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

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

The Human Blood Fluke, Schistosoma mansoni, Harbors Bacteria Throughout the Parasite's Life Cycle.

While symbiotic relationships between invertebrates and bacteria have been extensively described, studies of microbial communities inhabiting parasitic worms remain scarce. Exploring the microbiota associated with helminths responsible for major infectious diseases will inform on parasite biology, host-pathogen interactions, and disease pathophysiology. We investigated the presence of microorganisms inhabiting tissues of the human parasite Schistosoma mansoni. In situ hybridization using a pan-bacterial 16S rRNA gene probe revealed bacteria colonizing key developmental stages that were successfully removed after antibiotic treatment of live parasites. Understanding the composition and function of the S. mansoni-associated microbiota may lead to the development of novel microbiome-targeting control strategies.

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

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.

Complete representation of a tapeworm genome reveals chromosomes capped by centromeres, necessitating a dual role in segregation and protection.

BACKGROUND: Chromosome-level assemblies are indispensable for accurate gene prediction, synteny assessment, and understanding higher-order genome architecture. Reference and draft genomes of key helminth species have been published, but little is yet known about the biology of their chromosomes. Here, we present the complete genome of the tapeworm Hymenolepis microstoma, providing a reference quality, end-to-end assembly that represents the first fully assembled genome of a spiralian/lophotrochozoan, revealing new insights into chromosome evolution. RESULTS: Long-read sequencing and optical mapping data were added to previous short-read data enabling complete re-assembly into six chromosomes, consistent with karyology. Small genome size (169 Mb) and lack of haploid variation (1 SNP/3.2 Mb) contributed to exceptionally high contiguity with only 85 gaps remaining in regions of low complexity sequence. Resolution of repeat regions reveals novel gene expansions, micro-exon genes, and spliced leader trans-splicing, and illuminates the landscape of transposable elements, explaining observed length differences in sister chromatids. Syntenic comparison with other parasitic flatworms shows conserved ancestral linkage groups indicating that the H. microstoma karyotype evolved through fusion events. Strikingly, the assembly reveals that the chromosomes terminate in centromeric arrays, indicating that these motifs play a role not only in segregation, but also in protecting the linear integrity and full lengths of chromosomes. CONCLUSIONS: Despite strong conservation of canonical telomeres, our results show that they can be substituted by more complex, species-specific sequences, as represented by centromeres. The assembly provides a robust platform for investigations that require complete genome representation.

Histone methylation changes are required for life cycle progression in the human parasite Schistosoma mansoni.

Epigenetic mechanisms and chromatin structure play an important role in development. Their impact is therefore expected to be strong in parasites with complex life cycles and multiple, strikingly different, developmental stages, i.e. developmental plasticity. Some studies have already described how the chromatin structure, through histone modifications, varies from a developmental stage to another in a few unicellular parasites. While H3K4me3 profiles remain relatively constant, H3K27 trimethylation and bivalent methylation show strong variation. Inhibitors (A366 and GSK343) of H3K27 histone methyltransferase activity in S. mansoni efficiently blocked miracidium to sporocyst transition indicating that H3K27 trimethylation is required for life cycle progression. As S. mansoni is a multicellular parasite that significantly affects both the health and economy of endemic areas, a better understanding of fluke developmental processes within the definitive host will likely highlight novel disease control strategies. Towards this goal, we also studied H4K20me1 in female cercariae and adults. In particular, we found that bivalent trimethylation of H3K4 and H3K27 at the transcription start site of genes is a landmark of the cercarial stage. In cercariae, H3K27me3 presence and strong enrichment in H4K20me1 over long regions (10-100 kb) is associated with development related genes. Here, we provide a broad overview of the chromatin structure of a metazoan parasite throughout its most important lifecycle stages. The five developmental stages studied here present distinct chromatin structures, indicating that histone methylation plays an important role during development. Hence, components of the histone methylation (and demethylation) machinery may provide suitable Schistosomiasis control targets.

The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction.

Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5' ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct.

What helminth genomes have taught us about parasite evolution.

The genomes of more than 20 helminths have now been sequenced. Here we perform a meta-analysis of all sequenced genomes of nematodes and Platyhelminthes, and attempt to address the question of what are the defining characteristics of helminth genomes. We find that parasitic worms lack systems for surface antigenic variation, instead maintaining infections using their surfaces as the first line of defence against the host immune system, with several expanded gene families of genes associated with the surface and tegument. Parasite excretory/secretory products evolve rapidly, and proteases even more so, with each parasite exhibiting unique modifications of its protease repertoire. Endoparasitic flatworms show striking losses of metabolic capabilities, not matched by nematodes. All helminths do however exhibit an overall reduction in auxiliary metabolism (biogenesis of co-factors and vitamins). Overall, the prevailing pattern is that there are few commonalities between the genomes of independently evolved parasitic worms, with each parasite having undergone specific adaptations for their particular niche.

Proteomic and genetic analyses demonstrate that Plasmodium berghei blood stages export a large and diverse repertoire of proteins.

Malaria parasites actively remodel the infected red blood cell (irbc) by exporting proteins into the host cell cytoplasm. The human parasite Plasmodium falciparum exports particularly large numbers of proteins, including proteins that establish a vesicular network allowing the trafficking of proteins onto the surface of irbcs that are responsible for tissue sequestration. Like P. falciparum, the rodent parasite P. berghei ANKA sequesters via irbc interactions with the host receptor CD36. We have applied proteomic, genomic, and reverse-genetic approaches to identify P. berghei proteins potentially involved in the transport of proteins to the irbc surface. A comparative proteomics analysis of P. berghei non-sequestering and sequestering parasites was used to determine changes in the irbc membrane associated with sequestration. Subsequent tagging experiments identified 13 proteins (Plasmodium export element (PEXEL)-positive as well as PEXEL-negative) that are exported into the irbc cytoplasm and have distinct localization patterns: a dispersed and/or patchy distribution, a punctate vesicle-like pattern in the cytoplasm, or a distinct location at the irbc membrane. Members of the PEXEL-negative BIR and PEXEL-positive Pb-fam-3 show a dispersed localization in the irbc cytoplasm, but not at the irbc surface. Two of the identified exported proteins are transported to the irbc membrane and were named erythrocyte membrane associated proteins. EMAP1 is a member of the PEXEL-negative Pb-fam-1 family, and EMAP2 is a PEXEL-positive protein encoded by a single copy gene; neither protein plays a direct role in sequestration. Our observations clearly indicate that P. berghei traffics a diverse range of proteins to different cellular locations via mechanisms that are analogous to those employed by P. falciparum. This information can be exploited to generate transgenic humanized rodent P. berghei parasites expressing chimeric P. berghei/P. falciparum proteins on the surface of rodent irbc, thereby opening new avenues for in vivo screening adjunct therapies that block sequestration.

The parasitic nematode Strongyloides ratti exists predominantly as populations of long-lived asexual lineages.

Nematodes are important parasites of people and animals, and in natural ecosystems they are a major ecological force. Strongyloides ratti is a common parasitic nematode of wild rats and we have investigated its population genetics using single-worm, whole-genome sequencing. We find that S. ratti populations in the UK consist of mixtures of mainly asexual lineages that are widely dispersed across a host population. These parasite lineages are likely very old and may have originated in Asia from where rats originated. Genes that underly the parasitic phase of the parasite's life cycle are hyperdiverse compared with the rest of the genome, and this may allow the parasites to maximise their fitness in a diverse host population. These patterns of parasitic nematode population genetics have not been found before and may also apply to Strongyloides spp. that infect people, which will affect how we should approach their control.

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.

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 in vitro 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. IMPORTANCE 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.

WormBase in 2022-data, processes, and tools for analyzing Caenorhabditis elegans.

WormBase (www.wormbase.org) is the central repository for the genetics and genomics of the nematode Caenorhabditis elegans. We provide the research community with data and tools to facilitate the use of C. elegans and related nematodes as model organisms for studying human health, development, and many aspects of fundamental biology. Throughout our 22-year history, we have continued to evolve to reflect progress and innovation in the science and technologies involved in the study of C. elegans. We strive to incorporate new data types and richer data sets, and to provide integrated displays and services that avail the knowledge generated by the published nematode genetics literature. Here, we provide a broad overview of the current state of WormBase in terms of data type, curation workflows, analysis, and tools, including exciting new advances for analysis of single-cell data, text mining and visualization, and the new community collaboration forum. Concurrently, we continue the integration and harmonization of infrastructure, processes, and tools with the Alliance of Genome Resources, of which WormBase is a founding member.

The genome of the protist parasite Entamoeba histolytica.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.

Individual-level variations in malaria susceptibility and acquisition of clinical protection.

After decades of research, our understanding of when and why individuals infected with Plasmodium falciparum develop clinical malaria is still limited. Correlates of immune protection are often sought through prospective cohort studies, where measured host factors are correlated against the incidence of clinical disease over a set period of time. However, robustly inferring individual-level protection from these population-level findings has proved difficult due to small effect sizes and high levels of variance underlying such data. In order to better understand the nature of these inter-individual variations, we analysed the long-term malaria epidemiology of children ≤12 years old growing up under seasonal exposure to the parasite in the sub-location of Junju, Kenya. Despite the cohort's limited geographic expanse (ca. 3km x 10km), our data reveal a high degree of spatial and temporal variability in malaria prevalence and incidence rates, causing individuals to experience varying levels of exposure to the parasite at different times during their life. Analysing individual-level infection histories further reveal an unexpectedly high variability in the rate at which children experience clinical malaria episodes. Besides exposure to the parasite, measured as disease prevalence in the surrounding area, we find that the birth time of year has an independent effect on the individual's risk of experiencing a clinical episode. Furthermore, our analyses reveal that those children with a history of an above average number of episodes are more likely to experience further episodes during the upcoming transmission season. These findings are indicative of phenotypic differences in the rates by which children acquire clinical protection to malaria and offer important insights into the natural variability underlying malaria epidemiology.

Mapping Rora expression in resting and activated CD4+ T cells.

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 in general, but with an emphasis on Th2 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.

Genetics of mating and sex determination in the parasitic nematode Haemonchus contortus.

Genetic analysis of parasitic nematodes has been a neglected area of research and the basic genetics of this important group of pathogens are poorly understood. Haemonchus contortus is one of the most economically significant livestock parasites worldwide and is a key experimental model for the strongylid nematode group that includes many important human and animal pathogens. We have undertaken a study of the genetics and the mode of mating of this parasite using microsatellite markers. Inheritance studies with autosomal markers demonstrated obligate dioecious sexual reproduction and polyandrous mating that are reported here for the first time in a parasitic helminth and provide the parasite with a mechanism of increasing genetic diversity. The karyotype of the H. contortus, MHco3(ISE) isolate was determined as 2n = 11 or 12. We have developed a panel of microsatellite markers that are tightly linked on the X chromosome and have used them to determine the sex chromosomal karyotype as XO male and XX female. Haplotype analysis using the X-chromosomal markers also demonstrated polyandry, independent of the autosomal marker analysis, and enabled a more direct estimate of the number of male parental genotypes contributing to each brood. This work provides a basis for future forward genetic analysis on H. contortus and related parasitic nematodes.

GeneDB and Wikidata.

Publishing authoritative genomic annotation data, keeping it up to date, linking it to related information, and allowing community annotation is difficult and hard to support with limited resources. Here, we show how importing GeneDB annotation data into Wikidata allows for leveraging existing resources, integrating volunteer and scientific communities, and enriching the original information.

Evolutionary analysis of the most polymorphic gene family in falciparum malaria.

The var gene family of the human malaria parasite Plasmodium falciparum encode proteins that are crucial determinants of both pathogenesis and immune evasion and are highly polymorphic. Here we have assembled nearly complete var 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 var 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 var repertoire with respect to the ancestral genes within the Laverania 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.

Identification and expression profiling of microRNAs in Hymenolepis.

Tapeworms (cestodes) of the genus Hymenolepis are the causative agents of hymenolepiasis, a neglected zoonotic disease. Hymenolepis nana is the most prevalent human tapeworm, especially affecting children. The genomes of Hymenolepis microstoma and H. nana have been recently sequenced and assembled. MicroRNAs (miRNAs), a class of small non-coding RNAs, are principle regulators of gene expression at the post-transcriptional level and are involved in many different biological processes. In previous work, we experimentally identified miRNA genes in the cestodes Echinococcus, Taenia and Mesocestoides. However, current knowledge about miRNAs in Hymenolepis is limited. In this work we described for the first known time the expression profile of the miRNA complement in H. microstoma, and discovered miRNAs in H. nana. We found a reduced complement of 37 evolutionarily conserved miRNAs, putatively reflecting their low morphological complexity and parasitic lifestyle. We found high expression of a few miRNAs in the larval stage of H. microstoma that are conserved in other cestodes, suggesting that these miRNAs may have important roles in development, survival and for host-parasite interplay. We performed a comparative analysis of the identified miRNAs across the Cestoda and showed that most of the miRNAs in Hymenolepis are located in intergenic regions, implying that they are independently transcribed. We found a Hymenolepis-specific cluster composed of three members of the mir-36 family. Also, we found that one of the neighboring genes of mir-10 was a Hox gene as in most bilaterial species. This study provides a valuable resource for further experimental research in cestode biology that might lead to improved detection and control of these neglected parasites. The comprehensive identification and expression analysis of Hymenolepis miRNAs can help to identify novel biomarkers for diagnosis and/or novel therapeutic targets for the control of hymenolepiasis.

Genome-wide transcriptome profiling and spatial expression analyses identify signals and switches of development in tapeworms.

BACKGROUND: Tapeworms are agents of neglected tropical diseases responsible for significant health problems and economic loss. They also exhibit adaptations to a parasitic lifestyle that confound comparisons of their development with other animals. Identifying the genetic factors regulating their complex ontogeny is essential to understanding unique aspects of their biology and for advancing novel therapeutics. Here we use RNA sequencing to identify up-regulated signalling components, transcription factors and post-transcriptional/translational regulators (genes of interest, GOI) in the transcriptomes of Larvae and different regions of segmented worms in the tapeworm Hymenolepis microstoma and combine this with spatial gene expression analyses of a selection of genes. RESULTS: RNA-seq reads collectively mapped to 90% of the > 12,000 gene models in the H. microstoma v.2 genome assembly, demonstrating that the transcriptome profiles captured a high percentage of predicted genes. Contrasts made between the transcriptomes of Larvae and whole, adult worms, and between the Scolex-Neck, mature strobila and gravid strobila, resulted in 4.5-30% of the genes determined to be differentially expressed. Among these, we identified 190 unique GOI up-regulated in one or more contrasts, including a large range of zinc finger, homeobox and other transcription factors, components of Wnt, Notch, Hedgehog and TGF-ß/BMP signalling, and post-transcriptional regulators (e.g. Boule, Pumilio). Heatmap clusterings based on overall expression and on select groups of genes representing 'signals' and 'switches' showed that expression in the Scolex-Neck region is more similar to that of Larvae than to the mature or gravid regions of the adult worm, which was further reflected in large overlap of up-regulated GOI. CONCLUSIONS: Spatial expression analyses in Larvae and adult worms corroborated inferences made from quantitative RNA-seq data and in most cases indicated consistency with canonical roles of the genes in other animals, including free-living flatworms. Recapitulation of developmental factors up-regulated during larval metamorphosis suggests that strobilar growth involves many of the same underlying gene regulatory networks despite the significant disparity in developmental outcomes. The majority of genes identified were investigated in tapeworms for the first time, setting the stage for advancing our understanding of developmental genetics in an important group of flatworm parasites.