ASCARIS SUUM, A NEW FUNCTIONAL GENOMICS PLATFORM FOR NEMATODE PARASITES

Lead Research Organisation: Queen's University of Belfast
Department Name: Sch of Biological Sciences

Abstract

Nematodes (roundworms) are members of the Phylum Nematoda. There are >25,000 species of nematodes and they are outnumbered only by the Arthropods. Their success is due to their ability to occupy a diverse range of habitats. They can either be free-living or parasites of humans, animals and plants. Nematode parasites are a common source of human disease where 1 in 4 people carry at least one nematode species. Human parasitic diseases are mainly restricted to the developing world where poverty, inadequate health care provision, and poor living conditions favour their survival. Some of the most prevalent human nematode parasites live in the gasterointestinal (GI) system feeding on human tissue, blood and gut contents. For example, Ascaris lumbricoides is one of the largest GI nematodes and can cause serious health problems associated with intestinal blockage and impaired growth, especially in children. In livestock, nematode parasites can be a significant problem as they impact not only on the health and well-being of the animal but also on the productivity and subsequent profitability of the farming industry. Haemonchus contortus and Teladorsagia circumcincta are the most pathogenic nematodes of sheep and goats. They are blood-feeders and heavy infections can lead to severe anaemia and animal death. Nematodes are also problematic to the crop industry. They can infect food crops such as tomatoes and potatoes or utility grasses including football pitches or golf courses. Meloidogyne spp are a particular problem because they infect a wide range of plant hosts. They impair plant health by setting up feeding sites in the plant root, removing nutrients needed for plant growth.
Unfortunately the drugs that are available to treat parasitic nematode infections no longer work effectively or are environmentally toxic. We cannot continue to treat nematode infections with the current range of drugs and we must now actively seek new drug targets and develop novel drugs. This is particularly important to the UK as we have to ensure the sustainability of livestock farming for future food production.
This project aims to uncover novel drug targets for the treatment of nematode parasites of livestock. In this project we will collaborate with an animal-health pharmaceutical company to accelerate the chance of identifying a novel drug target at which new drugs could be directed. This approach will make use of scientific research skills and the expertise of the pharmaceutical industry in drug discovery. In order to find new drug targets for nematode parasites, we must first identify proteins that are important to nematode biology and survival. Recently there has been an increase in the availability of gene sequences for a number of important parasitic nematodes, like those described above. We will search through these sequences and find those which code for protein targets which may be essential to parasite survival. We will select sequences that are found in multiple parasites so that we can identify a drug target which could be used to treat multiple nematode diseases. We will then use a technique called RNA interference (RNAi) which allows us to switch off genes in the parasitic nematode to find out their function. For example, if we switch off a gene and the nematode dies or stops moving/feeding/reproducing then we have identified a good drug target candidate as the nematode can no longer infect or remain in its host. We will perform RNAi in a model nematode parasite (Ascaris suum) that we can easily collect from pig intestines at local abattoirs and maintain in the lab. Once we have switched off the target genes, we will determine the impact to the parasite by examining how they survive, behave, move, reproduce, and respond to stimulants. We will then select the five 'best' targets, based on their impact to nematode biology, and deliver these to the pharmaceutical industry who will develop drugs against them.

Technical Summary

Nematode parasites impose a significant burden on human/animal health and global food security. The agricultural impact of nematodiasis in the UK is ~£80 million/year, mainly attributed to animal death, morbidity, reduced productivity and reproductive impairment. Traditionally chemotherapeutics have been the foundation of nematode parasite control, but decreasing utility / treatment failures have exposed the rapid development of drug resistance, including multi-drug resistance such that current control options are not sustainable. As a result, novel chemotherapeutics are critical. In this study we will employ both contemporary and traditional approaches to identify and validate novel drug target candidates for exploitation by our industrial partner. Our target-identification strategy will be directed by the recent progress in parasitic nematode genomics/ transcriptomics and will score targets based on their conservation across the phylum, known druggability in other systems, key role in nematode biology and/or survival, and expression in therapeutically relevant lifecycle stages/tissues. Our approaches to functional validation combine two unique attributes offered by this industrial-partnership collaboration: we will use our newly developed adult A. suum RNA interference (RNAi) platform to investigate target function/importance to parasite biology; uniquely, we will interface these reverse genetic approaches with privileged access to the Merial/Sanofi chemical library, which will provide powerful tools to help steer early target prioritisation and to probe functional importance. The combination of both of these tools will provide a powerful and unique approach to animal parasite drug target validation. These efforts will provide the first target validation platform for an animal parasitic nematode and will deliver five functionally-validated drug target candidates for industrial exploitation.

Planned Impact

A. Commercial Private Sector
1. Pharma/Biotech will benefit through economic rewards associated with the provision of a new anthelmintic to a keen market. Our industrial partner (Merial) is committed to developing validated drug targets emerging from this project and exploiting these as screening tools for anthelmintic discovery. This project will also optimise a reverse genetics platform for validation of drug targets in nematode parasites, which will be available for exploitation by other pharma/biotech industries. Additional novel targets arising from this project, which are not of primary interest to Merial, will be available for validation by other industries. This is relevant to those developing treatments for both animal and human nematodiasis, and could provide opportunities for new jobs in the research/development/production pipeline.

2. Local and global farming enterprises will benefit through enhanced economic returns associated with healthier livestock that are more productive. The total income from farming in the UK was £4.7 billion in 2012 facilitating the employment of 481,000 personnel. On a global scale, nematode parasites are estimated to cost farmers $80 million / year. As a direct result of this project, livestock producers will receive economic gain through more effective drugs that are free from resistance. Novel anthelmintics will permit reduced drug treatments, and will enhance animal health, welfare and productivity. Increased productivity will drive economic success in all businesses involved in the 'farm to fork' production process, bolstering the competitiveness of the UK Agri-Food Industry.

B. Policy Makers
1. Stakeholders and policy makers will benefit through research-evidence that will inform policy development:: UK-based government bodies (DEFRA, DARD), Levy boards (EBLEX, BPEX), and other representatives of the Agri-Food industry will benefit as this project will provide an evidence-base for policy development and addreses EU-directed changes in agricultural legislation, e.g. the need for rapid drug-intervention to facilitate 'Sustainable Intensification'.

C. Public Sector
1. Farming communities in developing countries will benefit through enhanced quality of life: Nematode parasites have a devastating impact on agriculture in developing countries where many farmers and their families rely on livestock for income and food. Further, they infect >1/4 of the world's population causing serious morbidity. The goal of this project is the development of novel drugs for the treatment of nematode pathogens in livestock, which has the potential to improve quality of life through enhanced economic gain. Moreover the development of novel drugs for veterinary parasites which can be repurposed for use in humans will improve the health, well-being, and quality of life of those afflicted with nematodiasis.

2. Educational sector will benefit through greater understanding of the local and global significance of nematode pathogens: Local schools will benefit from this research by educating students and teachers about nematode biology, and through raised awareness of the importance of research in our society. In addition, research findings will form the basis of research-led teaching to university students at QUB. The host-institute will benefit through an enhanced research profile.

3. General public will benefit through job-creation and opportunity to promote sustainable prosperity: There is a growing demand from the consumer for organic and chemical residue-reduced food that is produced cost-effectively from animals maintained in a welfare-friendly environment. A novel, resistance-free anthelmintic will facilitate a reduction in drug use, therefore limiting food contamination and facilitating the organic farmer who is restricted to reduced treatment regimes. Also, the environmental impact associated with intensive drug use will be reduced.
 
Title Cover image International Journal for Parasitology 
Description Cover image for Volume 46, Issue 10 of International Journal for Parasitology - adult Ascaris suum. 
Type Of Art Image 
Year Produced 2015 
Impact International journal readership. Awareness of article/research focused on Ascaris suum. Publicity for RCUK funded research. 
URL http://www.sciencedirect.com/science/journal/00207519/45/11
 
Description Grant ongoing:
Reporting below on complete datasets only:
A1. In silico identification of nematode parasite NP-GPCRs [AIM 2]
We have identified orthologs of the152 predicted C. elegans neuropeptide receptors using Hidden Markov Models (HMM) to identify potential NP-GPCRs within the predicted protein datasets of 10 key nematode parasites (including Dirofilaria immitis, Haemonchus contortus and A. suum) Several key points emerge from these datasets: (i) parasitic nematodes have a reduced complement of C. elegans NP-GPCRs; (ii) several NP-GPCRs are conserved across the parasitic nematodes; (iii) Clade 2 nematodes have a reduced complement of NP-GPCRs.

A2. In silico NP-GPCR drug target prioritization [AIM 2]
We have designed a decision matrix suitable for identifying a cohort of putative NP-GPCR drug targets for further validation via the A. suum RNAi platform/other functional genomics platforms. This comprises data from three main sources (NP-GPCR conservation, C. elegans-derived phenotype, and life-stage specific expression) to prioritize GPCRs based on their perceived importance to nematode function and consequently their appeal as novel drug targets.
?Level 1 Prioritization:
2.1 Pan-phylum conservation of NP-GPCRs
Based on the HMM pan-phylum NP-GPCR identification dataset described above (point #1) several GPCRs have been prioritized: gnrr-1, ckr-2, frpr-19, C01F1.4, F59D12.1, pdfr-1, seb-3, daf-38, dmsr-2, dmsr-8, T11F9.1, H09F14.1.
?Level 2 Prioritization:
2.2 C. elegans phenotype
Based on phenotype data from C. elegans knockout/RNAi experiments several GPCRs have been prioritized: npr-5, -29, ckr-1, Y40C5A.4, fshr-1, npr-1, -4, -11, -12, -13, daf-38, dmsr-2, dmsr-4, fshr-1, F59D12.1, and pdfr-1.
?Level 3 Prioritization:
2.3 In silico identification of nematode parasite NP-GPCRs in life stages
Based on life-stage specific transcriptomic datasets for six (T. muris, T. spiralis, A. suum, D. immitis, N. americanus, G. pallida) of the 10 parasitic nematode species included in the neuropeptide GPCR identification. These data have enabled us to prioritise GPCRs based on their expression in all life-stages or therapeutically relevant life-stages of these key parasites.

In summary: NP-GPCRs emerging from prioritization include: pdfr-1, F59D12.1, npr-29, ckr-1, ckr-2 and frpr-19. pdfr-1 and F59D12.1, are common to all levels of prioritization - these will be channelled into the heterologous expression and functional genomics pipelines.

Manuscript in preparation for submission 2018: Atkinson, L.E., McCoy, C., Marks, N.J., Maule, A.G., Mousley, A. (2018) In silico drug target prioritization of neuropeptide GPCRs in parasitic nematodes. International Journal for Parasitology: Drugs and Drug Resistance (manuscript in preparation).

B1. In silico tissue specific RNA-Seq data analysis [AIM 1 and 2]
RNA-Seq was carried out on two specific regions of the A. suum reproductive apparatus (see previous reports for details/figures). Tissue specific differential expression analysis of NP-GPCRs and neuropeptide ligands revealed restricted groups of putative NP-GPCRs expressed on the ovijector, and neuropeptide ligands expressed in neuronal cells innervating the ovijector [FMRFamide-like peptides (flps) and neuropeptide-like proteins (nlps)]. These NP-GPCRs and neuropeptide ligands may represent putative receptor-ligand interactions and will feed into the A. suum functional genomics platform for NP-GPCR deorphanization. A number of key NP-GPCR-ligand interactions have been identified and are currently in RNAi/heterologous expression pipelines. This work is ongoing in collaboration with Prof. M. Mitreva and Dr B. Rosa at Washington University, St Louis, USA.

Manuscript in preparation for submission 2018: Atkinson, L.E., Rosa, BA., Marks, N.J., Maule, A.G., Mitreva, M. Mousley, A. (2018) Predicting ligand receptor interactions using tissue-specific RNAseq profiling in Ascaris suum. International Journal for Parasitology (manuscript in preparation).


C1. A. suum pseudocoelomic fluid (PCF) peptidomics - detection of neuropeptides in A. suum PCF to investigate other, non-synaptic routes of GPCR-ligand interaction [AIM 2].
Evidence from other nematode systems including C. elegans highlight the potential for extra-synaptic neuropeptide signaling whereby peptides released from specific neurons can act remotely (like hormones) to activate receptors expressed in distant cells and tissues. In a bid to characterize ligand interactions with ovijector-based NP-GPCRs which regulate parasite egg laying behavior, we have recently established a collaboration with Dr Lingjun Li (UW-Madison, USA), an expert in the detection of neuropeptides in crab haemolymph, to perform highly sensitive mass-spectrometry based peptidomic analyses of the A. suum PCF. PCF bathes the A. suum ovijector tissue and may represent an unexplored vehicle for neuropeptide signaling.
We have completed four rounds of PCF peptidomics in A. suum (n>40 females and n>20 males; single worm and pooled samples) in addition to a post-RNAi peptidomics analysis of PCF. Our results indicate that A. suum PCF contains neuropeptides including FLPs and NLPs, in addition to a number of anti-microbial peptides. This is the first neuropeptidomics profile of PCF from a nematode and is the first indication that extrasynaptic transmission may be an integral part of the functional connectome in nematodes. These data provide an additional layer of complexity to the ligand-receptor matching pipelines that we are currently employing and will further support novel target discovery.

Manuscript in preparation for submission 2018: Atkinson, L.E., Liu, Y., Li, Z, McKay, F., Marks, N.J., Maule, A.G., Li, L., Mousley, A. (2018) The neuropeptidome of Ascaris suum pseudocoelomic fluid. Analytical Chemistry (manuscript in preparation).
Exploitation Route ACADEMIC:
• Research: the outcomes from this work have been published (with additional publications in progress) and presented at national and international conferences. This has facilitated new collaborations and provided opportunity for a further funding stream, initially through PhD studentships, to continue the identification and validation of novel chemotherapeutic targets for nematode parasites.
Sectors Agriculture, Food and Drink,Education,Pharmaceuticals and Medical Biotechnology

 
Description Northern Ireland Pig Health Technical Group recommendation for EU Exceptional Adjustment Aid
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Participation in a advisory committee
 
Description Department for Employment and Learning PhD studentship
Amount £70,000 (GBP)
Organisation Government of Northern Ireland 
Department Department for Employment and Learning Northern Ireland (DELNI)
Sector Public
Country United Kingdom
Start 10/2016 
End 09/2019
 
Description Department for the Economy PhD studentship
Amount £70,000 (GBP)
Organisation Department for the Economy, Northern Ireland 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2020
 
Title Ascaris suum pseudocoelomic fluid peptidomics datasets 
Description These datasets represent the peptides expressed in Ascaris suum pseudocoelomic fluid 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? No  
Impact These datasets can be used to determine the peptides that are released extrasynaptically via volume transmission. In addition, these data are available to map to the predicted protein dataset in Ascaris suum. 
 
Title Ascaris suum tissue specific RNAseq datasets 
Description These datasets represent the genes expressed in distinct tissues of the pig roundworm parasite Ascaris suum. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? No  
Impact These datasets can be used for the differential expression analysis of any of the 18,000 genes present in A. suum. This enables the elucidation of fundamental biological pathways present in each tissue type and can infer the importance of specific genes and/or pathways to a tissue/organ essential to the parasite for reproduction and survival. 
 
Description Ascaris suum Microbiome analysis 
Organisation University of Washington
Department Genome Institute
Country United States 
Sector Academic/University 
PI Contribution Collaboration with Makedonka Mitreva at McDonnell Genome Institute (Washington University at St Louis) on gDNA 16S profile and RNAseq analysis of Ascaris suum gut tissue/contents. QUB will provide the RNAseq / gDNA datasets to the Mitreva group for further analysis.
Collaborator Contribution McDonnell Genome Institute (Washington University at St Louis) will perform the bioinformatics analysis pipeline and help with data presentation and interpretation.
Impact Multidisciplinary collaboration. McDonnell Genome Institute (Washington University at St Louis) have expertise in computational biology, QUB have expertise in biology and physiology of Ascaris.
Start Year 2018
 
Description GPCR receptor deorphanisation in Parasitic Nematodes in collaboration with Prof Liliane Schoofs and Dr Isabel Beets 
Organisation University of Leuven
Country Belgium 
Sector Academic/University 
PI Contribution We are working towards deorphanisation of neuropeptide G-protein coupled receptors in parasitic nematodes. KU leuven have the technologies and expertise in heterologous expression and post-expression ligand-receptor interaction profiling. We have identified, cloned and sequenced the relevant GPCRs and are providing them in the most appropriate expression vector. KU leuven will express in a heterologous system and screen for ligand interaction. QUB have synthesised and will provide the species specific ligand library. We will author publications that will arise from the collaboration.
Collaborator Contribution KU leuven will express the target GPCRs in a heterologous system and screen for ligand interaction. They will provide data on ligand binding kinetics. Drs
Impact Collaboration is multi-disciplinary. Prof Schoofs and Dr Beets have expertise in heterologous expression systems and receptor-ligand deorphanisation platforms.
Start Year 2016
 
Description Parascaris Maintenance and Culture 
Organisation University of Kentucky
Country United States 
Sector Academic/University 
PI Contribution Academic input to research project resulting in publication (co-author)
Collaborator Contribution Research partner carried out the research
Impact Publication (listed)
Start Year 2018
 
Description Peptidomic analysis of Ascaris suum pseudodoelomic fluid in collaboration with Dr Lingjun Li 
Organisation University of Wisconsin-Madison
Country United States 
Sector Academic/University 
PI Contribution We are characterising the peptidomic profile of Ascaris pseudocoelomic fluid. We are providing the biological material (PCF) from Ascaris suum and will analyse the data arising from the peptidomic analysis. We will author publications arising from the collaboration.
Collaborator Contribution Dr Li will undertake the peptidomic analysis of PCF fluid. Dr Li will also contribute to data analysis and publications raising from the collaboration.
Impact Collaboration is multi-disciplinary. Dr Li is a peptidomics expert within the School of Pharmacy at the University of Wisonsin-Madison.
Start Year 2017
 
Description RNAseq Analysis of Ascaris suum ovijector and gonopore tissue 
Organisation Washington University in St Louis
Country United States 
Sector Academic/University 
PI Contribution Collaboration with Makedonka Mitreva at McDonnell Genome Institute (Washington University at St Louis) on RNAseq analysis of Ascaris suum ovijector and gonopore tissue. QUB provided the RNAseq datasets to the Mitreva group for further analysis.
Collaborator Contribution Ongoing collaboration. Partners (Mitreva group) analysing RNAseq data
Impact Collaboration ongoing
Start Year 2016
 
Description Anthelmintics from Discovery to Resistance III (ORAL PRESENTATION L ATKINSON, FLORIDA, 30th JANUARY-2nd FEBRUARY: FLP-18 modulates reproductive function in Ascaris suum via extrasynaptic volume transmission? 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Our understanding of nematode nervous system function is founded on neural circuit synaptic interactions. However, other routes of non-synaptic communication are known in invertebrates including extrasynaptic (wireless) volume transmission (EVT), which enables close- or long-range communication where synaptic connections are absent. EVT is thought to be involved in biogenic amine signaling in Caenorhabditis elegans but has not yet been demonstrated in parasitic nematodes or for neuropeptide transmission. Neuropeptide signaling governs many important nematode functions and behaviors. The FMRFamide like peptides (FLPs; PGVLRFamides) encoded on flp-18 are expressed in multiple head neurons of Ascaris suum and have been implicated in locomotion, feeding and reproductive function via whole worm, pharyngeal and ovijector tissue bioassays. Although flp-18 encoded peptides are potent stimulators of the ovijector, they are expressed in head neurons that are anatomically distant from, and have no known synaptic connections to, the ovijector. Tissue specific RNA-Seq analysis reported here demonstrates that neurons innervating ovijector tissue do not express flp-18, and that the putative cognate FLP-18 G-protein coupled receptors (GPCRs) npr-4 and npr-5 are expressed in ovijector tissue. To investigate non-synaptic modes of FLP-18 signaling in A. suum we undertook a peptidomics analysis of the A. suum pseudocoelomic fluid (PCF), which bathes the nematode reproductive organs, including the ovijector. These studies identified multiple FLP-18 peptides in the circulating PCF of all nematodes examined (n=12). These data support the hypothesis that FLP-18 peptides modulate A. suum reproductive function non-synaptically via EVT. Establishing the role of non-synaptic (wireless) routes of neurosignaling in parasitic nematodes and their integration into a functional connectome provides an additional layer of complexity to the ligand-receptor matching processes that support new drug discovery programs.
Year(s) Of Engagement Activity 2018
 
Description BALLYCLARE ALC CAREERS EVENT (27TH JANUARY 2016) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact CAREERS EVENT FOR KEY STAGE 3, YEAR 6, 7 AND YEAR 8 SCHOOL LEAVERS. Purpose was to promote Biological Sciences pathways at Queen's University Belfast and to engage students in BBSRC-funded research activity
Year(s) Of Engagement Activity 2016
 
Description BALMORAL SHOW INTERACTIVE DISPLAY 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Local Agricultural Show (18th May 2018). Communication of BBSRC funded research projects. Education of local agricultural personnel and public about the impact of agricultural parasites.
Year(s) Of Engagement Activity 2018
 
Description Invited Speaker Presentation - Ascaris suum: local problem, global issue, experimental model (UCLA, Los Angeles, USA) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited speaker presentation to research professionals at UCLA
Year(s) Of Engagement Activity 2018
 
Description Merial Animal Health PLC/Boehringer Ingelheim 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Members of QUB research team and BI Animal Health research and development collaboration team met to discuss progress and future plans (September 2017 UK; January 2018 GA, USA). Plans were made for research within the next 3 months and an arrangement was made for the next quarterly progress meeting.
Year(s) Of Engagement Activity 2017,2018
 
Description Merial Animal Health Plc/Boehringer Ingelheim 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Members of QUB research team and Merial Animal Health Research and Development collaboration team met to discuss progress and future plans. Plans were made for research within the next three months and an arrangement was made for the next quarterly progress meeting.
Year(s) Of Engagement Activity 2016,2017
 
Description Molecular Helminthology: An Integrated Approach (POSTER, 19-22 March 2017, Cape Cod, MA, USA) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Abstract - The future of novel drug target discovery for neglected tropical diseases is reliant on the development of functional genomics platforms in key pathogens. While progress in this area has been slow for nematode parasites, where many species are reported to be intractable to current reverse genetics methodologies, a handful appear compatible with advanced molecular tools. Bolstered by a wealth of genomic/transcriptomic information, and increasing affordability of Next Generation Sequencing, the discovery and validation of novel drug targets is becoming a less distant prospect in Ascaris suum where significant progress has been made in the development of functional genomics tools.
Neuropeptide signalling system biology has been a central focus of nematode drug target discovery. Integrating 'omics' resources in A. suum has provided new opportunities for neuropeptide G-protein coupled receptor (GPCR) deorphanisation. In this study we combined the experimental tractability of A. suum with RNA-seq to identify the neuropeptide/neuropeptide GPCR complements of two distinct, functionally important tissues: (i) the ovijector, and (ii) the body wall muscle. Tissue specific RNA-seq libraries were generated (n=3) and analysed using the Tuxedo pipeline. Overexpression and functional enrichment analysis revealed 871 genes overexpressed in the ovijector, including 33 genes involved in GPCR activity. A cohort of at least five FMRF-amide like peptide GPCRs (flp-GPCR) were identified in the ovijector tissue alongside six putative ligands (flps) in the body wall muscle containing the neuronal cell bodies which innervate ovijector tissue. Immunlocalisation studies unequivocally confirmed the presence of specific FLPs in the neuronal cell bodies innervating the ovijector tissue. These data will direct functional deorphanisation attempts in A. suum using RNA interference (RNAi) coupled with a post-RNAi ovijector bioassay. Together these 'omic' approaches bring us closer to the prospect of ex vivo GPCR deorphanisation in animal parasitic nematodes and provide fresh hope for novel drug target identification.
Year(s) Of Engagement Activity 2017
URL https://www.elsevier.com/events/conferences/molecular-helminthology-an-integrated-approach
 
Description Molecular Helminthology: An Integrated Approach, Cape Cod, MA, USA, 19-22 March 2017 (POSTER PRESENTATION, L. ATKINSON, An 'omics' approach to neuropeptide receptor deorphanisation in Ascaris suum) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The future of novel drug target discovery for neglected tropical diseases is reliant on the development of functional genomics platforms in key pathogens. While progress in this area has been slow for nematode parasites, where many species are reported to be intractable to current reverse genetics methodologies, a handful appear compatible with advanced molecular tools. Bolstered by a wealth of genomic/transcriptomic information, and increasing affordability of Next Generation Sequencing, the discovery and validation of novel drug targets is becoming a less distant prospect in Ascaris suum where significant progress has been made in the development of functional genomics tools.
Neuropeptide signalling system biology has been a central focus of nematode drug target discovery. Integrating 'omics' resources in A. suum has provided new opportunities for neuropeptide G-protein coupled receptor (GPCR) deorphanisation. In this study we combined the experimental tractability of A. suum with RNA-seq to identify the neuropeptide/neuropeptide GPCR complements of two distinct, functionally important tissues: (i) the ovijector, and (ii) the body wall muscle. Tissue specific RNA-seq libraries were generated (n=3) and analysed using the Tuxedo pipeline. Overexpression and functional enrichment analysis revealed 871 genes overexpressed in the ovijector, including 33 genes involved in GPCR activity. A cohort of at least five FMRF-amide like peptide GPCRs (flp-GPCR) were identified in the ovijector tissue alongside six putative ligands (flps) in the body wall muscle containing the neuronal cell bodies which innervate ovijector tissue. Immunlocalisation studies unequivocally confirmed the presence of specific FLPs in the neuronal cell bodies innervating the ovijector tissue. These data will direct functional deorphanisation attempts in A. suum using RNA interference (RNAi) coupled with a post-RNAi ovijector bioassay. Together these 'omic' approaches bring us closer to the prospect of ex vivo GPCR deorphanisation in animal parasitic nematodes and provide fresh hope for novel drug target identification.
Year(s) Of Engagement Activity 2017
 
Description Nematode Neuropeptides: From Sequence to Biology and Back (INVITED SPEAKER: Anthelmintics: Discovery to Resistance II, San Diego Feb 9th - 12th, 2016) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Nematode Neuropeptides: From Sequence to Biology and Back

Angela Mousley, Louise Atkinson, Paul McVeigh, Ciaran McCoy, Neil Warnock, Johnathan Dalzell, Nikki Marks, Aaron Maule

Parasitology Research Group, Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Belfast, UK

We have maintained a long-standing interest in helminth neurobiology at Queen's University Belfast that stemmed from the early work of Professor D.W. Halton, and continued through research activity in the Maule and Marks laboratories. In the early days, localisation of neuropeptides through immunocytochemical techniques in association with classical biochemical characterisation methods, PCR-based gene detection tools, and muscle-based functional assay systems
provided data on the importance of neuropeptides to helminth biology, and flagged the candidature of the neuropeptidergic system as an anthelmintic target. Despite this, our understanding of the biology of neuropeptides and their signalling systems, especially in key therapeutically relevant pathogens, is limited. The recent growth in genomic and transcriptomic datasets for parasitic helminths, and positive shift in the cost and accessibility of gene sequencing technologies, provides a welcome gateway to the identification of neuropeptides, their receptors and pathway components. In addition, progress in the development of parasite-focused reverse genetics tools offers the ability to manipulate target function in key pathogens and opportunities for novel anthelmintic discovery. This presentation will provide an overview of the efforts to progress the identification and validation of neuropeptidergic system-derived drug target candidates in nematode parasites, and highlight recent advances in our understanding of neuropeptide biology across the phylum Nematoda.
Year(s) Of Engagement Activity 2016
 
Description Networking Workshop - Improving Clinical Helminth Infection Prevalence Data Using Molecular Diagnostics (Bangkok, Thailand) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Networking event delivery of research presentation (30 mins)
Year(s) Of Engagement Activity 2019
 
Description Oral Presentation: American Society of Tropical Medicine and Hygiene: 64th Annual Meeting, Philadelphia, USA, 25-29 October 2015; RNAi competency in adult Ascaris suum - potent, persistent and reproducible knockdown 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact RNAi competency in adult Ascaris suum - potent, persistent and reproducible knockdown
Year(s) Of Engagement Activity 2015
 
Description Parasitic Nematode Functional Genomics - fit for purpose? (INVITED SPEAKER: AAVP 2016 Annual Meeting San Antonio, USA, 5-9th August 2016) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Parasitic Nematode Functional Genomics - fit for purpose?
Angela Mousley, Louise Atkinson, Ciaran McCoy, Nikki Marks, Aaron Maule
Parasitology Research Group, Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Belfast, UK
The recent growth in genomic, transcriptomic and proteomic datasets for parasitic nematodes provides a welcome gateway to the identification of putative drug targets, vaccine candidates, and diagnostic biomarkers. Indeed, omics-directed approaches to drug target identification have become widely adopted drug-finding strategies for human therapeutics, and have begun in earnest for nematode parasites where a cohort of 'druggable' targets, believed to have chemotherapeutic appeal due to their predicted 'essentiality', have been identified and prioritized in key nematode pathogens including Haemonchus contortus, Ascaris suum and Brugia malayi through in silico analyses. Despite this, a key hurdle to the exploitation of putative targets is the absence of validation tools that allow the manipulation of target function in therapeutically-relevant pathogens. Reverse genetics tools have advanced to the stage where sophisticated methods of transgenesis, gene silencing (RNA interference), and genome editing (CRISPR/Cas9 technology) are established experimental tools that are being applied to probe the biology of many organisms. Their application to nematode parasites has been eagerly awaited; however translation of these technologies has either been difficult or is in early stages of development, such that their potential to novel drug discovery in the parasitology discipline is yet to be realised. This presentation provides an overview of the genetic manipulation tools that are currently available for use in parasitic nematodes, and evaluates the advantages and limitations of these tools to the discovery of novel control targets.
Year(s) Of Engagement Activity 2016
 
Description Primary Life Sciences Scheme (Jan-April 2015; Dec-March 2016) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Primary Life Sciences Scheme (Jan-April 2015; Dec-March 2016)
Worm watch: program delivered to primary level students to engage in scientific research. The purpose of the program was to engage the students in scientific research (specifically worm biology) and to promote STEM in primary schools. The teacher responsible for the class has requested that the scheme is continued in the future.
Year(s) Of Engagement Activity 2015,2016
 
Description Primary Life Sciences Scheme (March-June 2017; March-June 2018) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Primary Life Sciences Scheme (March-June 2017) Worm watch: program delivered to primary level students to engage in scientific research. The purpose of the program was to engage the students in scientific research (specifically worm biology) and to promote STEM in primary schools. The teacher responsible for the class has requested that the scheme is continued in the future.
Year(s) Of Engagement Activity 2017,2018
 
Description Royal Society Conference - Evolution and functional biology of neuropeptide signalling: from genomes to behaviour (INVITED SPEAKER, UK, 13-14th March 2017; The development of Functional Genomics Platforms for nematode pathogens: informing the biology of neuropeptides and their receptors) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Abstract:
Traditional approaches to the discovery, localisation and functional characterisation of nematode neuropeptides have included immunocytochemical techniques, classical biochemical characterisation methods, PCR-based gene detection tools, and muscle-based physiology assays. The data generated highlighted the importance of neuropeptides to nematode biology, and flagged the candidature of the neuropeptidergic system as a putative anthelmintic target. More recently, these datasets have been enhanced by the omic-analyses of nematode genome, transcriptome and peptidome datasets enabling the identification and prioritization of neuropeptides and their receptors that exhibit therapeutic appeal, but are not yet validated. Unfortunately, the development of functional biology tools in nematode parasites has not kept pace. Indeed, a key hurdle to the exploitation of putative targets is the absence of tools that allow the elucidation of target function in therapeutically-relevant nematode pathogens. Broadly, reverse genetics is being applied to probe the biology of many organisms through sophisticated methods of transgenesis, gene silencing (RNA interference), and genome editing (CRISPR/Cas9 technology). The application of these experimental tools to nematode parasites has been eagerly awaited; however their translation has either been difficult or is in early stages of development, such that their impact on novel drug discovery for the control of nematode pathogens is yet to be realised. This presentation provides an overview of Functional Genomics Platforms that are currently available for use in parasitic nematodes, and describes progress in their application to the understanding of the neuropeptidergic system.
Year(s) Of Engagement Activity 2017
URL https://royalsociety.org/science-events-and-lectures/2017/03/neuropeptide-signalling/
 
Description Royal Society Conference - Evolution and functional biology of neuropeptide signalling: from genomes to behaviour (POSTER, UK, 13-14th March 2017; The development of Functional Genomics Platforms for nematode pathogens: informing the biology of neuropeptides and their receptors) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Abstract - The neuropeptidergic system is a putative drug target repository for the control of nematode pathogens. Omics approaches have identified >250 nematode neuropeptides, and a number of neuropeptide G-protein coupled receptors (GPCRs). In vivo deorphanisation in nematode parasites has proven difficult; progress is reliant on the development of reverse genetics methodologies which allow putative ligand-receptor pairs to be identified. Ascaris suum offers an opportunity for a novel approach to neuropeptide receptor deorphanisation in a nematode parasite, through the integration of tissue-specific omics-derived data and the ability to probe target-, and tissue-specific gene function. This study describes the identification of neuropeptide/neuropeptide GPCR complements using RNAseq of two distinct A. suum tissues, the ovijector, and body wall muscle (nerve process originating in the body wall innervate the ovijector). 871 genes are overexpressed in the ovijector, including 33 genes involved in GPCR activity. Within one of the largest families of nematode neuropeptides (FMRF-amide like peptides, FLPs), at least five putative flp-GPCRs were identified in the ovijector, and six flps in the body wall that are hypothesised to be their cognate ligands. These data will direct functional deorphanisation attempts in A. suum using RNA interference (RNAi) coupled with a post-RNAi ovijector bioassay.
Year(s) Of Engagement Activity 2017
URL https://www.elsevier.com/events/conferences/molecular-helminthology-an-integrated-approach
 
Description STEMNET / SOCIETY OF BIOLOGY STEM CAREERS EVENT (24TH FEBRUARY 2016) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Lecture on the Applications of DNA technology followed by careers event. Purpose was to engage students in current research activity and to promote interest in STEM subjects. Students were engaged with careers advice.
Year(s) Of Engagement Activity 2016
 
Description The Neuropeptidergic System of Parasitic Nematodes: receptor deorphanisation approaches (INVITED SPEAKER: KU LEUVEN BELGIUM, 19-21 SEPTEMBER 2017) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The Neuropeptidergic System of Parasitic Nematodes: receptor deorphanisation approaches
Year(s) Of Engagement Activity 2017
 
Description UK/THAILAND CONFERENCE - One Health Approach to Parasite Control in South East Asia (INVITED SPEAKER, BANGKOK, THAILAND, 19-21 JUNE 2017; Ascaris suum: local problem, global issue, experimental model) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Ascaris suum: local problem, global issue, experimental model
Year(s) Of Engagement Activity 2017
 
Description UK/THAILAND RESEARCH GROUP MEETING - One Health Approach to Parasite Control in South East Asia 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact Research meeting designed to discuss, plan and disseminate research.
Year(s) Of Engagement Activity 2018
 
Description WAAVP conference (Liverpool UK 16-20 August 2015; Ascaris suum: A Novel Functional Genomics Platform for Nematode Parasites with Potential for Organism-, Tissue- and Cell-level Drug Target Validation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Abstract:
Year(s) Of Engagement Activity 2015
 
Description Workshop/Training Course: Helminth Genomics and Transcriptomics Bioinformatics Course, The Genome Institute, Washington University, St. Louis, USA (September 2015) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Genomics, Transcriptomics and Bioinformatics workshop
Year(s) Of Engagement Activity 2015