An integrated model of host-parasite interactions in coccidian parasites

BACKGROUND: Poultry and livestock production is a key economic component of the farming industry, providing high quality, safe and healthy food for UK consumers. Maintaining the health and welfare of food producing animals is not only ethically desirable, but has implications for food quality, human health and ultimately the economic sustainability of the industry. Poultry and livestock are susceptible to parasitic diseases, including some highly prevalent protozoan parasites (Coccidia) which cause severe and chronic diarrhoea, weight loss and abortion. The most important Coccidia include the Eimeria, which frequently affect poultry, and Cryptosporidium and Toxoplasma, which affect livestock including cattle, sheep and pigs. For some e.g. Eimeria, preventative drug treatments are available, but resistance is a major concern and for others no drugs are available. Vaccination is a more attractive option, but for both Eimeria and Toxoplasma current vaccines are based on live attenuated parasites which have inherent problems of production and in some cases reliability; no vaccination options are available for Cryptosporidium.

AIMS: This proposal aims to develop a comparative model of the complex molecular processes involved in the process of cell invasion in three widespread and economically important coccidian parasites and to validate this model with a series of functional tests. Our analysis will provide a platform to enhance our understanding of the biology of these parasites, provide a wealth of data to the wider scientific community in an easily accessible form and assist in the discovery of novel biological candidates that in future can be exploited by industry to help control these diseases in poultry and livestock.

APPROACH: Although coccidian parasites are quite diverse they all share one important common feature: the sexual stage of replication produces environmentally resistant oocysts which, when ingested by a new host break-open, releasing an invasive stage (sporozoite) which penetrates the cells of the gut and establishes a new infection; commonly all food producing animals can become infected by this route. We propose to target our research on these shared biological features and will do so by focussing our attention on the interaction of the sporozoites with the host cell. We have recently acquired the genetic information and technologies to undertake a large-scale analysis of thousands and genes and proteins and analyse their activation states (systems biology) during the invasion of parasites into cells. We will exploit this ability to analyse and compare the interaction of our three coccidian species. Using these data we will produce a detailed biological model describing the events which occur during the first few hours of sporozoite invasion, enabling us to understand both common and unique features of this process. We will use these data, alongside bioinformatics analysis of proteins that show distinctive patterns of expression, modulation and co-regulation, to focus on targets for which we will then acquire further functional information. For the most promising protein candidates we will verify their importance in the invasion process by further analysis of their interactions with the host cell. These candidates will then be tested biologically to verify their role in sporozoite cell invasion by determining what happens when these host-parasite interactions are artificially blocked, or the host cells with which they interact are modified.

OUTCOME: The outcome of the work will be to enhance our understanding of the biology of three economically important parasite species and provide the basic biological knowledge to identify novel targets for subsequent commercial development. In the long term this will increase UK competitiveness in the animal health market, improve animal welfare and help to guarantee safe and healthy food for the public.

We will undertake a comparative systems analysis of sporozoite host-cell invasion in three important coccidian parasites of livestock and poultry, Eimeria tenella, Cryptosporidium parvum and Toxoplasma gondii. These data will be used to develop an integrated model of sporozoite-host cell interactions, identifying both parasite-specific processes and those common across the Coccidia. The dynamics of gene and protein expression and protein activation will be analysed using state-of-art transcriptomics and quantitative label-free mass-spectrometry-based proteomics and phosphoproteomics. For each species we will acquire systems data simultaneously from parasite and host cells during the attachment and invasion of sporozoites, parasitophorous vacuole formation, early and late parasite growth and release of zoites (where applicable). We will use these data to (1) develop a comparative systems model of coccidian sporozoite invasion from which we can identify specific targets for validation and functional analysis, concentrating particularly on the hypothesis that Coccidia share a core set of universal invasion mechanisms; (2) provide a resource to the wider scientific community by disseminating the data via a publicly available platform (www.eupathdb.org). Focussing on shared responses between each species identified from our bioinformatics, we will undertake functional studies including (a) immunolocalisation of proteins during invasion and parasite growth (b) targeting specific parasite proteins using in vitro invasion blocking assays (c) targeting specific host response pathways by RNAi (d) exploring proposed interacting parasite-host networks by targeted MS-based protein pull-down experiments. This proposal will reveal a deeper understanding of the mechanisms of invasion, host response and colonisation of epithelial cells by coccidian parasites and result in the discovery of novel biological candidates for exploitation in partnership with industry.

This proposal will contribute to a new understanding of the biology of three economically important parasitic diseases of commercial livestock. Our research will result in the discovery of novel biological candidates that will be exploited in collaboration with an industry partner through an Industrial Partnership Award (IPA) to develop improved methods of control for coccidian diseases in poultry, sheep, cattle and pigs. Outcomes will assist in increasing UK competitiveness in the global animal health market, improving animal welfare and helping to guarantee safe,healthy food for the public.

The following (a) beneficiaries have been identified; (b) methods of how they will benefit; and (c) what will be done to ensure that they have the opportunity to benefit from this research.

BENEFICIARY ONE
(a) Animal health pharmaceutical industries concerned with the development of new therapies for commercially important poultry and livestock; and/or, pharmaceutical industries with an interest in human health in the area of zoonotic protozoan disease.
(b) Outcomes will benefit the commercial sector (i) by identifying a range of clearly defined candidate molecules involved in the early stages of colonisation of the gut by coccidian parasites. Candidates will be from 3 of the most important coccidian infection of poultry and livestock, Eimeria, Cryptosporidium and Toxoplasma; (ii) by revealing a deeper understanding of the mechanisms of invasion, host response and colonisation of epithelial cells by these parasites and the generation of large datasets of transcriptional, proteomic and functional information available for data mining and additional target discovery.
(c) This proposal encourages commercial investment by (i) focussing, where possible, on core mechanisms/targets that are shared across all 3 species, thus making commercial investment as attractive as possible by maximising potential returns on product development; (ii) by providing functional information downstream from the systems analysis to enable IP to be sought for candidate targets. This boosts prospects of obtaining the investment required to move the R&D into the industry sector. IP will therefore be protected throughout to enhance value and maximise opportunities for collaborative research or licensing.

BENEFICIARY TWO
(a) The UK animal health sector; poultry and livestock producers and animal health professionals. Coccidian parasites cause ten of millions of pounds in lost production and there is a need for alternative/new vaccines and improved therapeutics in this area.
(b) By working with industry utilising their strengths in product development and experimental and field trials.
(c) By sharing the costs of the development of new control methods with the commercial sector we aim to make the route to new innovations becoming available in the field as rapid as possible.

BENEFICIARY THREE
(a) Skills, training and knowledge economy
(b) The PDRAs and any undergraduate, postgraduate or intern students that contribute to the project will develop key interdisciplinary skills that will be extremely valuable for UK industry and contribute to the knowledge economy and increase the economic competitiveness of the UK.
(c) The PDRAs will have the opportunity to spend time in placements with our industry partner.

BENEFICIARY FOUR
(a) International Development
(b) Coccidian parasites are a major source of lost animal production in developing counties and zoonotic Coccidia, such as Cryptosporidium are a major cause of human diarrhoeal disease.
(c) Improved methods of control, especially the wider availability of suitably priced vaccines, would provide a boost to the farming sector in developing countries. Both academic institutions have strong links with developing countries and it is likely that PGR students from these countries will become associated with the research programme resulting in training and knowledge exchange.