Fasciola hepatica extracellular vesicles - the key to parasite control?

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

Abstract

Fasciolosis is a common, and economically important, disease of livestock. It is caused by a parasitic flatworm called Fasciola hepatica (also known as the liver fluke) that infects more than 300 million cattle and 250 million sheep worldwide resulting in losses of over $3 billion to global agriculture through lost productivity. It is also widespread in the UK, and costs the cattle farming industry alone around £23 million each year as a result of poor animal condition and a significant reduction of milk and meat yields. Although traditionally regarded as a disease of livestock, fasciolosis is an emerging disease of humans with an estimated 2.4 million people infected worldwide. The drug of choice against liver fluke infection is triclabendazole. However, just like the drug-resistant bacteria that are wreaking havoc in our hospitals, triclabendazole-resistant fluke are now rapidly spreading throughout the UK/Ireland, continental Europe and Australia. This has left farmers with little to combat the disease as most remaining drugs are ineffective against the immature flukes that cause most damage to the infected animal. With unprecedented outbreaks of liver fluke infection predicted to occur in the UK over the next 60 years, the development of new strategies for F. hepatica control is most urgent. The spread of drug resistance, together with heightened consumer concerns about the presence of chemical residues in food, has fuelled the search for anti-Fasciola vaccines. Despite some early successes there are still no commercially-available vaccines against F. hepatica. This is most likely due to the striking ability of the parasite to influence the host immune response. Fasciola is an accomplished immune-modulator, directing the host immune response away from the type that is most damaging to them - a Th1 response - and regulating the response to create an environment that optimises successful feeding and reproduction - a Th2 response. We have found that specific molecules released by the parasites are responsible for this immune-modulation and we believe that targeting the release of these may be the key to parasite control. Recent research has shown that molecules can be transferred from one cell to another by being packaged into sacs called extracellular vesicles (EVs). Our collaborator Dr Antonio Marcilla (University of Valencia) recently found that EVs are also released from F. hepatica and we now know that they contain many known immunomodulators. Thus, we propose that preventing the release of EVs from the parasite will stop the transfer of the immunomodulatory molecules packaged inside to host immune cells. This will allow a Th1 immune response to prevail leading to expulsion of the parasite. To achieve this we will use a new technique called RNA interference (RNAi) to "switch off" particular molecules that are involved in packaging and release of EVs from the parasite. We will then be able to determine if this approach can give the host`s immune response a boost and eliminate the parasite. This is a multidisciplinary project which will build on recent discoveries in Fasciola biology and advances in technology. As such, we have assembled a strong network of national and international collaborators who will provide considerable support and expertise in RNAi (Prof Maule), immunology (Prof Dalton and Dr Donnelly) and EV biology (Dr Marcilla). We envisage that specific targets will emerge from this research for control of liver fluke infections (by new drugs or vaccines) that will be commercially attractive and transferable to other parasitic infections of humans and animals.

Technical Summary

The liver fluke Fasciola hepatica remains a major threat to UK agriculture. Control of fluke infection relies on a single drug, triclabendazole. However triclabendazole resistance is spreading rapidly leaving farmers with little to combat the infection. To ensure its long-term survival Fasciola secretes molecules that stimulate host macrophages and dendritic cells to drive Th2 responses whilst suppressing the development of host-protective Th1 inflammation. We have discovered that F. hepatica packages known immunomodulators (and other RNA and protein molecules) into extracellular vesicles (EVs) which are released by the parasite. We propose that EVs are an important route for transfer of signalling molecules from parasite-to-host immune cells and that inhibiting the production of Fasciola EVs would prevent modulation of the host immune response and lead to Th1-driven expulsion of the parasite. Accordingly, we have three major objectives:

(1) What effector molecules do Fasciola EVs deliver to host immune cells?
We will profile the protein cargo packaged within Fasciola EVs using advanced proteomics techniques. We have found that Fasciola EVs contain a variety of RNA molecules, consistent with mRNA, microRNAs and other non-coding RNA. We will obtain a comprehensive picture of the RNA complement within the parasite EVs using next generation sequencing.

(2) How is EV biogenesis and release regulated in Fasciola?
Our proteomics studies of F. hepatica have identified several proteins involved in EV biogenesis and release. Here we will use RNA interference (RNAi) to silence selected targets and determine their role in EV formation using a range of biochemical and cellular assays.

(3) Does inhibiting Fasciola EV production lead to Th1-driven expulsion from the host?
Here we will use our murine model of fluke infection to investigate the effect of inhibiting Fasciola EV production on host immune cell function and the resolution of infection.

Planned Impact

Helminth (worm) pathogens cause >55% of all animal diseases and pose a major threat to global food security by affecting livestock growth rates, fertility, meat quality, wool or milk production, and sometimes causing death. The increasing demand for the supply of cheap but quality food, the demand for ethical welfare production systems and the concern regarding outbreaks of serious animal diseases has put a greater emphasis on controlling these infections. However, control of many economically important species, such as Fasciola hepatica (the focus of this project) with existing anthelmintics is unsustainable due to the emergence of drug-resistant parasites. Given the scale of the problem, it is imperative that new strategies to control parasitic helminth infection are discovered. Investigating the mechanism of extracellular vesicle (EV) biogenesis and release in Fasciola will primarily benefit the academic community but validation of this pathway as a target for parasite control, as this project aims to achieve, will be of great interest to the UK industrial sector with a view towards immunological (vaccines) or pharmacological (drugs) intervention. Potential beneficiaries of this research include, amongst others: [1] Public Sector: agencies in the UK such as EBLEX (the organisation for the English beef and sheep industry) and DairyCo and the Agriculture and Horticulture Development Board would gain tremendously from our research which has the potential to shape future agricultural policy; [2] Industry: pharmaceutical companies such as Pfizer and Merial Animal Health Ltd (which has funded my research in the past) would have an interest in the commercial development of drugs/vaccines targeting the Fasciola EV pathway. By feeding knowledge into the pharmaceutical industry, any commercialisation is likely to be pursued within the UK, which would increase wealth and foster economic competitiveness; [3] Charities and philanthropic organisations involved in anti-parasite research, such as the Bill & Melinda Gates Foundation, would benefit from a greater understanding of host-parasite interactions; [4] Academia: UK and overseas universities, including QUB's medical and life sciences students at the undergraduate, masters and doctoral levels. Our research strategy is to conduct multidisciplinary investigation by exploiting advances in technology. This can only be achieved through collaboration. Our research capacity will be enhanced by our network of internationally-recognised collaborators who are expert in their respective fields; [5] Local students: students of Queen's University Belfast will benefit from improved, research-led teaching excellence and from the resources available to them by the strengthened research position of the Institute for Global Food Security at Queens University. Local sixth form students will also benefit through increased access to work experience opportunities and outreach programmes aimed at promoting life sciences in local schools.

Publications

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Alvarado R (2017) The immune modulatory peptide FhHDM-1 secreted by the helminth Fasciola hepatica prevents NLRP3 inflammasome activation by inhibiting endolysosomal acidification in macrophages. in FASEB journal : official publication of the Federation of American Societies for Experimental Biology

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Cwiklinski K (2019) The cathepsin-like cysteine peptidases of trematodes of the genus Fasciola. in Advances in parasitology

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De La Torre-Escudero E (2016) Extracellular Vesicle Biogenesis in Helminths: More than One Route to the Surface? in Trends in parasitology

 
Description We have identified the cargo packaged into vesicles shed by the parasite Fasciola hepatica and also those molecules displayed on the vesicle surface which are likely to participate in host-parasite interactions. We identified mechanisms that govern how the vesicles are taken up by host immune cells and how this might be blocked for therapeutic benefit. We know also have a better understanding of the various mechanisms by which parasites produce these vesicles which was much more complex that originally thought.
Exploitation Route Function of the identified molecules can now be investigated using functional genomics techniques and assessed as drug targets/vaccine candidates. Surface-exposed molecules could be targeted by blocking antibodies to prevent uptake into host immune cells.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

URL http://www.ncbi.nlm.nih.gov/pubmed/26486420
 
Description Dr Mark Robinson joined teachnical working group of Animal Health Ireland
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
URL http://animalhealthireland.ie/?page_id=411
 
Description DEL PhD studentships
Amount £60,000 (GBP)
Organisation Government of Northern Ireland 
Department Department for Employment and Learning Northern Ireland (DELNI)
Sector Public
Country United Kingdom
Start 10/2015 
End 09/2018
 
Description QUB - Uruguay collaboration 
Organisation University of the Republic
Department Faculty of Medicine
Country Uruguay 
Sector Academic/University 
PI Contribution Provision of unique RNAseq libraries from isolated parasite-derived extracellular vesicles.
Collaborator Contribution Bioinformatics analysis of the RNAseq libraries
Impact Early stage collaboration - no outputs as yet.
Start Year 2016
 
Description QUB-Valencia 
Organisation University of Valencia
Country Spain 
Sector Academic/University 
PI Contribution Access to expertise/protocol development and biological data.
Collaborator Contribution Access to expertise & facilities.
Impact Cwiklinski K, de la Torre-Escudero E, Trelis M, Bernal D, Dufresne PJ, Brennan GP, O'Neill S, Tort J, Paterson S, Marcilla A, Dalton JP, Robinson MW. The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis. Mol Cell Proteomics. 2015 Dec;14(12):3258-73. doi: 10.1074/mcp.M115.053934.
Start Year 2014
 
Description Abattoir visits 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Industry/Business
Results and Impact Met with animal producers/area managers of several meat processing plants to educate about our BBSRC-funded research and how it might benefit the industry.
Year(s) Of Engagement Activity 2013,2014,2015
 
Description GRC meeting: host-parasite interactions 
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 Invited research presentation "Helminth extracellular vesicles - the key to parasite control". Gordon Research Conference: Biology of Host-Parasite Interactions, Salve Regina University, Newport RI, USA.
Year(s) Of Engagement Activity 2014
 
Description GRC meeting_ extracellular vesicles 
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 Poster presentation of our research "The surface proteome of extracellular vesicles from a helminth pathogen" at the Gordon Research Conference: Extracellular vesicles, Sunday River, Newry, MA, USA, 21st-26th August 2016. The event was attended by approx. 200 delegates including Nobel Laureate Randy Schekman. The event raised the research profile of my group and that of extracellular vesicle biology in parasites, an emerging topic in the wider field. Many researchers were particularly taken with our unique approach for studying the surface proteome of extracellular vesicles.
Year(s) Of Engagement Activity 2016
URL https://www.grc.org/programs.aspx?id=17267
 
Description Hydra 2019 conference presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave a talk at entitled "Spilling your guts: extracellular vesicles contribute to haemoglobin digestion by Fasciola hepatica" at the Parasitic Helminths - New Perspectives in Biology and Infection. Bratsera Hotel, Hydra, Greece, September 1st-6th 2019. The presentation sparked debate from the audience and led to requests for collaboration/joint publication.
Year(s) Of Engagement Activity 2019
 
Description IGFS newsletter 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Project was publicized in the institute newsletter. This has a wide readership including politicians, educators, researchers, industry.
Year(s) Of Engagement Activity 2013
 
Description Invited presentation at workshop 
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 Delivered an invited talk entitled "The molecular and cellular origins of extracellular vesicles released by Fasciola hepatica" at the Helminth Extracellular Vesicles Workshop. Bratsera Hotel, Hydra, Greece, August 31st-September 1st, 2019. The workshope brought together experts in the area of helminth EV research with the aim of stimulating collaboration in the field and preparation of a "position paper" aimed at standardizing research methods/reporting and helping new researchers enter the field.
Year(s) Of Engagement Activity 2019
 
Description Presentation at Helminth meeting - Thailand 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact GRCF funded workshop entitled "A One Health Approach to Helminth Control in South East Asia" held at Mahidol University, Bangkok, Thailand, 19th 22nd June 2017. Intention was to network with potential collaborators from across south-east Asia with a view to submitting future collaborative grant applications.
Year(s) Of Engagement Activity 2017
 
Description Presentation at workshop in Shanghai, China 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Third sector organisations
Results and Impact GRCF/British council funded workshop entitled "Neglected helminth diseases of China and the impact of urbanisation" held at Fudan University, Shanghai, China, 29th-31st January 2018. Intention was to network with potential collaborators from across China with a view to submitting future collaborative grant applications.
Year(s) Of Engagement Activity 2018
 
Description Presentations at Irish Society for parasitology meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact PDRA Dr Eduardo de la Torre Escudero and PhD student Adam Bennett gave research talks at this event. This served to raise the profile of this project at a local level and give the both these early career researchers valuable presentation experience.
Year(s) Of Engagement Activity 2016
 
Description QUB school open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Presentation and discussions with students from local/national schools. Aim was to raise profile of the university and BBSRC-funded research. Feedback from participating schools was positive with many subsequent applicants for biological science degrees.
Year(s) Of Engagement Activity 2014,2015
 
Description Research presentation at conference 
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 Research presentation made by a PhD student at the joint meeting of the BSPP/ISP/BAVP/EVPC, Brussels, May 3rd-4th 2018.

BENNETT, A., DE LA TORRE ESCUDERO, E. & ROBINSON, M.W. (2018). Tracing the cellular origins of Fasciola hepatica extracellular vesicles.

It reached a broad audience (>100 in attendance) with interest generated in the work by industry and academics. Industry contacts made that may lead to joint funding applications (e.g. BBSRC IPA).
Year(s) Of Engagement Activity 2018
URL https://www.bspp.be/scientific-meeting-2018/
 
Description Student presentation at the British Society for Parasitology meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Presentation by David Smith on work spun-out from some of the proteomics analysis conducted during this project:

SMITH, D., TIKHONOVA, I., DRYSDALE, O.C., DVORAK, J., ROBINSON, M.W., CWIKLINSKI, K. & DALTON, J.P. (2016). Unexpected activity of a novel kunitz-type parasite inhibitor: inhibition of cathepsins and not serine proteases. British Society for Parasitology Spring Meeting, Imperial College London, April 11th-13th 2016.
Year(s) Of Engagement Activity 2016
 
Description UK Veterinary Vaccinology Conference 2017 
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 I was part of the organising committee for the BBSRC UK Veterinary Vaccinology Conference (16th-17th January 2017, Riddel Hall, Queens University, Belfast). The event was a great success and attracted 140 delegates to see a strong line up of internationally-recognised speakers at Queens.
Year(s) Of Engagement Activity 2017
URL http://www.vetvaccnet.ac.uk/veterinary-vaccinology-network-conference-2017
 
Description WAAVP conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation at 2015 WAAVP conference in Liverpool. Purpose was to disseminate research findings and the outcome was raised awareness of my research group nationally and internationally.
Year(s) Of Engagement Activity 2015
URL http://www.waavp2015.com/