Biology subjects

Devaney, E.

Publications and source records attributed to Devaney, E..

12 recordsLinked to original sources

bioRxiv2024

A secreted helminth microRNA suppresses gastrointestinal cell differentiation required for innate immunity

Pathogens have developed multiple strategies to modulate host immune defense mechanisms. Understanding how this is achieved has potential to inform novel therapeutics for diseases caused by immune dysfunction. Parasitic helminths are masters of immune evasion, via release of secreted products, resulting in chronic infection. Helminths secrete small regulatory microRNA (miRNAs), which can interact with host cells. Here we show that a single parasite miRNA (miR-5352), conserved across gastrointestinal (GI) nematodes, suppresses IL-13-induced GI epithelial cell differentiation and cytokine responses, and promotes stem cell maintenance. Mechanistically, this is achieved through targeted repression of critical host factors, including Klf-4 and the IL-22 receptor, together with modulation of Wnt and Notch signalling pathways. Nematode miR-5352 shows seed sequence conservation with mammalian miR-92a family members, indicating that through convergent evolution, GI nematodes exploit a host miRNA regulatory network to suppress host innate responses, promote tissue regeneration and establish a favourable environment for chronic infection.

microbiology↗

bioRxiv2024

Gene transfer of human cardiomyopathy beta-MyHC mutant R403Qdirectly alters intact cardiac myocyte calcium homeostasis and causes hyper-contractility

The R403Q mutation of human cardiac {beta}-myosin heavy chain was the first missense mutation of a sarcomeric protein identified as being causal for hypertrophic cardiomyopathy (HCM), in humans. The direct effect of the R403Q mutant myosin on intracellular calcium homeostasis and contractility is not fully known. Here we have used in vitro gene transfer of the R403Q mutant human {beta}-myosin to study its direct effects on single intact adult cardiac myocyte contractility and calcium homeostasis. In the first experiments, adult cardiac myocytes transduced with the R403Q mutant myosin recombinant viral vectors were compared to myocytes transduced with wild-type human {beta}-myosin (wtMYH7). Efficiency of gene transfer was high in both groups (>98%) and the degree of stoichiometric myofilament incorporation of either the mutant or normal myosin was comparable at [~]40% in quiescent myocytes in primary culture. Sarcomere structure and cellular morphology were unaffected by R403Q myosin expression and myofilament incorporation. Functionally, in electrically paced cardiac myocytes, the R403Q mutant myosin caused a significant increase in intracellular calcium concentration and myocyte hyper-contractility. At the sub-cellular myofilament level, the mutant myosin increased the calcium sensitivity of steady state isometric tension development and increased isometric cross-bridge cycling kinetics. R403Q myocytes became arrhythmic after {beta}-adrenergic stimulation with spontaneous calcium transients and contractions in between electrical stimuli. These results indicate that human R403Q mutant myosin directly alters myofilament function and intracellular calcium cycling. Elevated calcium levels may provide a trigger for the ensuing hypertrophy and susceptibility to arrhythmia that are characteristic of HCM.

physiology↗

bioRxiv2024

Chromosomal genome assembly resolves drug resistance loci in the parasitic nematode Teladorsagia circumcincta

The parasitic nematode Teladorsagia circumcincta is one of the most important pathogens of sheep and goat farming in temperate climates worldwide and can rapidly evolve resistance to drugs used to control it. To understand the genetics of drug resistance, we have generated a highly contiguous genome assembly for the UK T. circumcincta isolate, MTci2. Assembly using PacBio long-reads and Hi-C long-molecule scaffolding together with manual curation resulted in a 573 Mb assembly (N50 = 84 Mb, n = 1,286) with five autosomal and one sex-linked chromosomal-scale scaffolds consistent with its karyotype. The genome resource was further improved via annotation of 22,948 genes, with manual curation of over 3,200 of these, resulting in a robust and near complete resource (96.3% complete protein BUSCOs) to support basic and applied research on this important veterinary pathogen. Genome-wide analyses of drug resistance, combining evidence from three distinct experiments, identified selection around known candidate genes for benzimidazole, levamisole and ivermectin resistance, as well as novel regions associated with ivermectin and moxidectin resistance. These insights into contemporary and historic genetic selection further emphasise the importance of contiguous genome assemblies in interpreting genome-wide genetic variation associated with drug resistance and identified key loci to prioritise in developing diagnostic markers of anthelmintic resistance to support parasite control. AUTHOR SUMMARYUnderstanding the genetics of anthelmintic resistance is a critical part of the sustainable control of parasitic worms. Here, we have generated a chromosome-scale genome assembly for the gastrointestinal nematode, Teladorsagia circumcincta, one of the most important pathogens of sheep in temperate climates worldwide. This genome and its annotation offers a substantial improvement over existing genetic resources, and will enable new insight into the biology for this parasite and new opportunities to characterise therapeutic targets such as drug and vaccine candidates. We used this resource to map genetic variation associated with resistance to multiple anthelmintic drug classes used as the primary means of parasite control, confirming known candidate genes and variants (i.e., beta-tubulin isotypes 1 and 2 associated with benzimidazole resistance, and acr-8 associated with levamisole resistance, and pgp-9 copy number variation, of which overexpression is associated broadly with anthelmintic resistance) and revealing new regions of the genome associated with drug treatment responses (i.e. 34-38 Mb on chromosome 5 associated with ivermectin resistance). Our results highlight the importance of a contiguous genome assembly and the use of complementary experimental approaches to reveal the impact of drug-mediated selection, both historically and directly in response to treatment, on genome-wide genetic variation.

genomics↗

bioRxiv2023

Tuft cell-derived acetylcholine is an effector of type 2 immunity and directly targets helminth parasites in the gut lumen

Upon parasitic helminth infection, activated intestinal tuft cells secrete IL-25, which initiates a type 2 immune response during which lamina propria ILC2s produce IL-13. This causes epithelial remodelling, including tuft cell hyperplasia with an unknown function. We describe a novel cholinergic effector function of tuft cells, which we show are the only epithelial cells expressing Choline Acetyltransferase (ChAT). During parasite infections, mice with epithelial-specific deletion of ChAT have increased worm burden and faecal egg counts although they are able to mount a comparable type 2 immune response. Mechanistically, IL-13-amplified tuft cells release acetylcholine (ACh) into the gut lumen. We demonstrate a direct effect of ACh on worms, reducing their viability and fecundity via helminth muscarinic ACh receptors, with effects promoted by inhibition of acetylcholinesterase, an helminth-secreted enzyme. Thus, tuft cells are sentinels in naive mice, and their amplification upon helminth infections serves an additional type 2 immune response effector function.

immunology↗

bioRxiv2022

Allele specific PCR for a major marker of levamisole resistance in Haemonchus contortus

Haemonchus contortus is a haematophagous parasitic nematode that infects small ruminants and causes significant animal health concerns and economic losses within the livestock industry on a global scale. Treatment primarily depends on broad-spectrum anthelmintics, however, resistance is established or rapidly emerging against all major drug classes. Levamisole (LEV) remains an important treatment option for parasite control, as resistance to LEV is less prevalent than to members of other major classes of anthelmintics. LEV is an acetylcholine receptor (AChR) agonist that, when bound, results in paralysis of the worm. Numerous studies implicated the AChR sub-unit, ACR-8, in LEV sensitivity and in particular, the presence of a truncated acr-8 transcript or a deletion in the acr-8 locus in some resistant isolates. Recently, a single non-synonymous SNP in acr-8 conferring a serine-to-threonine substitution (S168T) was identified that was strongly associated with LEV resistance. Here, we investigate the role of genetic variation at the acr-8 locus in a controlled genetic cross between the LEV susceptible MHco3(ISE) and LEV resistant MHco18(UGA 2004) isolates of H. contortus. Using single worm PCR assays, we found that the presence of S168T was strongly associated with LEV resistance in the parental isolates and F3 progeny of the genetic cross surviving LEV treatment. We developed and optimised an allele-specific PCR assay for the detection of S168T and validated the assay using laboratory isolates and field samples that were phenotyped for LEV resistance. In the LEV-resistant field population, a high proportion (>75%) of L3 encoded the S168T variant, whereas the variant was absent in the susceptible isolates studied. These data further support the potential role of acr-8 S168T in LEV resistance, with the allele-specific PCR providing an important step towards establishing a sensitive molecular diagnostic test for LEV resistance.

microbiology↗

bioRxiv2021

Genomic landscape of drug response reveals novel mediators of anthelmintic resistance

Understanding the genetic basis of anthelmintic drug resistance in parasitic nematodes is key to improving the efficacy and sustainability of parasite control. Here, we use a genetic cross in a natural host-parasite system to simultaneously map resistance loci for the three major classes of anthelmintics. This approach identifies novel alleles for resistance to benzimidazoles and levamisole and implicates the transcription factor, cky-1, in ivermectin resistance. This gene is within a locus under selection in ivermectin resistant populations worldwide; functional validation using knockout experiments supports a role for cky-1 overexpression in ivermectin resistance. Our work demonstrates the feasibility of high-resolution forward genetics in a parasitic nematode, and identifies variants for the development of molecular diagnostics to combat drug resistance in the field. One-Sentence SummaryGenetic mapping of known and novel anthelmintic resistance-associated alleles in a multi-drug resistant parasitic nematode

genomics↗

bioRxiv2021

Transcriptomic analyses implicate neuronal plasticity and chloride homeostasis in ivermectin resistance and recovery in a parasitic nematode

The antiparasitic drug ivermectin plays an essential role in human and animal health globally. However, ivermectin resistance is widespread in veterinary helminths and there are growing concerns of sub-optimal responses to treatment in related helminths of humans. Despite decades of research, the genetic mechanisms underlying ivermectin resistance are poorly understood in parasitic helminths. This reflects significant uncertainty regarding the mode of action of ivermectin in parasitic helminths, and the genetic complexity of these organisms; parasitic helminths have large, rapidly evolving genomes and differences in evolutionary history and genetic background can confound comparisons between resistant and susceptible populations. We undertook a controlled genetic cross of a multi-drug resistant and a susceptible reference isolate of Haemonchus contortus, an economically important gastrointestinal nematode of sheep, and ivermectin-selected the F2 population for comparison with an untreated F2 control. RNA-seq analyses of male and female adults of all populations identified high transcriptomic differentiation between parental isolates, which was significantly reduced in the F2, allowing differences associated specifically with ivermectin resistance to be identified. In all resistant populations, there was constitutive upregulation of a single gene, HCON_00155390:cky-1, a putative pharyngeal-expressed transcription factor, in a narrow locus on chromosome V previously shown to be under ivermectin selection. In addition, we detected sex-specific differences in gene expression between resistant and susceptible populations, including constitutive upregulation of a P-glycoprotein, HCON_00162780:pgp-11, in resistant males only. After ivermectin selection, we identified differential expression of genes with roles in neuronal function and chloride homeostasis, which is consistent with an adaptive response to ivermectin-induced hyperpolarisation of neuromuscular cells. Overall, we show the utility of a genetic cross to identify differences in gene expression that are specific to ivermectin selection and provide a framework to better understand ivermectin resistance and recovery in parasitic helminths. Author SummaryParasitic helminths (worms) infect people and animals throughout the world and are largely controlled with mass administration of anthelmintic drugs. There are a very limited number of anthelmintics available and parasitic helminths can rapidly develop resistance to these drugs. Ivermectin is a widely used anthelmintic in both humans and animals, but resistance is now widespread in the veterinary field. We crossed ivermectin resistant and ivermectin susceptible parasitic helminths and treated them with ivermectin or left them as untreated controls. This provided resistant and susceptible populations with a similar genetic background with which to study differences in gene expression associated with ivermectin resistance. We identified upregulation of a gene with no previous association with drug resistance (HCON_00155390:cky-1) in male and female worms in all resistant populations. This gene is thought to be expressed in the helminth pharynx (mouthpart) and, in mammals, plays a role in controlling nerve function and protecting nerves from damage. This is consistent with the known effects of ivermectin in inhibiting helminth feeding through pharyngeal paralysis and implicates a novel mechanism that allows resistant worms to survive treatment.

genomics↗

bioRxiv2020

Extensive genomic and transcriptomic variation defines the chromosome-scale assembly of Haemonchus contortus, a model gastrointestinal worm

BackgroundHaemonchus contortus is a globally distributed and economically important gastrointestinal pathogen of small ruminants, and has become the key nematode model for studying anthelmintic resistance and other parasite-specific traits among a wider group of parasites including major human pathogens. Two draft genome assemblies for H. contortus were reported in 2013, however, both were highly fragmented, incomplete, and differed from one another in important respects. While the introduction of long-read sequencing has significantly increased the rate of production and contiguity of de novo genome assemblies broadly, achieving high quality genome assemblies for small, genetically diverse, outcrossing eukaryotic organisms such as H. contortus remains a significant challenge. ResultsHere, we report using PacBio long read and OpGen and 10X Genomics long-molecule methods to generate a highly contiguous 283.4 Mbp chromosome-scale genome assembly including a resolved sex chromosome. We show a remarkable pattern of almost complete conservation of chromosome content (synteny) with Caenorhabditis elegans, but almost no conservation of gene order. Long-read transcriptome sequence data has allowed us to define coordinated transcriptional regulation throughout the life cycle of the parasite, and refine our understanding of cis- and trans-splicing relative to that observed in C. elegans. Finally, we use this assembly to give a comprehensive picture of chromosome-wide genetic diversity both within a single isolate and globally. ConclusionsThe H. contortus MHco3(ISE).N1 genome assembly presented here represents the most contiguous and resolved nematode assembly outside of the Caenorhabditis genus to date, together with one of the highest-quality set of predicted gene features. These data provide a high-quality comparison for understanding the evolution and genomics of Caenorhabditis and other nematodes, and extends the experimental tractability of this model parasitic nematode in understanding pathogen biology, drug discovery and vaccine development, and important adaptive traits such as drug resistance.

genomics↗

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↗

bioRxiv2018

A major locus for ivermectin resistance in a parasitic nematode

BackgroundInfections with helminths cause an enormous disease burden in billions of animals and plants worldwide. Large scale use of anthelmintics has driven the evolution of resistance in a number of species that infect livestock and companion animals, and there are growing concerns regarding the reduced efficacy in some human-infective helminths. Understanding the mechanisms by which resistance evolves is the focus of increasing interest; robust genetic analysis of helminths is challenging, and although many candidate genes have been proposed, the genetic basis of resistance remains poorly resolved. ResultsHere, we present a genome-wide analysis of two genetic crosses between ivermectin resistant and sensitive isolates of the parasitic nematode Haemonchus contortus, an economically important gastrointestinal parasite of small ruminants and a model for anthelmintic research. Whole genome sequencing of parental populations, and key stages throughout the crosses, identified extensive genomic diversity that differentiates populations, but after backcrossing and selection, a single genomic quantitative trait locus (QTL) localised on chromosome V was revealed to be associated with ivermectin resistance. This QTL was common between the two geographically and genetically divergent resistant populations and did not include any leading candidate genes, suggestive of a previously uncharacterised mechanism and/or driver of resistance. Despite limited resolution due to low recombination in this region, population genetic analyses and novel evolutionary models supported strong selection at this Q.TL, driven by at least partial dominance of the resistant allele, and that large resistance-associated haplotype blocks were enriched in response to selection. ConclusionsWe have described the genetic architecture and mode of ivermectin selection, revealing a major genomic locus associated with ivermectin resistance, the most conclusive evidence to date in any parasitic nematode. This study highlights a novel genome-wide approach to the analysis of a genetic cross in non-model organisms with extreme genetic diversity, and the importance of a high quality reference genome in interpreting the signals of selection so identified.

genomics↗

bioRxiv2017

A genome resequencing-based genetic map reveals the recombination landscape of an outbred parasitic nematode in the presence of polyploidy and polyandry

The parasitic nematode Haemonchus contortus is an economically and clinically important pathogen of small ruminants, and a model system for understanding the mechanisms and evolution of traits such as anthelmintic resistance. Anthelmintic resistance is widespread and is a major threat to the sustainability of livestock agriculture globally; however, little is known about the genome architecture and parameters such as recombination that will ultimately influence the rate at which resistance may evolve and spread. Here we performed a genetic cross between two divergent strains of H. contortus, and subsequently used whole-genome re-sequencing of a female worm and her brood to identify the distribution of genome-wide variation that characterises these strains. Using a novel bioinformatic approach to identify variants that segregate as expected in a pseudo-testcross, we characterised linkage groups and estimated genetic distances between markers to generate a chromosome-scale F1 genetic map composed of 1,618 SNPs. We exploited this map to reveal the recombination landscape, the first for any parasitic helminth species, demonstrating extensive variation in recombination rate within and between chromosomes. Analyses of these data also revealed the extent of polyandry, whereby at least eight males were found to have contributed to the genetic variation of the progeny analysed. Triploid offspring were also identified, which we hypothesise are the result of nondisjunction during female meiosis or polyspermy. These results expand our knowledge of the genetics of parasitic helminths and the unusual life-history of H. contortus, and will enable more precise characterisation of the evolution and inheritance of genetic traits such as anthelmintic resistance. This study also demonstrates the feasibility of whole-genome resequencing data to directly construct a genetic map in a single generation cross from a non-inbred non-model organism with a complex lifecycle.\n\nAuthor summaryRecombination is a key genetic process, responsible for the generation of novel genotypes and subsequent phenotypic variation as a result of crossing over between homologous chromosomes. Populations of strongylid nematodes, such as the gastrointestinal parasites that infect livestock and humans, are genetically very diverse, but little is known about patterns of recombination across the genome and how this may contribute to the genetics and evolution of these pathogens. In this study, we performed a genetic cross to quantify recombination in the barbers pole worm, Haemonchus contortus, an important parasite of sheep and goats. The reproductive traits of this worm make standard genetic crosses challenging, but by generating whole-genome sequence data from a female worm and her offspring, we identified genetic variants that act as though they come from a single mating cross, allowing the use of standard statistical approaches to build a genetic map and explore the distribution and rates of recombination throughout the genome. A number of genetic signatures associated with H. contortus life history traits were revealed in this analysis: we extend our understanding of multiple paternity (polyandry) in this species, and provide evidence and explanation for sporadic increases in chromosome complements (polyploidy) among the progeny. The resulting genetic map will aid in population genomic studies in general and enhance ongoing efforts to understand the genetic basis of resistance to the drugs used to control these worms, as well as for related species that infect humans throughout the world.

genomics↗

bioRxiv2017

Genomic And Transcriptomic Profiling Of Nematode Parasites Surviving After Vaccine Exposure

Some nematode species are economically important parasites of livestock, while others are important human pathogens causing some of the most important neglected tropical diseases. In both humans and animals, anthelmintic drug administration is the main control strategy, but the emergence of drug-resistant worms has stimulated the development of alternative control approaches. Among these, vaccination is considered to be a sustainable and cost effective strategy. Currently, Barbervax(R) for the ruminant strongylid Haemonchus contortus is the only registered subunit vaccine for a nematode parasite, although a vaccine for the human hookworm Necator americanus is undergoing clinical trials (HOOKVAC consortium). As both these vaccines comprise a limited number of proteins there is potential for selection of nematodes with altered sequence or expression of the vaccine antigens. Here we compared the transcriptome of H. contortus populations from sheep vaccinated with Barbervax(R) with worms from control animals. Barbervax(R) antigens are native integral membrane proteins isolated from the brush border of the intestinal cells of the adult parasite and many of them are proteases. Our findings provide no evidence for changes in expression of genes encoding Barbervax(R) antigens in the surviving parasite populations. However, surviving parasites from vaccinated animals showed increased expression of other proteases and regulators of lysosome trafficking, and displayed up-regulated lipid storage and defecation abilities that may have circumvented the vaccine effect. Implications for other potential vaccines for human and veterinary nematodes are discussed.

genomics↗