RNA interference in adult Ascaris suum – an opportunity for the development of a functional genomics platform that supports organism-, tissue- and cell-based biology in a nematode parasite

McCoy, Ciaran J.; Warnock, Neil D.; Atkinson, Louise E.; Atcheson, Erwan; Martin, R. J.; Robertson, Alan P.; Maule, Aaron G.; Marks, Nikki J.; Mousley, Angela

doi:10.1016/j.ijpara.2015.05.003

Cited by 45 publications

(40 citation statements)

References 26 publications

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“…S1). Variable transcript knockdown was also observed in other A. suum tissues injected IP with dsRNA in a previous study (McCoy et al, 2015), which may reflect differences in cellular uptake, relative abundances of target transcripts, or insufficient duration of the experiment (although 24 h was sufficient for other targets here).…”

Section: Resultssupporting

confidence: 61%

“…Previous research showed that IP injection of dsRNA caused transcript knockdown in adult A. suum tissues (including intestinal/hypodermis tissue), but intestine-specific effects were not delineated (McCoy et al, 2015). We performed IP treatments with otherwise intact worms (intact head and tail) and observed significant knockdown of target transcripts from the middle region of the intestine (associated with reproductive tissue) using either dsRNA or hsiRNA (hpo-19, 4 μg; Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The small size, biological complexity, and experimentally intractable nature of most parasitic nematodes hinder research aimed at elucidating essential functions in these pathogens. However, recently developed ‘omics’ resources (Jex et al, 2011; Jasmer et al, 2014; Rosa et al, 2014, 2015; Wang et al, 2015; Gao et al, 2016) and experimental capabilities (Chen et al, 2011; Jasmer et al, 2014; McCoy et al, 2015) established for the large round worm of swine and humans, Ascaris suum , continue to build on this global pathogen’s importance as a facile model system to conduct research on essential cellular functions in parasitic nematodes.…”

Section: Introductionmentioning

confidence: 99%

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Direct experimental manipulation of intestinal cells in Ascaris suum , with minor influences on the global transcriptome

Rosa

McNulty

Mitreva

et al. 2017

International Journal for Parasitology

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Ascaris suum provides a powerful model for studying parasitic nematodes, including individual tissues such as the intestine, an established target for anthelmintic treatments. Here, we add a valuable experimental component to our existing functional, proteomic, transcriptomic and phylogenomic studies of the Ascaris suum intestine, by developing a method to manipulate intestinal cell functions via direct delivery of experimental treatments (in this case, double-stranded (ds)RNA) to the apical intestinal membrane. We developed an intestinal perfusion method for direct, controlled delivery of dsRNA/heterogeneous small interfering (hsi) RNA into the intestinal lumen for experimentation. RNA-Seq (22 samples) was used to assess influences of the method on global intestinal gene expression. Successful mRNA-specific knockdown in intestinal cells of adult A. suum was accomplished with this new experimental method. Global transcriptional profiling confirmed that targeted transcripts were knocked down more significantly than any others, with only 12 (0.07% of all genes) or 238 (1.3%) off-target gene transcripts consistently differentially regulated by dsRNA treatment or the perfusion experimental design, respectively (after 24 h). The system supports controlled, effective delivery of treatments (dsRNA/hsiRNA) to the apical intestinal membrane with relatively minor off-target effects, and builds on our experimental model to dissect A. suum intestinal cell functions with broad relevance to parasitic nematodes.

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Section: Resultssupporting

confidence: 61%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Direct experimental manipulation of intestinal cells in Ascaris suum , with minor influences on the global transcriptome

Rosa

McNulty

Mitreva

et al. 2017

International Journal for Parasitology

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“…Despite there being a published genome of B. malayi (38), functional and genomic studies of the modes of action of anthelmintics in filaria have been difficult, and there have been only a few successful reports of RNAi experiments in B. malayi (34,(39)(40)(41)(42)(43)(44)(45)(46)(47). Here, we are able to describe a reliable RNAi method, using soaking in dsRNA that we have coupled with movement phenotyping and patch clamp to study the effects of cholinergic anthelmintics on adult B. malayi in vitro.…”

Section: Discussionmentioning

confidence: 99%

Functional genomics in Brugia malayi reveal diverse muscle nAChRs and differences between cholinergic anthelmintics

Verma

Kashyap

Robertson

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Many techniques for studying functional genomics of important target sites of anthelmintics have been restricted to Caenorhabditis elegans because they have failed when applied to animal parasites. To overcome these limitations, we have focused our research on the human nematode parasite Brugia malayi, which causes elephantiasis. Here, we combine single-cell PCR, whole muscle cell patch clamp, motility phenotyping (Worminator), and dsRNA for RNAi for functional genomic studies that have revealed, in vivo, four different muscle nAChRs (M-, L-, P-, and N-). The cholinergic anthelmintics had different selectivities for these receptors. We show that motility and patch-clamp responses to levamisole and pyrantel, but not morantel or nicotine, require the unc-38 and/or unc-29 genes. Derquantel behaved as a competitive antagonist and distinguished M-nAChRs activated by morantel (K b 13.9 nM), P-nAChRs activated by pyrantel (K b 126 nM), and L-nAChRs activated by levamisole (K b 0.96 μM) and bephenium. Derquantel was a noncompetitive antagonist of nicotine, revealing N-type nAChRs. The presence of four diverse nAChRs on muscle is perhaps surprising and not predicted from the C. elegans model. The diverse nAChRs represent distinguishable drug targets with different functions: Knockdown of unc-38+unc-29 (L-and/or P-receptors) inhibited motility but knockdown of acr-16+acr-26 (M-and/or N-receptors) did not.T he neglected tropical diseases caused by nematode parasites affect some 2 billion people globally, and effective vaccines are not available. Although these parasites by themselves do not produce high mortality, they produce significant morbidity that affects worker productivity and increase susceptibility to AIDS and malaria, and they are strongly associated with poverty in endemic countries (1-4). There are only a limited number of classes of anthelmintic drugs that are used to treat these diseases, and there are concerns about the development of resistance (5). There is a pressing need to advance the study of anthelmintic drugs.Many useful studies on modes of action of anthelmintic drugs have been conducted on the model nematode, Caenorhabditis elegans, but this is not a parasite: It is separated evolutionarily by millions of years from parasitic nematodes and it does not possess "parasitism genes" (6). This separation increases the impetus for anthelmintic studies using real parasites, but until now many techniques developed in C. elegans have not have been tractable when applied to animal parasites (7).The neuromuscular systems of nematodes, which control motility and support feeding, growth, and reproduction, are governed by significant parts of their gene pools (8, 9). Their neuromuscular systems are unlike those of vertebrates because nematode muscles send processes to the motor neurons to form synapses, rather than the other way around. Membrane ion-channels, which regulate nematode neuromuscular systems, are target sites of major classes of anthelmintic drugs, including the cholinergic anthelmintics levamisole,...

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“…Worms were maintained in Roswell Park Memorial Institute-1640 (RPMI-1640) medium, and viability was maintained in both systems for over two weeks. Dmitryjuk et al (2014) sustained adult A. suum in Ascaris ringer's solution (ARS) for 20 hours without any nutrient, while McCoy et al (2015) maintained A. suum for eight days in ARS without any nutrient. In contrast, only two studies have reported the in vitro maintenance of Parascaris spp.…”

Section: Introductionmentioning

confidence: 99%

Long live the worms: methods for maintaining and assessing the viability of intestinal stages of Parascaris spp. in vitro

et al. 2018

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In vitro maintenance of helminth parasites enables a variety of molecular, pharmaceutical and immunological analyses. Currently, the nutritional and environmental in vitro requirements of the equine ascarid parasite, Parascaris spp., have not been determined. Additionally, an objective method for assessing viability of Parascaris spp. intestinal stages does not exist. The purpose of this study was to ascertain the in vitro requirements of intestinal stages of Parascaris spp., and to develop a viability assessment method. A total of 1045 worms were maintained in a total of 212 cultures. Worms obtained from naturally infected foals at necropsy were immediately placed in culture flasks containing 200 mL of culture media. A variety of media types, nutrient supplementation and environmental conditions were examined. A motility-based scoring system was used to assess worm viability. Worms maintained in Roswell Park Memorial Institute-1640 had significantly better viability than any other media (P < 0.0001) and all media types supplemented with any of the nutrients examined (P < 0.0001). The use of a platform rocker also significantly improved viability (P = 0.0305). This is the first study to examine the requirements for maintaining Parascaris spp. intestinal stages in vitro and to evaluate their viability based on movement using an objective scoring system.

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RNA interference in adult Ascaris suum – an opportunity for the development of a functional genomics platform that supports organism-, tissue- and cell-based biology in a nematode parasite

Abstract: Graphical abstract

Cited by 45 publications

References 26 publications

Direct experimental manipulation of intestinal cells in Ascaris suum , with minor influences on the global transcriptome

Direct experimental manipulation of intestinal cells in Ascaris suum , with minor influences on the global transcriptome

Functional genomics in Brugia malayi reveal diverse muscle nAChRs and differences between cholinergic anthelmintics

Long live the worms: methods for maintaining and assessing the viability of intestinal stages of Parascaris spp. in vitro

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