Fasciola hepatica extracellular vesicles - the key to parasite control?

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.

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.

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.