Biology subjects

Berriman, M.

Publications and source records attributed to Berriman, M..

At least 55 records · Page 3Linked to original sources

bioRxiv2019

Genomic and transcriptomic evidence for descent from Plasmodium and loss of blood schizogony in Hepatocystis parasites from naturally infected red colobus monkeys

Hepatocystis is a genus of single-celled parasites infecting monkeys, bats and squirrels. Although thought to descend from malaria parasites (Plasmodium spp.), Hepatocystis spp. are thought not to undergo replication in the blood - the part of the Plasmodium life cycle which causes the symptoms of malaria. Furthermore, Hepatocystis is transmitted by midges, not mosquitoes. Comparative genomics of Hepatocystis and Plasmodium species therefore presents an opportunity to better understand some of the most important aspects of malaria parasite biology. We were able to generate a draft genome for Hepatocystis using DNA sequencing reads from the blood of a naturally infected red colobus monkey. We provide robust phylogenetic support for Hepatocystis as a sister group to Plasmodium parasites infecting rodents. We show transcriptomic support for a lack of replication in the blood and genomic support for a complete loss of a family of genes involved in red blood cell invasion. Our analyses highlight the rapid evolution of genes involved in parasite vector stages, revealing genes that may be critical for interactions between malaria parasites and mosquitoes.

genomics↗

bioRxiv2019

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

Plasmodium parasites undergo several major developmental transitions during their complex lifecycle, which are enabled by precisely ordered gene expression programs. Transcriptomes from the 48-hour 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%. 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 75% of the genome transcribed into mRNAs. These datasets allowed us to refine the 5 and 3 untranslated regions (UTRs), which can be variable, long (>1,000 nt), and often overlap those of adjacent transcripts. We also find that transcription from bidirectional promoters frequently results in non-coding, antisense transcripts. By capturing the 5 ends of mRNAs, we reveal both constant and dynamic use of transcriptional start sites across the intraerythrocytic developmental cycle resulting in an updated view of the P. falciparum transcriptome.

genomics↗

bioRxiv2019

Ecological divergence and hybridization of Neotropical Leishmania parasites

The tropical Andes is an important natural laboratory to understand speciation and diversification in many taxa. Here, we examined the evolutionary history of parasites of the Leishmania braziliensis species complex based on whole genome sequencing of 67 isolates from 47 localities in Peru. We firstly show the origin of near-clonal Andean Leishmania lineages that diverged from admixed Amazonian ancestors, accompanied by a significant reduction in genome diversity and large structural variations implicated in host-parasite interactions. Beside a clear dichotomy between Andean and Amazonian species, patterns of population structure were strongly associated with biogeographical origin. Molecular clock analyses 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 support a wider model on trypanosomatid evolution where major parasite lineages emerge through ecological fitting. Second, genome-scale analyses provided evidence of meiotic recombination between Andean and Amazonian Leishmania species, resulting in full-genome hybrids. The mitochondrial genome of these hybrids consisted of homogeneous uniparental maxicircles, but minicircles originated from both parental species, leaving a mosaic ancestry of minicircle-encoded guide RNA genes. We further show that mitochondrial minicircles - but not maxicircles - show a similar evolutionary pattern as the nuclear genome, suggesting that biparental inheritance of minicircles is universal and may be important to alleviate maxicircle-nuclear incompatibilities. By comparing full nuclear and mitochondrial genome ancestries, our data expands our appreciation on the genetic consequences of diversification and hybridization in parasitic protozoa.

genomics↗

bioRxiv2019

Population genomic evidence that human and animal infections in Africa come from the same populations of Dracunculus medinensis

BackgroundGuinea worm - Dracunculus medinensis - was historically one of the major parasites of humans and has been known since antiquity. Now, Guinea worm is on the brink of eradication, as efforts to interrupt transmission have reduced the annual burden of disease from millions of infections per year in the 1980s to only 30 human cases reported globally last year. Despite the enormous success of eradication efforts to date, one complication has arisen. Over the last few years, hundreds of dogs have been found infected with this previously apparently anthroponotic parasite, almost all in Chad. Moreover, the relative numbers of infections in humans and dogs suggests that dogs may be key in maintaining transmission in that country.\n\nResultsIn an effort to shed light on this peculiar epidemiology of Guinea worm in Chad, we have sequenced and compared the genomes of worms from dog, human and other animal infections. Confirming previous work with other molecular markers, we show that all of these worms are D. medinensis, and that the same population of worms are causing both infections, can confirm the suspected transmission between host species and detect signs of a population bottleneck due to the eradication efforts. The diversity of worms in Chad appears to exclude the possibility that there were no, or very few, worms present in the country during a 10-year absence of reported cases.\n\nConclusionsThis work reinforces the importance of adequate surveillance of both human and dog populations in the Guinea worm eradication campaign and suggests that control programs should stay aware of the possible emergence of unusual epidemiology as they approach elimination.

genomics↗

bioRxiv2019

Single-cell atlas of the first intra-mammalian developmental stage of the human parasite Schistosoma mansoni

Over 250 million people suffer from schistosomiasis, a tropical disease caused by parasitic flatworms known as schistosomes. Humans become infected by free-swimming, water-borne larvae, which penetrate the skin. The earliest intra-mammalian stage, called the schistosomulum, undergoes a series of developmental transitions. These changes are critical for the parasite to adapt to its new environment as it navigates through host tissues to reach its niche, where it will grow to reproductive maturity. Unravelling the mechanisms that drive intra-mammalian development requires knowledge of the spatial organisation and transcriptional dynamics of different cell types that comprise the schistomulum body. To fill these important knowledge gaps, we performed single-cell RNA sequencing on two-day old schistosomula of Schistosoma mansoni. We identified likely gene expression profiles for muscle, nervous system, tegument, parenchymal/primordial gut cells, and stem cells. In addition, we validated cell markers for all these clusters by in situ hybridisation in schistosomula and adult parasites. Taken together, this study provides a comprehensive cell-type atlas for the early intra-mammalian stage of this devastating metazoan parasite.

genomics↗

bioRxiv2019

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

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.\n\nAuthor SummaryThe life cycle of the parasitic flatworm Schistosoma mansoni is split between snail and mammalian (often human) hosts. An infection can last for more than 10 years, during which time the parasite physically interacts with its mammalian host as it moves through the bloodstream, travelling through the lungs and liver, to eventually establish a chronic infection in the blood vessels around the host gut. Throughout this complex journey, the parasite develops from a relatively simple larval form into a more complex, sexually reproducing adult. To understand the molecular basis of parasite interactions with the host during this complex journey we have produced genome-wide expression data from developing parasites. The parasites were collected from experimentally-infected mice over its developmental time-course from the poorly studied lung stage, to the fully mature egg-laying adult worm. The data highlight many genes involved in processes known to be associated with key stages of the infection. In addition, the gene expression data provide a unique view of interactions between the parasite and the immune system in the lung, including novel players in host-parasite interactions. A detailed understanding of these processes may provide new opportunities to design intervention strategies, particularly those focussed on the early stages of the infection that are not targeted by current chemotherapy.

microbiology↗

bioRxiv2019

Global genome diversity of the Leishmania donovani complex

Protozoan parasites of the Leishmania donovani complex - L. donovani and L. infantum - 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. L. donovani isolates separated into five groups that largely coincide with geographical origin but vary greatly in diversity. In contrast, the majority of L. infantum 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 Leishmania donovani complex.

genomics↗

bioRxiv2019

Rora regulates activated T helper cells during inflammation

The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment.

immunology↗

bioRxiv2019

Genomes of intracellular Leishmania parasites directly sequenced from patients

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

microbiology↗

bioRxiv2019

Evaluation of DNA extraction methods on individual helminth egg and larval stages for whole genome sequencing

Whole genome sequencing is being rapidly applied to the study of helminth genomes, including de novo genome assembly, population genetics, and diagnostic applications. Although late-stage juvenile and adult parasites typically produce sufficient DNA for molecular analyses, these parasitic stages are almost always inaccessible in the live host; immature life stages found in the environment for which samples can be collected non-invasively offer a potential alternative, however, these samples are typically yield very low quantities of DNA, can be environmentally resistant, and are susceptible to contamination, often from bacterial or host DNA. Here, we have tested five low-input DNA extraction protocols together with a low-input sequencing library protocol to assess the feasibility of whole genome sequencing of individual immature helminth samples. These approaches do not use whole genome amplification, a common but costly approach to increase the yield of low input samples. We first tested individual parasites from two species spotted onto FTA cards - egg and L1 stages of Haemonchus contortus and miracidia of Schistosoma mansoni - before further testing on an additional six species - Ancylostoma caninum, Ascaridia dissimilis, Dirofilaria immitis, Dracunculus medinensis, Strongyloides stercoralis, and Trichuris muris - with an optimal protocol. Whole genome sequencing followed by analyses to determine the proportion of on- and off-target mapping revealed successful sample preparations for six of the eight species tested with variation between species, and within species but between life stages, described. These results demonstrate the feasibility of whole genome sequencing of individual parasites, and highlight a new avenue towards generating sensitive, specific, and information-rich data for the diagnosis and surveillance of helminths.

genomics↗

bioRxiv2019

A library of cell-surface and secreted proteins from Schistosoma mansoni identifies early serological markers of infection

Schistosomiasis is a major global health problem caused by blood-dwelling parasitic worms and is currently treated by the mass administration of the drug praziquantel. Appropriate drug treatment strategies are informed by diagnostics that establish the prevalence and intensity of infection, which, in regions of low transmission should be highly sensitive. To identify sensitive new serological markers of Schistosoma mansoni infections, we have compiled a recombinant protein library of 115 parasite cell surface and secreted proteins expressed in mammalian cells. The vast majority of them were shown to be immunoreactive and to contain heat-labile conformational epitopes when tested against pooled human sera from endemic regions. After probing the library against a time series of sera samples from experimental infections in mice, we identified several markers of infection, the majority of which belong to the saposin-domain-containing and cathepsin families of proteins. These new markers will be a valuable tool to detect ongoing and previous S. mansoni infections, including in regions of low transmission. We envisage that this new recombinant protein resource will be used in a wide range of cellular and molecular assays to further our understanding of Schistosoma biology.

immunology↗

bioRxiv2019

The Malaria Cell Atlas: a comprehensive reference of single parasite transcriptomes across the complete Plasmodium life cycle

Malaria parasites adopt a remarkable variety of morphological life stages as they transition through multiple mammalian host and mosquito vector environments. Here we profile the single-cell transcriptomes of thousands of individual parasites, deriving the first high-resolution transcriptional atlas of the entire Plasmodium berghei life cycle. We then use our atlas to precisely define developmental stages of single cells from three different human malaria parasite species, including parasites isolated directly from infected individuals. The Malaria Cell Atlas provides both a comprehensive view of gene usage in a complex eukaryotic parasite and an open access reference data set for the study of malaria parasites.

genomics↗

bioRxiv2019

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

Parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae) cause widespread and devastating human diseases, ranging from self-healing but disfiguring cutaneous lesions to destructive mucocutaneous presentations or usually fatal visceral disease. 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 evolutionary history of admixture that produced these hybrid populations remain unclear, as most of the relevant literature examines only a limited number (typically fewer than 10) genetic loci. Here, we use whole-genome sequence data to investigate Ethiopian L. donovani isolates previously characterised 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 sequence. The current findings demonstrate important differences. We propose 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 startling 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 evidence describing how recombination, and gene flow, shape natural populations of Leishmania.\n\nAuthor SummaryLeishmaniasis is a spectrum of diseases caused by the protozoan parasite Leishmania. It is transmitted by sandfly insect vectors and is responsible for an enormous burden of human suffering. In this manuscript we examine Leishmania isolates from Ethiopia that cause the most serious form of the disease, namely visceral leishmaniasis, which is usually fatal without treatment. Historically the general view was that such parasites reproduce clonally, so that their progeny are genetically identical to the founding cells. This view has changed over time and it is increasingly clear that recombination between genetically different Leishmania parasites occurs. The implication is that new biological traits such as virulence, resistance to drug treatments or the ability to infect new species of sandfly could emerge. The frequency and underlying mechanism of such recombination in natural isolates is poorly understood. Here we perform a detailed whole genome analysis on a cohort of hybrid isolates from Ethiopia together with their potential parents to assess the genetic nature of hybrids in more detail. Results reveal a complex pattern of mating and inbreeding indicative of multiple mating events that has likely shaped the epidemiology of the disease agent. We also show that some hybrids have very different relative amounts of DNA (polysomy) the implications of which are discussed. Together the results contribute to a fuller understanding of the nature of genetic recombination in natural populations of Leishmania.

genomics↗

bioRxiv2018

The global diversity of a major parasitic nematode is shaped by human intervention and climatic adaptation

The gastrointestinal parasite Haemonchus contortus is an haematophagous parasitic nematode of veterinary interest and a model for the study of drug resistance mechanisms or host-parasite interactions. To understand its evolutionary history, and its ability to adapt in the face of climatic and drug pressure, we have performed an extensive survey of genome-wide diversity using single-worm whole genome sequencing of 223 individuals sampled from 19 isolates spanning five continents. The pattern of global diversity is driven by an African origin for the species, together with contemporary dispersal that is consistent with modern human movement, with evidence for parasites spreading during the transatlantic slave trade and colonisation of Australia presented. Strong selective sweeps were identified in independent populations each surrounding the {beta}-tubulin locus, a target of benzimidazole anthelmintic drug treatment used widely to control H. contortus infections. These signatures of selection were further supported by signals of diversifying selection enriched in genes involved in response to drugs, as well as other anthelmintic-associated biological functions including pharyngeal pumping and oviposition. From these analyses, we identify some known, and previously undescribed, candidate genes that may play a role in ivermectin resistance. Finally, we describe genetic signatures of climate-driven adaptation, revealing a gene acting as an epigenetic regulator and components of the dauer pathway may play a role in adaptation in the face of climatic fluctuations. These results begin to define genetic adaptation to climate for the first time in a parasitic nematode, and provides insight into the ongoing expansion in the range of Haemonchus contortus, which may have consequences for the management of this parasite.

genomics↗

bioRxiv2018

Whole genome sequencing and morphological analysis of the human-infecting schistosome emerging in Europe reveals a complex admixture between Schistosoma haematobium and Schistosoma bovis parasites.

Schistosomes cause schistosomiasis, the worlds second most important parasitic disease after malaria. A peculiar feature of schistosomes is their ability to produce viable and fertile hybrids. Originally only present in the tropics, schistosomiasis is now also endemic in Europe. Based on two genetic markers the European species had been identified as a hybrid between the ruminant-infective Schistosoma bovis and the human-infective Schistosoma haematobium.\n\nHere we describe for the first time the genomic composition of the European schistosome hybrid (77% of S. haematobium and 23% of S. bovis origins), its morphometric parameters and its compatibility with the European vector snail and intermediate host Compatibility is a key parameter for the parasites life cycle progression. We also show that egg morphology (a classical diagnostic parameter) does not allow for differential diagnosis while genetic tests do so. Additionally, we performed genome assembly improvement and annotation of S. bovis, the parental species for which no satisfactory genome assembly was available.\n\nFor the first time since the discovery of hybrid schistosomes, these results reveal at the whole genomic level a complex admixture of parental genomes highlighting (i) the high permeability of schistosomes to other species alleles, and (ii) the importance of hybrid formation for pushing species boundaries not only conceptionally but also geographically.

genomics↗

bioRxiv2018

Exclusive dependence of IL-10Rα signalling on intestinal microbiota homeostasis and control of whipworm infection

The whipworm Trichuris trichiura is a soil-transmitted helminth that dwells in the epithelium of the caecum and proximal colon of their hosts causing the human disease, trichuriasis. Trichuriasis is characterized by colitis attributed to the inflammatory response elicited by the parasite while tunnelling through intestinal epithelial cells (IECs).\n\nThe IL-10 family of receptors, comprising combinations of subunits IL-10R, IL-10R{beta}, IL-22R and IL-28R, modulates intestinal inflammatory responses. Here we carefully dissected the role of these subunits in the resistance of mice to infection with T. muris, a mouse model of the human whipworm T. trichiura. Our findings demonstrate that whilst IL-22R and IL-28R are dispensable in the host response to whipworms, IL-10 signalling through IL-10R and IL-10R{beta} is essential to control caecal pathology, worm expulsion and survival during T. muris infections. We show that deficiency of IL-10, IL-10R and IL-10R{beta} results in dysbiosis of the caecal microbiota characterised by expanded populations of opportunistic bacteria of the families Enterococcaceae and Enterobacteriaceae. Moreover, breakdown of the epithelial barrier after whipworm infection in IL-10, IL-10R and IL-10R{beta}-deficient mice, allows the translocation of these opportunistic pathogens or their excretory products to the liver causing organ failure and lethal disease. Importantly, bone marrow chimera experiments indicate that signalling through IL-10R and IL-10R{beta} in haematopoietic cells, but not IECs, is crucial to control worm expulsion and immunopathology. These findings are supported by worm expulsion upon infection of conditional mutant mice for the IL-10R on IECs. Our findings emphasize the pivotal role of systemic IL-10R signalling on immune cells in promoting microbiota homeostasis and maintaining the intestinal epithelial barrier, thus preventing immunopathology during whipworms infections.\n\nAuthor summaryThe human gut is home to millions of bacteria, collectively called the microbiota, and also to parasites that include whipworms. The interactions between gut cells, the microbiota and whipworms define conditions for balanced parasitism. Cells lining the gut host whipworms but also interact with gut immune cells to deploy measures that control or expel whipworms whilst maintaining a barrier to prevent microbial translocation. Whipworms affect the composition of the microbiota, which in turn impacts the condition of the gut lining and the way in which immune cells are activated. In order to avoid tissue damage and disease, these interactions are tightly regulated. Here we show that signalling through a member of the IL-10 receptor family, IL-10R, in gut immune cells is critical for regulating of these interactions. Lack of this receptor on gut immune cells results in persistence of whipworms in the gut accompanied by an uncontrolled inflammation that destroys the gut lining. This tissue damage is accompanied by the overgrowth of members of the microbiota that act as opportunistic pathogens. Furthermore, the destruction of the gut barrier allows these bacteria to reach the liver where they cause organ failure and fatal disease.

immunology↗

bioRxiv2018

Functional genomics and programmed genome editing of omega-1 of the blood fluke Schistosoma mansoni

CRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 ({omega}1) of Schistosoma mansoni as a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of {omega}1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5-and 3-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within {omega}1. CRISPResso analysis of amplicons spanning the DSB revealed [~]4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding {omega}1 were reduced >80% and lysates of {omega}1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the {omega}1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of {omega}1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding {omega}1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of {omega}1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of {omega}1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

molecular biology↗

bioRxiv2018

Meta-analysis of RNA-seq studies reveals genes responsible for life stage-dominant functions in Schistosoma mansoni

BackgroundSince the genome of the parasitic flatworm Schistosoma mansoni was sequenced in 2009, various RNA-seq studies have been conducted to investigate differential gene expression between certain life stages. Based on these studies, the overview of gene expression in all life stages can improve our understanding of S. mansoni genome biology.\n\nMethodspublicly available RNA-seq data covering all life stages and gonads were mapped to the latest S. mansoni genome. Read counts were normalised across all samples and differential expression analysis was preformed using the generalized linear model (GLM) approach.\n\nResultswe revealed for the first time the dissimilarities among all life stages. Genes that are abundantly-expressed in all life stages, as well as those preferentially-expressed in certain stage(s), were determined. The latter reveals genes responsible for stage-dominant functions of the parasite, which can be a guidance for the investigation and annotation of gene functions. In addition, distinct differential expression patterns were observed between adjacent life stages, which not only correlate well with original individual studies, but also provide additional information on changes in gene expression during parasite transitions. Furthermore, thirteen novel housekeeping genes across all life stages were identified, which is valuable for quantitative studies (e.g., qPCR).\n\nConclusionsthe metaanalysis provides valuable information on the expression and potential functions of S. mansoni genes across all life stages, and can facilitate basic as well as applied research for the community.

developmental biology↗