Biology subjects

Sanders, M.

Publications and source records attributed to Sanders, M..

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bioRxiv2021

Daily rhythms in the transcriptomes of the human parasite Schistosoma mansoni

BackgroundThe consequences of the earths daily rotation have led to 24-hour biological rhythms in most organisms. Even parasites have daily rhythms, which, when in synchrony with host rhythms, optimize their fitness. Understanding these rhythms may enable development of novel control strategies that take advantage of rhythmic vulnerabilities. Recent work on blood-dwelling protozoan parasites has revealed daily rhythms in gene expression, physiology, drug sensitivity and the presence of an intrinsic circadian clock. However, similar studies on metazoan parasites are lacking. The aims of this study were to investigate if a metazoan parasite has daily molecular oscillations, whether they give us insight into how these longer-lived organisms can survive host daily cycles over a life-span of many years and to determine whether canonical metazoan 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 serious neglected tropical disease schistosomiasis. ResultsUsing round-the-clock transcriptomics of male and female adult worms we discovered that [~]2% of its genes followed a daily pattern of expression. Rhythmic processes included a night-time stress response and a day-time metabolic rush hour. 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 drug targets and a vaccine candidate. These 24hr rhythms may be driven by host rhythms and/or generated by a circadian clock. However, core clock genes are missing and orthologues of secondary clock genes show no 24hr rhythmicity in transcript abundance. ConclusionsThe daily rhythms identified here reveal temporally compartmentalised internal processes and host interactions over the daily cycle, including processes 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 Metazoa. Most importantly, knowing which gene transcripts oscillate at this temporal scale is relevant to functional genomic studies that will lead to the development and delivery of therapeutics against schistosomiasis.

systems biology↗

bioRxiv2021

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 anti-leishmanial compound

The implementation of prospective drug resistance (DR) studies in the R&D pipelines is a common practice for many infectious diseases, but not for Neglected Tropical Diseases. 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 GSK Leishbox to treat VL. We experimentally selected resistance to TCMDC-143345 in vitro and characterized resistant parasites at 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 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/resistance of L. donovani to TCMDC-143345. ImportanceHumans 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.

microbiology↗

bioRxiv2020

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

At least 250 million people worldwide suffer from schistosomiasis, caused by Schistosoma worms. Genome sequences for several Schistosoma species are available, including a high-quality annotated reference for Schistosoma mansoni. 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 S. mansoni, to produce somatic mutations in the omega-1 ({omega}1) gene. Here, we employed CRISPR-Cas9 to introduce somatic mutations in a second gene, SULT-OR, a sulfotransferase expressed in the parasitic stages of S. mansoni, in which mutations confer resistance to the drug oxamniquine. A 262-bp PCR product spanning the region targeted by the gRNA against SULT-OR 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 SULT-OR transcription, or leading to mRNA degradation via the nonsense-mediated mRNA decay pathway. However, no SULT-OR 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 SULT-OR. 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 SULT-OR gene to derive an oxamniquine-resistant stable transgenic line.

molecular biology↗

bioRxiv2020

Decreased torsinA or LINC Complex Function Rescues a Laminopathy in Caenorhabditis elegans

The function of the nucleus depends on the integrity of the nuclear lamina, an intermediate filament network associated with the linker of nucleoskeleton and cytoskeleton (LINC) complex spanning the nuclear envelope. In turn, the AAA+ ATPase torsinA regulates force transmission from the cytoskeleton to the nucleus. In humans, mutations affecting nuclear envelope-associated proteins cause laminopathies, including progeria, myopathy, and dystonia. We report that decreasing the function of the C. elegans torsinA homolog, OOC-5, rescues the sterility and premature aging caused by a null mutation in the single worm lamin homolog, lmn-1. Loss of OOC-5 activity prevents nuclear collapse in lmn-1 mutants by disrupting the function of the LINC complex. These results suggest that LINC complex-transmitted forces damage nuclei with a compromised nuclear lamina. One Sentence SummaryInhibiting LINC complex activity prevents a progeric syndrome in C. elegans.

developmental biology↗

bioRxiv2020

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

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 was 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.

microbiology↗

bioRxiv2019

The Phlebotomus papatasi transcriptomic response to trypanosomatid-contaminated blood is robust but non-specific

Leishmaniasis, caused by parasites of the genus Leishmania, is a disease that effects 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. In this work we wanted to identify fly transcriptomic signatures associated with infection success or failure. We used next generation sequencing to describe the transcriptome of the sand fly Phlebotomus papatasi when fed with blood alone or with blood containing one of three trypanosomatids: Leishmania major, Leishmania donovani and Herpetomonas muscarum: a parasite not transmitted to humans. Of these, only L. major was able to successfully establish an infection in P. papatasi. However, the transcriptional signatures observed were not specific to success or failure of infection but a generalised response to the blood meal. This implies that sand flies perceive Leishmania as just a 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. Authors summaryLeishmania are parasites that cause leishmaniasis, a group of serious diseases that affect millions of people, mainly across the subtropics and tropics. They are transmitted to humans by phlebotomine sand flies. However, despite establishment in the insects midgut being key to transmission, early infection events inside the insect are still unclear. Here, we study the gene expression response of the insect vector to a Leishmania parasite that is able to establish infection (L. major) one that is unable to do so (L. donovani) as well as one that is not a natural parasite of sand flies (Herpetomonas muscarum). We found that responses following any of the infected blood meals was very similar to uninfected blood meal. However, changes post-blood meal from day 1 to day 9 were dramatic. As a blood feeding insect can accumulate three times its weight in one blood meal, this seems to be the most important physiological change rather than the presence of the parasite. The latter might be just one in a number of microbes the insect encounters. This result will generate new thinking around the concept of stopping transmission by controlling the parasite inside the insect.

immunology↗

bioRxiv2019

The mutational profile and clonal landscape of the inflammatory bowel disease affected colon

Summary paragraphInflammatory bowel disease (IBD) is a chronic inflammatory disease associated with increased risk of gastrointestinal cancers. Here, we whole-genome sequenced 447 colonic crypts from 46 IBD patients, and compared these to 412 crypts from 41 non-IBD controls. The average mutation rate of affected colonic epithelial cells is 2.4-fold that of healthy colon and this increase is mostly driven by acceleration of mutational processes ubiquitously observed in normal colon. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimeter-scale clonal expansions. We discovered non-synonymous mutations in ARID1A, FBXW7, PIGR and ZC3H12A, and genes in the interleukin 17 and Toll-like receptor pathways, under positive selection in IBD. These results suggest distinct selection mechanisms in the colitis-affected colon and that somatic mutations potentially play a causal role in IBD pathogenesis.

genetics↗

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

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

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

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↗

bioRxiv2017

Leishmania naiffi and Leishmania guyanensis reference genomes highlight genome structure and gene content evolution in the Viannia subgenus

The unicellular protozoan parasite Leishmania causes the neglected tropical disease leishmaniasis, affecting 12 million people in 98 countries. In South America where the Viannia subgenus predominates, so far only L. (Viannia) braziliensis and L. (V.) panamensis 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, L. (V.) naiffi and L. (V.) guyanensis 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 L. braziliensis 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 L. guyanensis and L. naiffi with ten other Viannia genomes revealed four traits common to all Viannia: aneuploidy, 22 orthologous groups of genes absent in other Leishmania subgenera, elevated TATE transposon copies, and a high NADH-dependent fumarate reductase gene copy number. Within the Viannia, there were limited structural changes in genome architecture specific to individual species: a 45 Kb amplification on chromosome 34 was present in all bar L. lainsoni, L. naiffi had a higher copy number of the virulence factor leishmanolysin, and laboratory isolate L. shawi 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 Viannia has uncovered new insights into the origin and evolution of this subgenus and can help improve diagnostics for leishmaniasis surveillance.

genomics↗

bioRxiv2016

Genomes of an entire Plasmodium subgenus reveal paths to virulent human malaria

Plasmodium falciparum, the most virulent agent of human malaria, shares a recent common ancestor with the gorilla parasite P. 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 confidently for the first time 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.

evolutionary biology↗

bioRxiv2016

Complete avian malaria parasite genomes reveal host-specific parasite evolution in birds and mammals

Avian malaria parasites are prevalent around the world, and infect a wide diversity of bird species. Here we report the sequencing and analysis of high quality draft genome sequences for two avian malaria species, Plasmodium relictum and Plasmodium gallinaceum. We identify 50 genes that are specific to avian malaria, located in an otherwise conserved core of the genome that shares gene synteny with all other sequenced malaria genomes. Phylogenetic analysis suggests that the avian malaria species form an outgroup to the mammalian Plasmodium species and using amino acid divergence between species, we estimate the avian and mammalian-infective lineages diverged in the order of 10 million years ago. Consistent with their phylogenetic position, we identify orthologs of genes that had previously appeared to be restricted to the clades of parasites containing P. falciparum and P. vivax - the species with the greatest impact on human health. From these orthologs, we explore differential diversifying selection across the genus and show that the avian lineage is remarkable in the extent to which invasion related genes are evolving. The subtelomeres of the P. relictum and P. gallinaceum genomes contain several novel gene families, including an expanded surf multigene family. We also identify an expansion of reticulocyte binding protein homologs in P. relictum and within these proteins, we detect distinct regions that are specific to non-human primate, humans, rodent and avian hosts. For the first time in the Plasmodium lineage we find evidence of transposable elements, including several hundred fragments of LTR-retrotransposons in both species and an apparently complete LTR-retrotransposon in the genome of P. gallinaceum.

genomics↗