likely to develop severe symptoms and tended to be infected for a longer period. Thus, parasites with more pathogenic EMP1 variants are more common in patients with a naive immune status, and/or adverse inflammatory host responses to first infections favor the growth of EPCR-binding parasites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.69040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102065PMC
April 2021

Application of single-cell transcriptomics to kinetoplastid research.

Parasitology 2021 Mar 8:1-14. Epub 2021 Mar 8.

Wellcome Centre for Integrative Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, UK.

Kinetoplastid parasites are responsible for both human and animal diseases across the globe where they have a great impact on health and economic well-being. Many species and life cycle stages are difficult to study due to limitations in isolation and culture, as well as to their existence as heterogeneous populations in hosts and vectors. Single-cell transcriptomics (scRNA-seq) has the capacity to overcome many of these difficulties, and can be leveraged to disentangle heterogeneous populations, highlight genes crucial for propagation through the life cycle, and enable detailed analysis of host–parasite interactions. Here, we provide a review of studies that have applied scRNA-seq to protozoan parasites so far. In addition, we provide an overview of sample preparation and technology choice considerations when planning scRNA-seq experiments, as well as challenges faced when analysing the large amounts of data generated. Finally, we highlight areas of kinetoplastid research that could benefit from scRNA-seq technologies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1017/S003118202100041XDOI Listing
March 2021

Essential Genes of the Parasitic Apicomplexa.

Trends Parasitol 2021 Apr 5;37(4):304-316. Epub 2021 Jan 5.

Center for Global Health and Infectious Diseases and USF Genomics Program, College of Public Health, University of South Florida, 3720 Spectrum Boulevard, Suite 404, Tampa, FL 33612, USA. Electronic address:

Genome-scale mutagenesis screens for genes essential for apicomplexan parasite survival have been completed in three species: Plasmodium falciparum, the major human malaria parasite, Plasmodium berghei, a model rodent malaria parasite, and the more distantly related Toxoplasma gondii, the causative agent of toxoplasmosis. These three species share 2606 single-copy orthologs, 1500 of which have essentiality data in all three screens. In this review, we explore the overlap between these datasets to define the core essential genes of the phylum Apicomplexa. We further discuss the implications of these groundbreaking studies for understanding apicomplexan parasite biology, and we identify promising areas of focus for developing new pan-apicomplexan parasite interventions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pt.2020.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954859PMC
April 2021

Machine learning approaches classify clinical malaria outcomes based on haematological parameters.

BMC Med 2020 11 30;18(1):375. Epub 2020 Nov 30.

Institute of Infection, Immunity & Inflammation, MVLS, University of Glasgow, Glasgow, UK.

Background: Malaria is still a major global health burden, with more than 3.2 billion people in 91 countries remaining at risk of the disease. Accurately distinguishing malaria from other diseases, especially uncomplicated malaria (UM) from non-malarial infections (nMI), remains a challenge. Furthermore, the success of rapid diagnostic tests (RDTs) is threatened by Pfhrp2/3 deletions and decreased sensitivity at low parasitaemia. Analysis of haematological indices can be used to support the identification of possible malaria cases for further diagnosis, especially in travellers returning from endemic areas. As a new application for precision medicine, we aimed to evaluate machine learning (ML) approaches that can accurately classify nMI, UM, and severe malaria (SM) using haematological parameters.

Methods: We obtained haematological data from 2,207 participants collected in Ghana: nMI (n = 978), SM (n = 526), and UM (n = 703). Six different ML approaches were tested, to select the best approach. An artificial neural network (ANN) with three hidden layers was used for multi-classification of UM, SM, and uMI. Binary classifiers were developed to further identify the parameters that can distinguish UM or SM from nMI. Local interpretable model-agnostic explanations (LIME) were used to explain the binary classifiers.

Results: The multi-classification model had greater than 85% training and testing accuracy to distinguish clinical malaria from nMI. To distinguish UM from nMI, our approach identified platelet counts, red blood cell (RBC) counts, lymphocyte counts, and percentages as the top classifiers of UM with 0.801 test accuracy (AUC = 0.866 and F1 score = 0.747). To distinguish SM from nMI, the classifier had a test accuracy of 0.96 (AUC = 0.983 and F1 score = 0.944) with mean platelet volume and mean cell volume being the unique classifiers of SM. Random forest was used to confirm the classifications, and it showed that platelet and RBC counts were the major classifiers of UM, regardless of possible confounders such as patient age and sampling location.

Conclusion: The study provides proof of concept methods that classify UM and SM from nMI, showing that the ML approach is a feasible tool for clinical decision support. In the future, ML approaches could be incorporated into clinical decision-support algorithms for the diagnosis of acute febrile illness and monitoring response to acute SM treatment particularly in endemic settings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12916-020-01823-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702702PMC
November 2020

Increased circulation time of Plasmodium falciparum underlies persistent asymptomatic infection in the dry season.

Nat Med 2020 12 26;26(12):1929-1940. Epub 2020 Oct 26.

Mali International Center of Excellence in Research, University of Sciences, Techniques and Technologies of Bamako, Bamako, Mali.

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquito vectors to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the human host or compromising host survival, is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of individuals with febrile malaria in the transmission season, coinciding with longer circulation within each replicative cycle of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication but rather to increased splenic clearance of longer-circulating infected erythrocytes, which likely maintain parasitemias below clinical and immunological radar. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-020-1084-0DOI Listing
December 2020

Tolerance induction in memory CD4 T cells is partial and reversible.

Immunology 2021 Jan 27;162(1):68-83. Epub 2020 Oct 27.

Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, UK.

Memory T cells respond rapidly in part because they are less reliant on a heightened levels of costimulatory molecules. This enables rapid control of secondary infecting pathogens but presents challenges to efforts to control or silence memory CD4 T cells, for example in antigen-specific tolerance strategies for autoimmunity. We have examined the transcriptional and functional consequences of reactivating memory CD4 T cells in the absence of an adjuvant. We find that memory CD4 T cells generated by infection or immunisation survive secondary activation with antigen delivered without adjuvant, regardless of their location in secondary lymphoid organs or peripheral tissues. These cells were, however, functionally altered following a tertiary immunisation with antigen and adjuvant, proliferating poorly but maintaining their ability to produce inflammatory cytokines. Transcriptional and cell cycle analysis of these memory CD4 T cells suggests they are unable to commit fully to cell division potentially because of low expression of DNA repair enzymes. In contrast, these memory CD4 T cells could proliferate following tertiary reactivation by viral re-infection. These data indicate that antigen-specific tolerogenic strategies must examine multiple parameters of Tcell function, and provide insight into the molecular mechanisms that may lead to deletional tolerance of memory CD4 T cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/imm.13263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730012PMC
January 2021

β2 Integrins differentially regulate γδ T cell subset thymic development and peripheral maintenance.

Proc Natl Acad Sci U S A 2020 09 26;117(36):22367-22377. Epub 2020 Aug 26.

Institute of Infection, Immunity & Inflammation, University of Glasgow, G12 8TA Glasgow, United Kingdom;

The γδ T cells reside predominantly at barrier sites and play essential roles in immune protection against infection and cancer. Despite recent advances in the development of γδ T cell immunotherapy, our understanding of the basic biology of these cells, including how their numbers are regulated in vivo, remains poor. This is particularly true for tissue-resident γδ T cells. We have identified the β family of integrins as regulators of γδ T cells. β-integrin-deficient mice displayed a striking increase in numbers of IL-17-producing Vγ6Vδ1 γδ T cells in the lungs, uterus, and circulation. Thymic development of this population was normal. However, single-cell RNA sequencing revealed the enrichment of genes associated with T cell survival and proliferation specifically in β-integrin-deficient IL-17 cells compared to their wild-type counterparts. Indeed, β-integrin-deficient Vγ6 cells from the lungs showed reduced apoptosis ex vivo, suggesting that increased survival contributes to the accumulation of these cells in β-integrin-deficient tissues. Furthermore, our data revealed an unexpected role for β integrins in promoting the thymic development of the IFNγ-producing CD27 Vγ4 γδ T cell subset. Together, our data reveal that β integrins are important regulators of γδ T cell homeostasis, inhibiting the survival of IL-17-producing Vγ6Vδ1 cells and promoting the thymic development of the IFNγ-producing Vγ4 subset. Our study introduces unprecedented mechanisms of control for γδ T cell subsets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1921930117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486781PMC
September 2020

The Impact of Malaria Parasites on Dendritic Cell-T Cell Interaction.

Front Immunol 2020 24;11:1597. Epub 2020 Jul 24.

Institute of Infection, Immunity & Inflammation, University of Glasgow, Glasgow, United Kingdom.

Malaria is caused by apicomplexan parasites of the genus . While infection continues to pose a risk for the majority of the global population, the burden of disease mainly resides in Sub-Saharan Africa. Although immunity develops against disease, this requires years of persistent exposure and is not associated with protection against infection. Repeat infections occur due to the parasite's ability to disrupt or evade the host immune responses. However, despite many years of study, the mechanisms of this disruption remain unclear. Previous studies have demonstrated a parasite-induced failure in dendritic cell (DCs) function affecting the generation of helper T cell responses. These T cells fail to help B cell responses, reducing the production of antibodies that are necessary to control malaria infection. This review focuses on our current understanding of the effect of parasite on DC function, DC-T cell interaction, and T cell activation. A better understanding of how parasites disrupt DC-T cell interactions will lead to new targets and approaches to reinstate adaptive immune responses and enhance parasite immunity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.01597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393936PMC
April 2021

Testing the impact of a single nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor on experimental cerebral malaria in mice.

Sci Rep 2020 08 12;10(1):13630. Epub 2020 Aug 12.

Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 5625 Fishers Lane, Room 4S04, Rockville, MD, USA.

Cerebral malaria (CM) is the deadliest form of severe Plasmodium infections. Currently, we have limited understanding of the mechanisms by which Plasmodium parasites induce CM. The mouse model of CM, experimental CM (ECM), induced by infection with the rodent parasite, Plasmodium berghei ANKA (PbANKA) has been extensively used to study the pathophysiology of CM. Recent genomic analyses revealed that the coding regions of PbANKA and the closely related Plasmodium berghei NK65 (PbNK65), that does not cause ECM, differ in only 21 single nucleotide polymorphysims (SNPs). Thus, the SNP-containing genes might contribute to the pathogenesis of ECM. Although the majority of these SNPs are located in genes of unknown function, one SNP is located in the DNA binding site of a member of the Plasmodium ApiAP2 transcription factor family, that we recently showed functions as a virulence factor alternating the host's immune response to the parasite. Here, we investigated the impact of this SNP on the development of ECM. Our results using CRISPR-Cas9 engineered parasites indicate that despite its immune modulatory function, the SNP is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific differences in ECM phenotypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-70617-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424516PMC
August 2020

Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.

Nat Med 2020 08 29;26(8):1295-1306. Epub 2020 Jun 29.

Research into Inflammatory Arthritis Centre Versus Arthritis (RACE), .

Immune-regulatory mechanisms of drug-free remission in rheumatoid arthritis (RA) are unknown. We hypothesized that synovial tissue macrophages (STM), which persist in remission, contribute to joint homeostasis. We used single-cell transcriptomics to profile 32,000 STMs and identified phenotypic changes in patients with early/active RA, treatment-refractory/active RA and RA in sustained remission. Each clinical state was characterized by different frequencies of nine discrete phenotypic clusters within four distinct STM subpopulations with diverse homeostatic, regulatory and inflammatory functions. This cellular atlas, combined with deep-phenotypic, spatial and functional analyses of synovial biopsy fluorescent activated cell sorted STMs, revealed two STM subpopulations (MerTKTREM2 and MerTKLYVE1) with unique remission transcriptomic signatures enriched in negative regulators of inflammation. These STMs were potent producers of inflammation-resolving lipid mediators and induced the repair response of synovial fibroblasts in vitro. A low proportion of MerTK STMs in remission was associated with increased risk of disease flare after treatment cessation. Therapeutic modulation of MerTK STM subpopulations could therefore be a potential treatment strategy for RA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-020-0939-8DOI Listing
August 2020

Refining the transcriptome of the human malaria parasite Plasmodium falciparum using amplification-free RNA-seq.

BMC Genomics 2020 Jun 8;21(1):395. Epub 2020 Jun 8.

Department of Biochemistry & Molecular Biology and Huck Center for Malaria Research, Pennsylvania State University, University Park, PA, 16802, USA.

Background: Plasmodium parasites undergo several major developmental transitions during their complex lifecycle, which are enabled by precisely ordered gene expression programs. Transcriptomes from the 48-h blood stages of the major human malaria parasite Plasmodium falciparum have been described using cDNA microarrays and RNA-seq, but these assays have not always performed well within non-coding regions, where the AT-content is often 90-95%.

Results: We developed a directional, amplification-free RNA-seq protocol (DAFT-seq) to reduce bias against AT-rich cDNA, which we have applied to three strains of P. falciparum (3D7, HB3 and IT). While strain-specific differences were detected, overall there is strong conservation between the transcriptional profiles. For the 3D7 reference strain, transcription was detected from 89% of the genome, with over 78% of the genome transcribed into mRNAs. We also find that transcription from bidirectional promoters frequently results in non-coding, antisense transcripts. These datasets allowed us to refine the 5' and 3' untranslated regions (UTRs), which can be variable, long (> 1000 nt), and often overlap those of adjacent transcripts.

Conclusions: The approaches applied in this study allow a refined description of the transcriptional landscape of P. falciparum and demonstrate that very little of the densely packed P. falciparum genome is inactive or redundant. By capturing the 5' and 3' ends of mRNAs, we reveal both constant and dynamic use of transcriptional start sites across the intraerythrocytic developmental cycle that will be useful in guiding the definition of regulatory regions for use in future experimental gene expression studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12864-020-06787-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278070PMC
June 2020

Identification of a conserved var gene in different Plasmodium falciparum strains.

Malar J 2020 May 29;19(1):194. Epub 2020 May 29.

Institute of Tropical Medicine, University of Tuebingen, Wilhelmstr. 27, 72074, Tuebingen, Germany.

Background: The multicopy var gene family of Plasmodium falciparum is of crucial importance for pathogenesis and antigenic variation. So far only var2csa, the var gene responsible for placental malaria, was found to be highly conserved among all P. falciparum strains. Here, a new conserved 3D7 var gene (PF3D7_0617400) is identified in several field isolates.

Methods: DNA sequencing, transcriptional analysis, Cluster of Differentiation (CD) 36-receptor binding, indirect immunofluorescence with PF3D7_0617400-antibodies and quantification of surface reactivity against semi-immune sera were used to characterize an NF54 clone and a Gabonese field isolate clone (MOA C3) transcribing the gene. A population of 714 whole genome sequenced parasites was analysed to characterize the conservation of the locus in African and Asian isolates. The genetic diversity of two var2csa fragments was compared with the genetic diversity of 57 microsatellites fragments in field isolates.

Results: PFGA01_060022400 was identified in a Gabonese parasite isolate (MOA) from a chronic infection and found to be 99% identical with PF3D7_0617400 of the 3D7 genome strain. Transcriptional analysis and immunofluorescence showed expression of the gene in an NF54 and a MOA clone but CD36 binding assays and surface reactivity to semi-immune sera differed markedly in the two clones. Long-read Pacific bioscience whole genome sequencing showed that PFGA01_060022400 is located in the internal cluster of chromosome 6. The full length PFGA01_060022400 was detected in 36 of 714 P. falciparum isolates and 500 bp fragments were identified in more than 100 isolates. var2csa was in parts highly conserved (H = 0) but in other parts as variable (H = 0.86) as the 57 microsatellites markers (H = 0.8).

Conclusions: Individual var gene sequences exhibit conservation in the global parasite population suggesting that purifying selection may limit overall genetic diversity of some var genes. Notably, field and laboratory isolates expressing the same var gene exhibit markedly different phenotypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12936-020-03257-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260770PMC
May 2020

Evolutionary analysis of the most polymorphic gene family in malaria.

Wellcome Open Res 2019 3;4:193. Epub 2019 Dec 3.

Parasite Genetics, Wellcome Trust Sanger Institute, Hinxton, UK.

The gene family of the human malaria parasite encode proteins that are crucial determinants of both pathogenesis and immune evasion and are highly polymorphic. Here we have assembled nearly complete gene repertoires from 2398 field isolates and analysed a normalised set of 714 from across 12 countries. This therefore represents the first large scale attempt to catalogue the worldwide distribution of gene sequences We confirm the extreme polymorphism of this gene family but also demonstrate an unexpected level of sequence sharing both within and between continents. We show that this is likely due to both the remnants of selective sweeps as well as a worrying degree of recent gene flow across continents with implications for the spread of drug resistance. We also address the evolution of the repertoire with respect to the ancestral genes within the and show that diversity generated by recombination is concentrated in a number of hotspots. An analysis of the subdomain structure indicates that some existing definitions may need to be revised From the analysis of this data, we can now understand the way in which the family has evolved and how the diversity is continuously being generated. Finally, we demonstrate that because the genes are distributed across the genome, sequence sharing between genotypes acts as a useful population genetic marker.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12688/wellcomeopenres.15590.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001760PMC
December 2019

Transcriptional and genomic parallels between the monoxenous parasite Herpetomonas muscarum and Leishmania.

PLoS Genet 2019 11 11;15(11):e1008452. Epub 2019 Nov 11.

Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

Trypanosomatid parasites are causative agents of important human and animal diseases such as sleeping sickness and leishmaniasis. Most trypanosomatids are transmitted to their mammalian hosts by insects, often belonging to Diptera (or true flies). These are called dixenous trypanosomatids since they infect two different hosts, in contrast to those that infect just insects (monoxenous). However, it is still unclear whether dixenous and monoxenous trypanosomatids interact similarly with their insect host, as fly-monoxenous trypanosomatid interaction systems are rarely reported and under-studied-despite being common in nature. Here we present the genome of monoxenous trypanosomatid Herpetomonas muscarum and discuss its transcriptome during in vitro culture and during infection of its natural insect host Drosophila melanogaster. The H. muscarum genome is broadly syntenic with that of human parasite Leishmania major. We also found strong similarities between the H. muscarum transcriptome during fruit fly infection, and those of Leishmania during sand fly infections. Overall this suggests Drosophila-Herpetomonas is a suitable model for less accessible insect-trypanosomatid host-parasite systems such as sand fly-Leishmania.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1008452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6872171PMC
November 2019

Modelling pathogen load dynamics to elucidate mechanistic determinants of host-Plasmodium falciparum interactions.

Nat Microbiol 2019 09 17;4(9):1592-1602. Epub 2019 Jun 17.

Section of Paediatrics, Imperial College, London, UK.

During infection, increasing pathogen load stimulates both protective and harmful aspects of the host response. The dynamics of this interaction are hard to quantify in humans, but doing so could improve understanding of the mechanisms of disease and protection. We sought to model the contributions of the parasite multiplication rate and host response to observed parasite load in individual subjects infected with Plasmodium falciparum malaria, using only data obtained at the time of clinical presentation, and then to identify their mechanistic correlates. We predicted higher parasite multiplication rates and lower host responsiveness in cases of severe malaria, with severe anaemia being more insidious than cerebral malaria. We predicted that parasite-growth inhibition was associated with platelet consumption, lower expression of CXCL10 and type 1 interferon-associated genes, but increased cathepsin G and matrix metallopeptidase 9 expression. We found that cathepsin G and matrix metallopeptidase 9 directly inhibit parasite invasion into erythrocytes. The parasite multiplication rate was associated with host iron availability and higher complement factor H levels, lower expression of gametocyte-associated genes but higher expression of translation-associated genes in the parasite. Our findings demonstrate the potential of using explicit modelling of pathogen load dynamics to deepen understanding of host-pathogen interactions and identify mechanistic correlates of protection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-019-0474-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6708439PMC
September 2019

Progression of the canonical reference malaria parasite genome from 2002-2019.

Wellcome Open Res 2019 28;4:58. Epub 2019 May 28.

Parasite Genomics, Wellcome Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK.

Here we describe the ways in which the sequence and annotation of the reference genome has changed since its publication in 2002. As the malaria species responsible for the most deaths worldwide, the richness of annotation and accuracy of the sequence are important resources for the research community as well as the basis for interpreting the genomes of subsequently sequenced species. At the time of publication in 2002 over 60% of predicted genes had unknown functions. As of March 2019, this number has been significantly decreased to 33%. The reduction is due to the inclusion of genes that were subsequently characterised experimentally and genes with significant similarity to others with known functions. In addition, the structural annotation of genes has been significantly refined; 27% of gene structures have been changed since 2002, comprising changes in exon-intron boundaries, addition or deletion of exons and the addition or deletion of genes. The sequence has also undergone significant improvements. In addition to the correction of a large number of single-base and insertion or deletion errors, a major miss-assembly between the subtelomeres of chromosome 7 and 8 has been corrected. As the number of sequenced isolates continues to grow rapidly, a single reference genome will not be an adequate basis for interpreting intra-species sequence diversity. We therefore describe in this publication a population reference genome of , called Pfref1. This reference will enable the community to map to regions that are not present in the current assembly. 3D7 will continue to be maintained, with ongoing curation ensuring continual improvements in annotation quality.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12688/wellcomeopenres.15194.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484455PMC
May 2019

An Experimental Human Blood-Stage Model for Studying Plasmodium malariae Infection.

J Infect Dis 2020 03;221(6):948-955

QIMR Berghofer Medical Research Institute.

Background: Plasmodium malariae is considered a minor malaria parasite, although its global disease burden is underappreciated. The aim of this study was to develop an induced blood-stage malaria (IBSM) model of P. malariae to study parasite biology, diagnostic assays, and treatment.

Methods: This clinical trial involved 2 healthy subjects who were intravenously inoculated with cryopreserved P. malariae-infected erythrocytes. Subjects were treated with artemether-lumefantrine after development of clinical symptoms. Prior to antimalarial therapy, mosquito-feeding assays were performed to investigate transmission, and blood samples were collected for rapid diagnostic testing and parasite transcription profiling. Serial blood samples were collected for biomarker analysis.

Results: Both subjects experienced symptoms and signs typical of early malaria. Parasitemia was detected 7 days after inoculation, and parasite concentrations increased until antimalarial treatment was initiated 25 and 21 days after inoculation for subjects 1 and 2 respectively (peak parasitemia levels, 174 182 and 50 291 parasites/mL, respectively). The parasite clearance half-life following artemether-lumefantrine treatment was 6.7 hours. Mosquito transmission was observed for 1 subject, while in vivo parasite transcription and biomarkers were successfully profiled.

Conclusions: An IBSM model of P. malariae has been successfully developed and may be used to study the biology of, diagnostic testing for, and treatment of this neglected malaria species.

Clinical Trials Registration: ACTRN12617000048381.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/infdis/jiz102DOI Listing
March 2020

Integrated transcriptomic and proteomic analysis of pathogenic mycobacteria and their esx-1 mutants reveal secretion-dependent regulation of ESX-1 substrates and WhiB6 as a transcriptional regulator.

PLoS One 2019 23;14(1):e0211003. Epub 2019 Jan 23.

Pathogen Genomics Laboratory, BESE Division, King Abdullah University of Science and Technology (KAUST), Thuwal-Jeddah, Kingdom of Saudi Arabia.

The mycobacterial type VII secretion system ESX-1 is responsible for the secretion of a number of proteins that play important roles during host infection. The regulation of the expression of secreted proteins is often essential to establish successful infection. Using transcriptome sequencing, we found that the abrogation of ESX-1 function in Mycobacterium marinum leads to a pronounced increase in gene expression levels of the espA operon during the infection of macrophages. In addition, the disruption of ESX-1-mediated protein secretion also leads to a specific down-regulation of the ESX-1 substrates, but not of the structural components of this system, during growth in culture medium. This effect is observed in both M. marinum and M. tuberculosis. We established that down-regulation of ESX-1 substrates is the result of a regulatory process that is influenced by the putative transcriptional regulator whib6, which is located adjacent to the esx-1 locus. In addition, the overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in a significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Taken together, these data show that WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates and that ESX-1 substrates are regulated independently from the structural components, both during infection and as a result of active secretion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0211003PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343904PMC
October 2019

Low sulfated heparins target multiple proteins for central nervous system repair.

Glia 2019 04 26;67(4):668-687. Epub 2018 Dec 26.

Institute of Infection, Immunity, and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.

The lack of endogenous repair following spinal cord injury (SCI) accounts for the frequent permanent deficits for which effective treatments are absent. Previously, we demonstrated that low sulfated modified heparin mimetics (LS-mHeps) attenuate astrocytosis, suggesting they may represent a novel therapeutic approach. mHeps are glycomolecules with structural similarities to resident heparan sulfates (HS), which modulate cell signaling by both sequestering ligands, and acting as cofactors in the formation of ligand-receptor complexes. To explore whether mHeps can affect the myelination and neurite outgrowth necessary for repair after SCI, we created lesioned or demyelinated neural cell co-cultures and exposed them with a panel of mHeps with varying degrees and positions of their sulfate moieties. LS-mHep7 enhanced neurite outgrowth and myelination, whereas highly sulfated mHeps (HS-mHeps) had attenuating effects. LS-mHeps had no effects on myelination or neurite extension in developing, uninjured myelinating cultures, suggesting they might exert their proregenerating effects by modulating or sequestering inhibitory factors secreted after injury. To investigate this, we examined conditioned media from cultures using chemokine arrays and conducted an unbiased proteomics approach by applying TMT-LC/MS to mHep7 affinity purified conditioned media from these cultures. Multiple protein factors reported to play a role in damage or repair mechanisms were identified, including amyloid betaA4. Amyloid beta peptide (1-42) was validated as an important candidate by treating myelination cultures and shown to inhibit myelination. Thus, we propose that LS-mHeps exert multiple beneficial effects on mechanisms supporting enhanced repair, and represent novel candidates as therapeutics for CNS damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/glia.23562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492281PMC
April 2019

Schizont transcriptome variation among clinical isolates and laboratory-adapted clones of the malaria parasite Plasmodium falciparum.

BMC Genomics 2018 Dec 10;19(1):894. Epub 2018 Dec 10.

Pathogen Molecular Biology Department, London School of Hygiene and Tropical Medicine, London, UK.

Background: Malaria parasites are genetically polymorphic and phenotypically plastic. In studying transcriptome variation among parasites from different infections, it is challenging to overcome potentially confounding technical and biological variation between samples. We investigate variation in the major human parasite Plasmodium falciparum, generating RNA-seq data on multiple independent replicate sample preparations of merozoite-containing intra-erythrocytic schizonts from a panel of clinical isolates and from long-term laboratory-adapted clones, with a goal of robustly identifying differentially expressed genes.

Results: Analysis of biological sample replicates shows that increased numbers improve the true discovery rate of differentially expressed genes, and that six independent replicates of each parasite line allowed identification of most differences that could be detected with larger numbers. For highly expressed genes, focusing on the top quartile at schizont stages, there was more power to detect differences. Comparing cultured clinical isolates and laboratory-adapted clones, genes more highly expressed in the laboratory-adapted clones include those encoding an AP2 transcription factor (PF3D7_0420300), a ubiquitin-binding protein and two putative methyl transferases. In contrast, higher expression in clinical isolates was seen for the merozoite surface protein gene dblmsp2, proposed to be a marker of schizonts forming merozoites committed to sexual differentiation. Variable expression was extremely strongly, but not exclusively, associated with genes known to be targeted by Heterochromatin Protein 1. Clinical isolates show variable expression of several known merozoite invasion ligands, as well as other genes for which new RT-qPCR assays validate the quantitation and allow characterisation in samples with more limited material. Expression levels of these genes vary among schizont preparations of different clinical isolates in the first ex vivo cycle in patient erythrocytes, but mean levels are similar to those in continuously cultured clinical isolates.

Conclusions: Analysis of multiple biological sample replicates greatly improves identification of genes variably expressed between different cultured parasite lines. Clinical isolates recently established in culture show differences from long-term adapted clones in transcript levels of particular genes, and are suitable for analyses requiring biological replicates to understand parasite phenotypes and variable expression likely to be relevant in nature.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12864-018-5257-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6288915PMC
December 2018

Pre-clinical evaluation of a -based whole-sporozoite malaria vaccine candidate.

NPJ Vaccines 2018 27;3:54. Epub 2018 Nov 27.

1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal.

Whole-sporozoite vaccination/immunization induces high levels of protective immunity in both rodent models of malaria and in humans. Recently, we generated a transgenic line of the rodent malaria parasite () that expresses the () circumsporozoite protein (CS), and showed that this parasite line (Vac) was capable of (1) infecting and developing in human hepatocytes but not in human erythrocytes, and (2) inducing neutralizing antibodies against the human parasite. Here, we analyzed Vac in detail and developed tools necessary for its use in clinical studies. A microbiological contaminant-free Master Cell Bank of Vac parasites was generated through a process of cyclic propagation and clonal expansion in mice and mosquitoes and was genetically characterized. A highly sensitive qRT-PCR-based method was established that enables Vac parasite detection and quantification at low parasite densities in vivo. This method was employed in a biodistribution study in a rabbit model, revealing that the parasite is only present at the site of administration and in the liver up to 48 h post infection and is no longer detectable at any site 10 days after administration. An extensive toxicology investigation carried out in rabbits further showed the absence of Vac-related toxicity. In vivo drug sensitivity assays employing rodent models of infection showed that both the liver and the blood stage forms of Vac were completely eliminated by Malarone treatment. Collectively, our pre-clinical safety assessment demonstrates that Vac possesses all characteristics necessary to advance into clinical evaluation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41541-018-0091-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258718PMC
November 2018

A forward genetic screen reveals a primary role for Plasmodium falciparum Reticulocyte Binding Protein Homologue 2a and 2b in determining alternative erythrocyte invasion pathways.

PLoS Pathog 2018 11 29;14(11):e1007436. Epub 2018 Nov 29.

Malaria Programme, Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom.

Invasion of human erythrocytes is essential for Plasmodium falciparum parasite survival and pathogenesis, and is also a complex phenotype. While some later steps in invasion appear to be invariant and essential, the earlier steps of recognition are controlled by a series of redundant, and only partially understood, receptor-ligand interactions. Reverse genetic analysis of laboratory adapted strains has identified multiple genes that when deleted can alter invasion, but how the relative contributions of each gene translate to the phenotypes of clinical isolates is far from clear. We used a forward genetic approach to identify genes responsible for variable erythrocyte invasion by phenotyping the parents and progeny of previously generated experimental genetic crosses. Linkage analysis using whole genome sequencing data revealed a single major locus was responsible for the majority of phenotypic variation in two invasion pathways. This locus contained the PfRh2a and PfRh2b genes, members of one of the major invasion ligand gene families, but not widely thought to play such a prominent role in specifying invasion phenotypes. Variation in invasion pathways was linked to significant differences in PfRh2a and PfRh2b expression between parasite lines, and their role in specifying alternative invasion was confirmed by CRISPR-Cas9-mediated genome editing. Expansion of the analysis to a large set of clinical P. falciparum isolates revealed common deletions, suggesting that variation at this locus is a major cause of invasion phenotypic variation in the endemic setting. This work has implications for blood-stage vaccine development and will help inform the design and location of future large-scale studies of invasion in clinical isolates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1007436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289454PMC
November 2018

Multi-population genomic analysis of malaria parasites indicates local selection and differentiation at the gdv1 locus regulating sexual development.

Sci Rep 2018 10 25;8(1):15763. Epub 2018 Oct 25.

Pathogen Molecular Biology Department, London School of Hygiene and Tropical Medicine, Keppel St, London, UK.

Parasites infect hosts in widely varying environments, encountering diverse challenges for adaptation. To identify malaria parasite genes under locally divergent selection across a large endemic region with a wide spectrum of transmission intensity, genome sequences were obtained from 284 clinical Plasmodium falciparum infections from four newly sampled locations in Senegal, The Gambia, Mali and Guinea. Combining these with previous data from seven other sites in West Africa enabled a multi-population analysis to identify discrete loci under varying local selection. A genome-wide scan showed the most exceptional geographical divergence to be at the early gametocyte gene locus gdv1 which is essential for parasite sexual development and transmission. We identified a major structural dimorphism with alternative 1.5 kb and 1.0 kb sequence deletions at different positions of the 3'-intergenic region, in tight linkage disequilibrium with the most highly differentiated single nucleotide polymorphism, one of the alleles being very frequent in Senegal and The Gambia but rare in the other locations. Long non-coding RNA transcripts were previously shown to include the entire antisense of the gdv1 coding sequence and the portion of the intergenic region with allelic deletions, suggesting adaptive regulation of parasite sexual development and transmission in response to local conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-34078-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202401PMC
October 2018

Whole genome sequencing and microsatellite analysis of the Plasmodium falciparum E5 NF54 strain show that the var, rifin and stevor gene families follow Mendelian inheritance.

Malar J 2018 Oct 22;17(1):376. Epub 2018 Oct 22.

Institute of Tropical Medicine, University of Tuebingen, Wilhelmstr. 27, 72074, Tuebingen, Germany.

Background: Plasmodium falciparum exhibits a high degree of inter-isolate genetic diversity in its variant surface antigen (VSA) families: P. falciparum erythrocyte membrane protein 1, repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR). The role of recombination for the generation of this diversity is a subject of ongoing research. Here the genome of E5, a sibling of the 3D7 genome strain is presented. Short and long read whole genome sequencing (WGS) techniques (Ilumina, Pacific Bioscience) and a set of 84 microsatellites (MS) were employed to characterize the 3D7 and non-3D7 parts of the E5 genome. This is the first time that VSA genes in sibling parasites were analysed with long read sequencing technology.

Results: Of the 5733 E5 genes only 278 genes, mostly var and rifin/stevor genes, had no orthologues in the 3D7 genome. WGS and MS analysis revealed that chromosomal crossovers occurred at a rate of 0-3 per chromosome. var, stevor and rifin genes were inherited within the respective non-3D7 or 3D7 chromosomal context. 54 of the 84 MS PCR fragments correctly identified the respective MS as 3D7- or non-3D7 and this correlated with var and rifin/stevor gene inheritance in the adjacent chromosomal regions. E5 had 61 var and 189 rifin/stevor genes. One large non-chromosomal recombination event resulted in a new var gene on chromosome 14. The remainder of the E5 3D7-type subtelomeric and central regions were identical to 3D7.

Conclusions: The data show that the rifin/stevor and var gene families represent the most diverse compartments of the P. falciparum genome but that the majority of var genes are inherited without alterations within their respective parental chromosomal context. Furthermore, MS genotyping with 54 MS can successfully distinguish between two sibling progeny of a natural P. falciparum cross and thus can be used to investigate identity by descent in field isolates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12936-018-2503-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6198375PMC
October 2018

Genome organization and DNA accessibility control antigenic variation in trypanosomes.

Nature 2018 11 17;563(7729):121-125. Epub 2018 Oct 17.

Department of Veterinary Sciences, Experimental Parasitology, Ludwig-Maximilians-Universität München, Munich, Germany.

Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses-Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing-that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0619-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784898PMC
November 2018

Plasmodium vivax-like genome sequences shed new insights into Plasmodium vivax biology and evolution.

PLoS Biol 2018 08 24;16(8):e2006035. Epub 2018 Aug 24.

MIVEGEC, IRD, CNRS, University of Montpellier, Montpellier, France.

Although Plasmodium vivax is responsible for the majority of malaria infections outside Africa, little is known about its evolution and pathway to humans. Its closest genetic relative, P. vivax-like, was discovered in African great apes and is hypothesized to have given rise to P. vivax in humans. To unravel the evolutionary history and adaptation of P. vivax to different host environments, we generated using long- and short-read sequence technologies 2 new P. vivax-like reference genomes and 9 additional P. vivax-like genotypes. Analyses show that the genomes of P. vivax and P. vivax-like are highly similar and colinear within the core regions. Phylogenetic analyses clearly show that P. vivax-like parasites form a genetically distinct clade from P. vivax. Concerning the relative divergence dating, we show that the evolution of P. vivax in humans did not occur at the same time as the other agents of human malaria, thus suggesting that the transfer of Plasmodium parasites to humans happened several times independently over the history of the Homo genus. We further identify several key genes that exhibit signatures of positive selection exclusively in the human P. vivax parasites. Two of these genes have been identified to also be under positive selection in the other main human malaria agent, P. falciparum, thus suggesting their key role in the evolution of the ability of these parasites to infect humans or their anthropophilic vectors. Finally, we demonstrate that some gene families important for red blood cell (RBC) invasion (a key step of the life cycle of these parasites) have undergone lineage-specific evolution in the human parasite (e.g., reticulocyte-binding proteins [RBPs]).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.2006035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130868PMC
August 2018

Cyclin-dependent kinase 12 is a drug target for visceral leishmaniasis.

Nature 2018 08 25;560(7717):192-197. Epub 2018 Jul 25.

Global Health R&D, GlaxoSmithKline, Tres Cantos, Spain.

Visceral leishmaniasis causes considerable mortality and morbidity in many parts of the world. There is an urgent need for the development of new, effective treatments for this disease. Here we describe the development of an anti-leishmanial drug-like chemical series based on a pyrazolopyrimidine scaffold. The leading compound from this series (7, DDD853651/GSK3186899) is efficacious in a mouse model of visceral leishmaniasis, has suitable physicochemical, pharmacokinetic and toxicological properties for further development, and has been declared a preclinical candidate. Detailed mode-of-action studies indicate that compounds from this series act principally by inhibiting the parasite cdc-2-related kinase 12 (CRK12), thus defining a druggable target for visceral leishmaniasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0356-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402543PMC
August 2018

Integrated pathogen load and dual transcriptome analysis of systemic host-pathogen interactions in severe malaria.

Sci Transl Med 2018 06;10(447)

Section of Paediatrics, Imperial College, London W2 1PG, UK.

The pathogenesis of infectious diseases depends on the interaction of host and pathogen. In malaria, host and parasite processes can be assessed by dual RNA sequencing of blood from infected patients. We performed dual transcriptome analyses on samples from 46 malaria-infected Gambian children to reveal mechanisms driving the systemic pathophysiology of severe malaria. Integrating these transcriptomic data with estimates of parasite load and detailed clinical information allowed consideration of potentially confounding effects due to differing leukocyte proportions in blood, parasite developmental stage, and whole-body pathogen load. We report hundreds of human and parasite genes differentially expressed between severe and uncomplicated malaria, with distinct profiles associated with coma, hyperlactatemia, and thrombocytopenia. High expression of neutrophil granule-related genes was consistently associated with all severe malaria phenotypes. We observed severity-associated variation in the expression of parasite genes, which determine cytoadhesion to vascular endothelium, rigidity of infected erythrocytes, and parasite growth rate. Up to 99% of human differential gene expression in severe malaria was driven by differences in parasite load, whereas parasite gene expression showed little association with parasite load. Coexpression analyses revealed interactions between human and , with prominent co-regulation of translation genes in severe malaria between host and parasite. Multivariate analyses suggested that increased expression of granulopoiesis and interferon-γ-related genes, together with inadequate suppression of type 1 interferon signaling, best explained severity of infection. These findings provide a framework for understanding the contributions of host and parasite to the pathogenesis of severe malaria and identifying new treatments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aar3619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326353PMC
June 2018

Long read assemblies of geographically dispersed isolates reveal highly structured subtelomeres.

Wellcome Open Res 2018 3;3:52. Epub 2018 May 3.

Wellcome Sanger Institute, Hinxton, UK.

: Although thousands of clinical isolates of are being sequenced and analysed by short read technology, the data do not resolve the highly variable subtelomeric regions of the genomes that contain polymorphic gene families involved in immune evasion and pathogenesis. There is also no current standard definition of the boundaries of these variable subtelomeric regions. : Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated the genomes of 15 isolates, ten of which are newly cultured clinical isolates. We performed comparative analysis of the entire genome with particular emphasis on the subtelomeric regions and the internal genes clusters.   : The nearly complete sequence of these 15 isolates has enabled us to define a highly conserved core genome, to delineate the boundaries of the subtelomeric regions, and to compare these across isolates. We found highly structured variable regions in the genome. Some exported gene families purportedly involved in release of merozoites show copy number variation. As an example of ongoing genome evolution, we found a novel CLAG gene in six isolates.  We also found a novel gene that was relatively enriched in the South East Asian isolates compared to those from Africa. : These 15 manually curated new reference genome sequences with their nearly complete subtelomeric regions and fully assembled genes are an important new resource for the malaria research community. We report the overall conserved structure and pattern of important gene families and the more clearly defined subtelomeric regions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12688/wellcomeopenres.14571.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964635PMC
May 2018