Publications by authors named "Mandy Sanders"

93 Publications

Geographic Origin and Vertical Transmission of Leishmania infantum Parasites in Hunting Hounds, United States.

Emerg Infect Dis 2022 Jun;28(6):1211-1223

Vertical transmission of leishmaniasis is common but is difficult to study against the background of pervasive vector transmission. We present genomic data from dogs in the United States infected with Leishmania infantum parasites; these infections have persisted in the apparent absence of vector transmission. We demonstrate that these parasites were introduced from the Old World separately and more recently than L. infantum from South America. The parasite population shows unusual genetics consistent with a lack of meiosis: a high level of heterozygous sites shared across all isolates and no decrease in linkage with genomic distance between variants. Our data confirm that this parasite population has been evolving with little or no sexual reproduction. This demonstration of vertical transmission has profound implications for the population genetics of Leishmania parasites. When investigating transmission in complex natural settings, considering vertical transmission alongside vector transmission is vital.
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http://dx.doi.org/10.3201/eid2806.211746DOI Listing
June 2022

Defining the early stages of intestinal colonisation by whipworms.

Nat Commun 2022 04 1;13(1):1725. Epub 2022 Apr 1.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, CB10 1SA, UK.

Whipworms are large metazoan parasites that inhabit multi-intracellular epithelial tunnels in the large intestine of their hosts, causing chronic disease in humans and other mammals. How first-stage larvae invade host epithelia and establish infection remains unclear. Here we investigate early infection events using both Trichuris muris infections of mice and murine caecaloids, the first in-vitro system for whipworm infection and organoid model for live helminths. We show that larvae degrade mucus layers to access epithelial cells. In early syncytial tunnels, larvae are completely intracellular, woven through multiple live dividing cells. Using single-cell RNA sequencing of infected mouse caecum, we reveal that progression of infection results in cell damage and an expansion of enterocytes expressing of Isg15, potentially instigating the host immune response to the whipworm and tissue repair. Our results unravel intestinal epithelium invasion by whipworms and reveal specific host-parasite interactions that allow the whipworm to establish its multi-intracellular niche.
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http://dx.doi.org/10.1038/s41467-022-29334-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8976045PMC
April 2022

Genomic and Phenotypic Characterization of Experimentally Selected Resistant Leishmania donovani Reveals a Role for Dynamin-1-Like Protein in the Mechanism of Resistance to a Novel Antileishmanial Compound.

mBio 2022 Jan 11:e0326421. Epub 2022 Jan 11.

Molecular Parasitology Unit, Institute of Tropical Medicine, Antwerp, Belgium.

The implementation of prospective drug resistance (DR) studies in the research-and-development (R&D) pipeline is a common practice for many infectious diseases but not for neglected tropical diseases (NTDs). Here, we explored and demonstrated the importance of this approach using as paradigms Leishmania donovani, the etiological agent of visceral leishmaniasis (VL), and TCMDC-143345, a promising compound of the GlaxoSmithKline (GSK) "Leishbox" to treat VL. We experimentally selected resistance to TCMDC-143345 and characterized resistant parasites at the genomic and phenotypic levels. We found that it took more time to develop resistance to TCMDC-143345 than to other drugs in clinical use and that there was no cross-resistance to these drugs, suggesting a new and unique mechanism. By whole-genome sequencing, we found two mutations in the gene encoding the L. donovani dynamin-1-like protein (LdoDLP1) that were fixed at the highest drug pressure. Through phylogenetic analysis, we identified LdoDLP1 as a family member of the dynamin-related proteins, a group of proteins that impacts the shapes of biological membranes by mediating fusion and fission events, with a putative role in mitochondrial fission. We found that L. donovani lines genetically engineered to harbor the two identified LdoDLP1 mutations were resistant to TCMDC-143345 and displayed altered mitochondrial properties. By homology modeling, we showed how the two LdoDLP1 mutations may influence protein structure and function. Taken together, our data reveal a clear involvement of LdoDLP1 in the adaptation/reduced susceptibility of L. donovani to TCMDC-143345. Humans and their pathogens are continuously locked in a molecular arms race during which the eventual emergence of pathogen drug resistance (DR) seems inevitable. For neglected tropical diseases (NTDs), DR is generally studied retrospectively once it has already been established in clinical settings. We previously recommended to keep one step ahead in the host-pathogen arms race and implement prospective DR studies in the R&D pipeline, a common practice for many infectious diseases but not for NTDs. Here, using Leishmania donovani, the etiological agent of visceral leishmaniasis (VL), and TCMDC-143345, a promising compound of the GSK Leishbox to treat VL, as paradigms, we experimentally selected resistance to the compound and proceeded to genomic and phenotypic characterization of DR parasites. The results gathered in the present study suggest a new DR mechanism involving the L. donovani dynamin-1-like protein (LdoDLP1) and demonstrate the practical relevance of prospective DR studies.
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http://dx.doi.org/10.1128/mbio.03264-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8749414PMC
January 2022

Daily rhythms in gene expression of the human parasite Schistosoma mansoni.

BMC Biol 2021 12 2;19(1):255. Epub 2021 Dec 2.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, UK.

Background: The consequences of the earth's daily rotation have led to 24-h biological rhythms in most organisms. Even some parasites are known to have daily rhythms, which, when in synchrony with host rhythms, can optimise their fitness. Understanding these rhythms may enable the development of control strategies that take advantage of rhythmic vulnerabilities. Recent work on protozoan parasites has revealed 24-h rhythms in gene expression, drug sensitivity and the presence of an intrinsic circadian clock; however, similar studies on metazoan parasites are lacking. To address this, we investigated if a metazoan parasite has daily molecular oscillations, whether they reveal how these longer-lived organisms can survive host daily cycles over a lifespan of many years and if animal circadian clock genes are present and rhythmic. We addressed these questions using the human blood fluke Schistosoma mansoni that lives in the vasculature for decades and causes the tropical disease schistosomiasis.

Results: Using round-the-clock transcriptomics of male and female adult worms collected from experimentally infected mice, we discovered that ~ 2% of its genes followed a daily pattern of expression. Rhythmic processes included a stress response during the host's active phase and a 'peak in metabolic activity' during the host's resting phase. Transcriptional profiles in the female reproductive system were mirrored by daily patterns in egg laying (eggs are the main drivers of the host pathology). Genes cycling with the highest amplitudes include predicted drug targets and a vaccine candidate. These 24-h rhythms may be driven by host rhythms and/or generated by a circadian clock; however, orthologs of core clock genes are missing and secondary clock genes show no 24-h rhythmicity.

Conclusions: There are daily rhythms in the transcriptomes of adult S. mansoni, but they appear less pronounced than in other organisms. The rhythms reveal temporally compartmentalised internal processes and host interactions relevant to within-host survival and between-host transmission. Our findings suggest that if these daily rhythms are generated by an intrinsic circadian clock then the oscillatory mechanism must be distinct from that in other animals. We have shown which transcripts oscillate at this temporal scale and this will benefit the development and delivery of treatments against schistosomiasis.
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http://dx.doi.org/10.1186/s12915-021-01189-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8638415PMC
December 2021

Long-read assembly and comparative evidence-based reanalysis of genome sequences reveal expanded transporter repertoire and duplication of entire chromosome ends including subtelomeric regions.

Genome Res 2022 01 11;32(1):203-213. Epub 2021 Nov 11.

Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia 30602, USA.

Cryptosporidiosis is a leading cause of waterborne diarrheal disease globally and an important contributor to mortality in infants and the immunosuppressed. Despite its importance, the community has only had access to a good, but incomplete, IOWA reference genome sequence. Incomplete reference sequences hamper annotation, experimental design, and interpretation. We have generated a new IOWA genome assembly supported by Pacific Biosciences (PacBio) and Oxford Nanopore long-read technologies and a new comparative and consistent genome annotation for three closely related species: , , and We made 1926 annotation updates based on experimental evidence. They include new transporters, ncRNAs, introns, and altered gene structures. The new assembly and annotation revealed a complete methylase ortholog. Comparative annotation between , , and revealed that most "missing" orthologs are found, suggesting that the biological differences between the species must result from gene copy number variation, differences in gene regulation, and single-nucleotide variants (SNVs). Using the new assembly and annotation as reference, 190 genes are identified as evolving under positive selection, including many not detected previously. The new IOWA reference genome assembly is larger, gap free, and lacks ambiguous bases. This chromosomal assembly recovers all 16 chromosome ends, 13 of which are contiguously assembled. The three remaining chromosome ends are provisionally placed. These ends represent duplication of entire chromosome ends including subtelomeric regions revealing a new level of genome plasticity that will both inform and impact future research.
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http://dx.doi.org/10.1101/gr.275325.121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8744675PMC
January 2022

Endosymbiont Capture, a Repeated Process of Endosymbiont Transfer with Replacement in Trypanosomatids spp.

Pathogens 2021 Jun 4;10(6). Epub 2021 Jun 4.

Life Science Research Centre, Faculty of Science, University of Ostrava, 710 00 Ostrava, Czech Republic.

Trypanosomatids of the subfamily Strigomonadinae bear permanent intracellular bacterial symbionts acquired by the common ancestor of these flagellates. However, the cospeciation pattern inherent to such relationships was revealed to be broken upon the description of , which is sister to , but bears an endosymbiont genetically close to that of . Based on phylogenetic inferences, it was proposed that the bacterium from had been horizontally transferred to . Here, we sequenced the bacterial genomes from two isolates, including a new one from Papua New Guinea, and compared them with the published genome of the endosymbiont, revealing differences below the interspecific level. Our phylogenetic analyses confirmed that the endosymbionts of were obtained from and, in addition, demonstrated that this occurred more than once. We propose that coinfection of the same blowfly host and the phylogenetic relatedness of the trypanosomatids facilitate such transitions, whereas the drastic difference in the occurrence of the two trypanosomatid species determines the observed direction of this process. This phenomenon is analogous to organelle (mitochondrion/plastid) capture described in multicellular organisms and, thereafter, we name it endosymbiont capture.
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http://dx.doi.org/10.3390/pathogens10060702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8229890PMC
June 2021

Diversity and Within-Host Evolution of Leishmania donovani from Visceral Leishmaniasis Patients with and without HIV Coinfection in Northern Ethiopia.

mBio 2021 06 29;12(3):e0097121. Epub 2021 Jun 29.

Wellcome Sanger Institute, Hinxton, United Kingdom.

Visceral leishmaniasis (VL) is a fatal disease and a growing public health problem in East Africa, where Ethiopia has one of the highest VL burdens. The largest focus of VL in Ethiopia is driven by high prevalence in migrant agricultural workers and associated with a high rate of coinfection with HIV. This coinfection makes VL more difficult to treat successfully and is associated with a high rate of relapse, with VL/HIV patients frequently experiencing many relapses of VL before succumbing to this infection. We present genome-wide data on Leishmania donovani isolates from a longitudinal study of cohorts of VL and VL/HIV patients reporting to a single clinic in Ethiopia. Extensive clinical data allow us to investigate the influence of coinfection and relapse on the populations of parasites infecting these patients. We find that the same parasite population is responsible for both VL and VL/HIV infections and that, in most cases, disease relapse is caused by recrudescence of the population of parasites that caused primary VL. Complex, multiclonal infections are present in both primary and relapse cases, but the infrapopulation of parasites within a patient loses genetic diversity between primary disease presentation and subsequent relapses, presumably due to a population bottleneck induced by treatment. These data suggest that VL/HIV relapses are not caused by genetically distinct parasite infections or by reinfection. Treatment of VL does not lead to sterile cure, and in VL/HIV, the infecting parasites are able to reestablish after clinically successful treatment, leading to repeated relapse of VL. Visceral leishmaniasis (VL) is the second largest cause of deaths due to parasite infections and a growing problem in East Africa. In Ethiopia, it is particularly associated with migrant workers moving from regions of nonendemicity for seasonal agricultural work and is frequently found as a coinfection with HIV, which leads to frequent VL relapse following treatment. Insight into the process of relapse in these patients is thus key to controlling the VL epidemic in Ethiopia. We show that there is little genetic differentiation between the parasites infecting HIV-positive and HIV-negative VL patients. Moreover, we provide evidence that relapses are caused by the initially infecting parasite population and that treatment induces a loss of genetic diversity in this population. We propose that restoring functioning immunity and improving antiparasitic treatment may be key in breaking the cycle of relapsing VL in VL/HIV patients.
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http://dx.doi.org/10.1128/mBio.00971-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262925PMC
June 2021

Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite .

Wellcome Open Res 2020 20;5:178. Epub 2021 Jan 20.

Wellcome Sanger Institute, Hinxton, CB10 1SA, UK.

At least 250 million people worldwide suffer from schistosomiasis, caused by worms. Genome sequences for several species are available, including a high-quality annotated reference for . There is a pressing need to develop a reliable functional toolkit to translate these data into new biological insights and targets for intervention. CRISPR-Cas9 was recently demonstrated for the first time in , to produce somatic mutations in the ( ) gene. We employed CRISPR-Cas9 to introduce somatic mutations in a second gene, , a sulfotransferase expressed in the parasitic stages of , in which mutations confer resistance to the drug oxamniquine. A 262-bp PCR product spanning the region targeted by the gRNA against was amplified, and mutations identified in it by high-throughput sequencing. We found that 0.3-2.0% of aligned reads from CRISPR-Cas9-treated adult worms showed deletions spanning the predicted Cas9 cut site, compared to 0.1-0.2% for sporocysts, while deletions were extremely rare in eggs. The most common deletion observed in adults and sporocysts was a 34 bp-deletion directly upstream of the predicted cut site, but rarer deletions reaching as far as 102 bp upstream of the cut site were also detected. The CRISPR-Cas9-induced deletions, if homozygous, are predicted to cause resistance to oxamniquine by producing frameshifts, ablating transcription, or leading to mRNA degradation the nonsense-mediated mRNA decay pathway. However, no knock down at the mRNA level was observed, presumably because the cells in which CRISPR-Cas9 did induce mutations represented a small fraction of all cells expressing . Further optimisation of CRISPR-Cas protocols for different developmental stages and particular cell types, including germline cells, will contribute to the generation of a homozygous knock-out in any gene of interest, and in particular the gene to derive an oxamniquine-resistant stable transgenic line.
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http://dx.doi.org/10.12688/wellcomeopenres.16031.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405262.2PMC
January 2021

Mapping immune variation and gene switching in naive hosts infected with .

Elife 2021 03 2;10. Epub 2021 Mar 2.

Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, United Kingdom.

Falciparum malaria is clinically heterogeneous and the relative contribution of parasite and host in shaping disease severity remains unclear. We explored the interaction between inflammation and parasite variant surface antigen (VSA) expression, asking whether this relationship underpins the variation observed in controlled human malaria infection (CHMI). We uncovered marked heterogeneity in the host response to blood challenge; some volunteers remained quiescent, others triggered interferon-stimulated inflammation and some showed transcriptional evidence of myeloid cell suppression. Significantly, only inflammatory volunteers experienced hallmark symptoms of malaria. When we tracked temporal changes in parasite VSA expression to ask whether variants associated with severe disease rapidly expand in naive hosts, we found no transcriptional evidence to support this hypothesis. These data indicate that parasite variants that dominate severe malaria do not have an intrinsic growth or survival advantage; instead, they presumably rely upon infection-induced changes in their within-host environment for selection.
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http://dx.doi.org/10.7554/eLife.62800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924948PMC
March 2021

The Phlebotomus papatasi systemic transcriptional response to trypanosomatid-contaminated blood does not differ from the non-infected blood meal.

Parasit Vectors 2021 Jan 6;14(1):15. Epub 2021 Jan 6.

Department of Biochemistry, University of Oxford, South Parks Rd, Oxford, OX1 3QU, UK.

Background: Leishmaniasis, caused by parasites of the genus Leishmania, is a disease that affects up to 8 million people worldwide. Parasites are transmitted to human and animal hosts through the bite of an infected sand fly. Novel strategies for disease control require a better understanding of the key step for transmission, namely the establishment of infection inside the fly.

Methods: The aim of this work was to identify sand fly systemic transcriptomic signatures associated with Leishmania infection. We used next generation sequencing to describe the transcriptome of whole Phlebotomus papatasi sand flies when fed with blood alone (control) or with blood containing one of three trypanosomatids: Leishmania major, L. donovani and Herpetomonas muscarum, the latter being a parasite not transmitted to humans.

Results: Of the trypanosomatids studied, only L. major was able to successfully establish an infection in the host P. papatasi. However, the transcriptional signatures observed after each parasite-contaminated blood meal were not specific to success or failure of a specific infection and they did not differ from each other. The transcriptional signatures were also indistinguishable after a non-contaminated blood meal.

Conclusions: The results imply that sand flies perceive Leishmania as just one feature of their microbiome landscape and that any strategy to tackle transmission should focus on the response towards the blood meal rather than parasite establishment. Alternatively, Leishmania could suppress host responses. These results will generate new thinking around the concept of stopping transmission by controlling the parasite inside the insect.
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http://dx.doi.org/10.1186/s13071-020-04498-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789365PMC
January 2021

Ecological divergence and hybridization of Neotropical parasites.

Proc Natl Acad Sci U S A 2020 10 21;117(40):25159-25168. Epub 2020 Sep 21.

Department of Biomedical Sciences, Institute of Tropical Medicine, 2000 Antwerp, Belgium;

The tropical Andes are an important natural laboratory to understand speciation in many taxa. Here we examined the evolutionary history of parasites of the species complex based on whole-genome sequencing of 67 isolates from 47 localities in Peru. We first show the origin of Andean as a clade of near-clonal lineages that diverged from admixed Amazonian ancestors, accompanied by a significant reduction in genome diversity and large structural variations implicated in host-parasite interactions. Within the Andean species, patterns of population structure were strongly associated with biogeographical origin. Molecular clock and ecological niche modeling suggested that the history of diversification of the Andean lineages is limited to the Late Pleistocene and intimately associated with habitat contractions driven by climate change. These results suggest that changes in forestation over the past 150,000 y have influenced speciation and diversity of these Neotropical parasites. Second, genome-scale analyses provided evidence of meiotic-like recombination between Andean and Amazonian species, resulting in full-genome hybrids. The mitochondrial genome of these hybrids consisted of homogeneous uniparental maxicircles, but minicircles originated from both parental species. We further show that mitochondrial minicircles-but not maxicircles-show a similar evolutionary pattern to the nuclear genome, suggesting that compatibility between nuclear-encoded mitochondrial genes and minicircle-encoded guide RNA genes is essential to maintain efficient respiration. By comparing full nuclear and mitochondrial genome ancestries, our data expand our appreciation on the genetic consequences of diversification and hybridization in parasitic protozoa.
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http://dx.doi.org/10.1073/pnas.1920136117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547230PMC
October 2020

Evaluation of whole genome amplification and bioinformatic methods for the characterization of Leishmania genomes at a single cell level.

Sci Rep 2020 09 14;10(1):15043. Epub 2020 Sep 14.

Institute of Tropical Medicine Antwerp, Molecular Parasitology Unit, Antwerp, Belgium.

Here, we report a pilot study paving the way for further single cell genomics studies in Leishmania. First, the performances of two commercially available kits for Whole Genome Amplification (WGA), PicoPLEX and RepliG were compared on small amounts of Leishmania donovani DNA, testing their ability to preserve specific genetic variations, including aneuploidy levels and SNPs. We show here that the choice of WGA method should be determined by the planned downstream genetic analysis, PicoPLEX and RepliG performing better for aneuploidy and SNP calling, respectively. This comparison allowed us to evaluate and optimize corresponding bio-informatic methods. As PicoPLEX was shown to be the preferred method for studying single cell aneuploidy, this method was applied in a second step, on single cells of L. braziliensis, which were sorted by fluorescence activated cell sorting (FACS). Even sequencing depth was achieved in 28 single cells, allowing accurate somy estimation. A dominant karyotype with three aneuploid chromosomes was observed in 25 cells, while two different minor karyotypes were observed in the other cells. Our method thus allowed the detection of aneuploidy mosaicism, and provides a solid basis which can be further refined to concur with higher-throughput single cell genomic methods.
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http://dx.doi.org/10.1038/s41598-020-71882-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490275PMC
September 2020

Transcriptome of the parasitic flatworm Schistosoma mansoni during intra-mammalian development.

PLoS Negl Trop Dis 2020 05 6;14(5):e0007743. Epub 2020 May 6.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom.

Schistosomes are parasitic blood flukes that survive for many years within the mammalian host vasculature. How the parasites establish a chronic infection in the hostile bloodstream environment, whilst evading the host immune response is poorly understood. The parasite develops morphologically and grows as it migrates to its preferred vascular niche, avoiding or repairing damage from the host immune system. In this study, we investigated temporal changes in gene expression during the intra-mammalian development of Schistosoma mansoni. RNA-seq data were analysed from parasites developing in the lung through to egg-laying mature adult worms, providing a comprehensive picture of in vivo intra-mammalian development. Remarkably, genes involved in signalling pathways, developmental control, and adaptation to oxidative stress were up-regulated in the lung stage. The data also suggested a potential role in immune evasion for a previously uncharacterised gene. This study not only provides a large and comprehensive data resource for the research community, but also reveals new directions for further characterising host-parasite interactions that could ultimately lead to new control strategies for this neglected tropical disease pathogen.
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http://dx.doi.org/10.1371/journal.pntd.0007743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263636PMC
May 2020

Genomic analysis of natural intra-specific hybrids among Ethiopian isolates of Leishmania donovani.

PLoS Negl Trop Dis 2020 04 20;14(4):e0007143. Epub 2020 Apr 20.

Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.

Parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae) cause widespread and devastating human diseases. Visceral leishmaniasis due to Leishmania donovani is endemic in Ethiopia where it has also been responsible for major epidemics. The presence of hybrid genotypes has been widely reported in surveys of natural populations, genetic variation reported in a number of Leishmania species, and the extant capacity for genetic exchange demonstrated in laboratory experiments. However, patterns of recombination and the evolutionary history of admixture that produced these hybrid populations remain unclear. Here, we use whole-genome sequence data to investigate Ethiopian L. donovani isolates previously characterized as hybrids by microsatellite and multi-locus sequencing. To date there is only one previous study on a natural population of Leishmania hybrids based on whole-genome sequences. We propose that these hybrids originate from recombination between two different lineages of Ethiopian L. donovani occurring in the same region. Patterns of inheritance are more complex than previously reported with multiple, apparently independent, origins from similar parents that include backcrossing with parental types. Analysis indicates that hybrids are representative of at least three different histories. Furthermore, isolates were highly polysomic at the level of chromosomes with differences between parasites recovered from a recrudescent infection from a previously treated individual. The results demonstrate that recombination is a significant feature of natural populations and contributes to the growing body of data that shows how recombination, and gene flow, shape natural populations of Leishmania.
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http://dx.doi.org/10.1371/journal.pntd.0007143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237039PMC
April 2020

Global genome diversity of the complex.

Elife 2020 03 25;9. Epub 2020 Mar 25.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom.

Protozoan parasites of the complex - and - cause the fatal disease visceral leishmaniasis. We present the first comprehensive genome-wide global study, with 151 cultured field isolates representing most of the geographical distribution. isolates separated into five groups that largely coincide with geographical origin but vary greatly in diversity. In contrast, the majority of samples fell into one globally-distributed group with little diversity. This picture is complicated by several hybrid lineages. Identified genetic groups vary in heterozygosity and levels of linkage, suggesting different recombination histories. We characterise chromosome-specific patterns of aneuploidy and identified extensive structural variation, including known and suspected drug resistance loci. This study reveals greater genetic diversity than suggested by geographically-focused studies, provides a resource of genomic variation for future work and sets the scene for a new understanding of the evolution and genetics of the complex.
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http://dx.doi.org/10.7554/eLife.51243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105377PMC
March 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.
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http://dx.doi.org/10.12688/wellcomeopenres.15590.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001760PMC
December 2019

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

PLoS Negl Trop Dis 2019 12 12;13(12):e0007900. Epub 2019 Dec 12.

Institute of Tropical Medicine Antwerp, Molecular Parasitology Unit, Antwerp, Belgium.

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.
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http://dx.doi.org/10.1371/journal.pntd.0007900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6932831PMC
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.
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http://dx.doi.org/10.1371/journal.pgen.1008452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6872171PMC
November 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.
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http://dx.doi.org/10.12688/wellcomeopenres.15194.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484455PMC
May 2019

Whole genome sequencing of experimental hybrids supports meiosis-like sexual recombination in Leishmania.

PLoS Genet 2019 05 15;15(5):e1008042. Epub 2019 May 15.

Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

Hybrid genotypes have been repeatedly described among natural isolates of Leishmania, and the recovery of experimental hybrids from sand flies co-infected with different strains or species of Leishmania has formally demonstrated that members of the genus possess the machinery for genetic exchange. As neither gamete stages nor cell fusion events have been directly observed during parasite development in the vector, we have relied on a classical genetic analysis to determine if Leishmania has a true sexual cycle. Here, we used whole genome sequencing to follow the chromosomal inheritance patterns of experimental hybrids generated within and between different strains of L. major and L. infantum. We also generated and sequenced the first experimental hybrids in L. tropica. We found that in each case the parental somy and allele contributions matched the inheritance patterns expected under meiosis 97-99% of the time. The hybrids were equivalent to F1 progeny, heterozygous throughout most of the genome for the markers that were homozygous and different between the parents. Rare, non-Mendelian patterns of chromosomal inheritance were observed, including a gain or loss of somy, and loss of heterozygosity, that likely arose during meiosis or during mitotic divisions of the progeny clones in the fly or culture. While the interspecies hybrids appeared to be sterile, the intraspecies hybrids were able to produce backcross and outcross progeny. Analysis of 5 backcross and outcross progeny clones generated from an L. major F1 hybrid, as well as 17 progeny clones generated from backcrosses involving a natural hybrid of L. tropica, revealed genome wide patterns of recombination, demonstrating that classical crossing over occurs at meiosis, and allowed us to construct the first physical and genetic maps in Leishmania. Altogether, the findings provide strong evidence for meiosis-like sexual recombination in Leishmania, presenting clear opportunities for forward genetic analysis and positional cloning of important genes.
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http://dx.doi.org/10.1371/journal.pgen.1008042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519804PMC
May 2019

Repeated clinical malaria episodes are associated with modification of the immune system in children.

BMC Med 2019 03 13;17(1):60. Epub 2019 Mar 13.

Francis Crick Institute, London, UK.

Background: There are over 200 million reported cases of malaria each year, and most children living in endemic areas will experience multiple episodes of clinical disease before puberty. We set out to understand how frequent clinical malaria, which elicits a strong inflammatory response, affects the immune system and whether these modifications are observable in the absence of detectable parasitaemia.

Methods: We used a multi-dimensional approach comprising whole blood transcriptomic, cellular and plasma cytokine analyses on a cohort of children living with endemic malaria, but uninfected at sampling, who had been under active surveillance for malaria for 8 years. Children were categorised into two groups depending on the cumulative number of episodes experienced: high (≥ 8) or low (< 5).

Results: We observe that multiple episodes of malaria are associated with modification of the immune system. Children who had experienced a large number of episodes demonstrated upregulation of interferon-inducible genes, a clear increase in circulating levels of the immunoregulatory cytokine IL-10 and enhanced activation of neutrophils, B cells and CD8 T cells.

Conclusion: Transcriptomic analysis together with cytokine and immune cell profiling of peripheral blood can robustly detect immune differences between children with different numbers of prior malaria episodes. Multiple episodes of malaria are associated with modification of the immune system in children. Such immune modifications may have implications for the initiation of subsequent immune responses and the induction of vaccine-mediated protection.
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http://dx.doi.org/10.1186/s12916-019-1292-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6415347PMC
March 2019

Genomic and transcriptomic comparisons of closely related malaria parasites differing in virulence and sequestration pattern.

Wellcome Open Res 2018 6;3:142. Epub 2018 Dec 6.

Malaria Immunology laboratory, Francis Crick Institute, London, NW1 1AT, UK.

Malaria parasite species differ greatly in the harm they do to humans. While kills hundreds of thousands per year, kills much less often and is relatively benign. Strains of the rodent malaria parasite show phenotypic variation in virulence during infections of laboratory mice. This make it an excellent species to study genes which may be responsible for this trait. By understanding the mechanisms which underlie differences in virulence we can learn how parasites adapt to their hosts and how we might prevent disease. Here we present a complete reference genome sequence for a more virulent strain, PcCB, and perform a detailed comparison with the genome of the less virulent PcAS strain. We found the greatest variation in the subtelomeric regions, in particular amongst the sequences of the gene family, which has been associated with virulence and establishment of chronic infection. Despite substantial variation at the sequence level, the repertoire of these genes has been largely maintained, highlighting the requirement for functional conservation as well as diversification in host-parasite interactions. However, a subset of genes, previously associated with increased virulence, were more highly expressed in PcCB, suggesting a role for this gene family in virulence differences between strains. We found that core genes involved in red blood cell invasion have been under positive selection and that the more virulent strain has a greater preference for reticulocytes, which has elsewhere been associated with increased virulence. These results provide the basis for a mechanistic understanding of the phenotypic differences between strains, which might ultimately be translated into a better understanding of malaria parasites affecting humans.
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http://dx.doi.org/10.12688/wellcomeopenres.14797.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6259598PMC
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.
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http://dx.doi.org/10.1038/s41541-018-0091-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258718PMC
November 2018

Variant antigen repertoires in populations and experimental infections can be profiled from deep sequence data using universal protein motifs.

Genome Res 2018 09 13;28(9):1383-1394. Epub 2018 Jul 13.

Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool L3 5RF, United Kingdom.

African trypanosomes are vector-borne hemoparasites of humans and animals. In the mammal, parasites evade the immune response through antigenic variation. Periodic switching of the variant surface glycoprotein (VSG) coat covering their cell surface allows sequential expansion of serologically distinct parasite clones. Trypanosome genomes contain many hundreds of genes, subject to rapid changes in nucleotide sequence, copy number, and chromosomal position. Thus, analyzing, or even quantifying, VSG diversity over space and time presents an enormous challenge to conventional techniques. Indeed, previous population genomic studies have overlooked this vital aspect of pathogen biology for lack of analytical tools. Here we present a method for analyzing population-scale VSG diversity in from deep sequencing data. Previously, we suggested that VSGs segregate into defined "phylotypes" that do not recombine. In our data set comprising 41 genome sequences from across Africa, these phylotypes are universal and exhaustive. Screening sequence contigs with diagnostic protein motifs accurately quantifies relative phylotype frequencies, providing a metric of VSG diversity, called the "variant antigen profile." We applied our metric to VSG expression in the tsetse fly, showing that certain, rare VSG phylotypes may be preferentially expressed in infective, metacyclic-stage parasites. Hence, variant antigen profiling accurately and rapidly determines the gene and transcript repertoire from sequence data, without need for manual curation or highly contiguous sequences. It offers a tractable approach to measuring VSG diversity across strains and during infections, which is imperative to understanding the host-parasite interaction at population and individual scales.
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http://dx.doi.org/10.1101/gr.234146.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120623PMC
September 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.
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http://dx.doi.org/10.12688/wellcomeopenres.14571.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964635PMC
May 2018

Genomes of all known members of a Plasmodium subgenus reveal paths to virulent human malaria.

Nat Microbiol 2018 06 21;3(6):687-697. Epub 2018 May 21.

Laboratoire MIVEGEC, UMR 5290-224 CNRS 5290-IRD224-UM, Montpellier, France.

Plasmodium falciparum, the most virulent agent of human malaria, shares a recent common ancestor with the gorilla parasite Plasmodium praefalciparum. Little is known about the other gorilla- and chimpanzee-infecting species in the same (Laverania) subgenus as P. falciparum, but none of them are capable of establishing repeated infection and transmission in humans. To elucidate underlying mechanisms and the evolutionary history of this subgenus, we have generated multiple genomes from all known Laverania species. The completeness of our dataset allows us to conclude that interspecific gene transfers, as well as convergent evolution, were important in the evolution of these species. Striking copy number and structural variations were observed within gene families and one, stevor, shows a host-specific sequence pattern. The complete genome sequence of the closest ancestor of P. falciparum enables us to estimate the timing of the beginning of speciation to be 40,000-60,000 years ago followed by a population bottleneck around 4,000-6,000 years ago. Our data allow us also to search in detail for the features of P. falciparum that made it the only member of the Laverania able to infect and spread in humans.
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http://dx.doi.org/10.1038/s41564-018-0162-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985962PMC
June 2018

and reference genomes highlight genome structure and gene evolution in the subgenus.

R Soc Open Sci 2018 Apr 25;5(4):172212. Epub 2018 Apr 25.

School of Mathematics, Applied Mathematics and Statistics, National University of Ireland, Galway, Republic of Ireland.

The unicellular protozoan parasite causes the neglected tropical disease leishmaniasis, affecting 12 million people in 98 countries. In South America, where the subgenus predominates, so far only () and () have been sequenced, assembled and annotated as reference genomes. Addressing this deficit in molecular information can inform species typing, epidemiological monitoring and clinical treatment. Here, () and () genomic DNA was sequenced to assemble these two genomes as draft references from short sequence reads. The methods used were tested using short sequence reads for M2904 against its published reference as a comparison. This assembly and annotation pipeline identified 70 additional genes not annotated on the original M2904 reference. Phylogenetic and evolutionary comparisons of and with 10 other genomes revealed four traits common to all : aneuploidy, 22 orthologous groups of genes absent in other subgenera, elevated TATE transposon copies and a high NADH-dependent fumarate reductase gene copy number. Within the , there were limited structural changes in genome architecture specific to individual species: a 45 Kb amplification on chromosome 34 was present in all bar , had a higher copy number of the virulence factor leishmanolysin, and laboratory isolate M8408 had a possible minichromosome derived from the 3' end of chromosome 34 This combination of genome assembly, phylogenetics and comparative analysis across an extended panel of diverse has uncovered new insights into the origin and evolution of this subgenus and can help improve diagnostics for leishmaniasis surveillance.
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http://dx.doi.org/10.1098/rsos.172212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5936940PMC
April 2018

Integrated genomic and metabolomic profiling of ISC1, an emerging Leishmania donovani population in the Indian subcontinent.

Infect Genet Evol 2018 08 19;62:170-178. Epub 2018 Apr 19.

Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium; Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium. Electronic address:

Leishmania donovani is the responsible agent for visceral leishmaniasis (VL) in the Indian subcontinent (ISC). The disease is lethal without treatment and causes 0.2 to 0.4 million cases each year. Recently, reports of VL in Nepalese hilly districts have increased as well as VL cases caused by L. donovani from the ISC1 genetic group, a new and emerging genotype. In this study, we perform for the first time an integrated, untargeted genomics and metabolomics approach to characterize ISC1, in comparison with the Core Group (CG), main population that drove the most recent outbreak of VL in the ISC. We show that the ISC1 population is very different from the CG, both at genome and metabolome levels. The genomic differences include SNPs, CNV and small indels in genes coding for known virulence factors, immunogens and surface proteins. Both genomic and metabolic approaches highlighted dissimilarities related to membrane lipids, the nucleotide salvage pathway and the urea cycle in ISC1 versus CG. Many of these pathways and molecules are important for the interaction with the host/extracellular environment. Altogether, our data predict major functional differences in ISC1 versus CG parasites, including virulence. Therefore, particular attention is required to monitor the fate of this emerging ISC1 population in the ISC, especially in a post-VL elimination context.
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http://dx.doi.org/10.1016/j.meegid.2018.04.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261844PMC
August 2018

Single-cell RNA-seq reveals hidden transcriptional variation in malaria parasites.

Elife 2018 03 27;7. Epub 2018 Mar 27.

Malaria Programme, Wellcome Sanger Institute, Cambridge, United Kingdom.

Single-cell RNA-sequencing is revolutionising our understanding of seemingly homogeneous cell populations but has not yet been widely applied to single-celled organisms. Transcriptional variation in unicellular malaria parasites from the genus is associated with critical phenotypes including red blood cell invasion and immune evasion, yet transcriptional variation at an individual parasite level has not been examined in depth. Here, we describe the adaptation of a single-cell RNA-sequencing (scRNA-seq) protocol to deconvolute transcriptional variation for more than 500 individual parasites of both rodent and human malaria comprising asexual and sexual life-cycle stages. We uncover previously hidden discrete transcriptional signatures during the pathogenic part of the life cycle, suggesting that expression over development is not as continuous as commonly thought. In transmission stages, we find novel, sex-specific roles for differential expression of contingency gene families that are usually associated with immune evasion and pathogenesis.
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http://dx.doi.org/10.7554/eLife.33105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5871331PMC
March 2018
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