Biochemical characterisation of the translocation process of RxLR-like effector proteins via tyrosine-O-sulphate modified cell surface receptors

The United Nations predict that by 2050 about 9-10 billion people will populate the world. To provide enough food for the growing world population requires that areas of food production that still have the capacity to grow, like aquaculture, will be exploited more rigorously in the future but also that losses due to diseases need to be reduced. Our aim is to understand early host pathogen interactions of oomycete parasites that heavily impact food production. Oomycetes are fungal-like organisms that cause diseases on plants and animals. For example different Saprolegnia species infects fish (Saprolegniosis) at different development levels. In order to cause disease these parasites need to deliver molecules into their host cells to manipulate the host in such a way that it can infect and propagate successfully. One group of such molecules are the so called effectors. These molecules do not only target specific host molecules but some also seem to highjack host transport mechanisms in order to enter the cells. We will characterise the molecular mechanisms that effector proteins use to translocate into host cells, since a detailed understanding of these processes will help us to develop methods/substances that can block the infection process. This will allow fish to fight off their oomycete attackers more efficiently. In addition, we would like to use the 'effector entering system' to develop an immersion vaccine whereby fish do not need to be handled and injected anymore. Such a vaccine would reduce stress in fish and the occurrence of vaccination associated diseases, which includes Saprolegniosis.

Oomycete RxLR- and RxLR-like proteins contain a conserved motif, Arg-Xaa-Leu-Arg, that is located ca. 40 amino acids after the signal peptide cleavage site. Recently, we showed that the effector protein SpHtp1 can enter host cells in a tyrosine-O-sulphate (Tys) dependent manner. The main aim of this proposal is to identify the receptors for SpHtp1 and another RxLR-like protein, SpHtp3 to understand how they facilitate the translocation. Different pull down approaches will be used to identify the involved receptors. Characterisation of the receptor-effector interaction will involve the determination of the binding constants to several synthetic peptides derived from the receptor sequences containing predicted Tys sites. The binding reactions will be thermodynamically analysed using isothermal titration calorimetry and time resolved by stopped-flow-fluorescence and circular-dichroism (CD) measurements together with Pfizer. With these approaches we will obtain information about the respective binding modes and will be able to map the receptor sequences involved. The soluble domains of the receptors will be synthesised using different recombinant expression systems to evaluate the effect of a large polypeptide environment and posttranslational modifications on the binding reaction. Antibodies directed against the receptors will be generated to verify the co-localisation of SpHtp1 and SpHtp3 with their respective counterparts using confocal-microscopy. To biologically verify the interaction we will generate a transgenic human HEK293 cell line (in which SpHtp1 and SpHtp3 do normally not translocate) that express the receptor(s) of the fish specific proteins SpHtp1 and SpHtp3. A 'proof of concept experiment' will be performed to investigate whether the RxLR translocation properties of SpHtp1 can be used to generate an immune response in live fish, which would allow future development of an immersion based vaccine for fish.

The United Nations estimates that by 2050 about 9-10 billon people will populate the world. To accommodate the growing food demand a more intensified food production is needed. However, more dense and intense production will increase the impact of pests on the yields of the respective food source. We aim to understand early host pathogen interaction of oomycete parasites that heavily impact on food production and to use this knowledge to develop control strategies. Our main focus lies with a group of pathogens belonging to the oomycetes. We study two species in particular: Phytophthora infestans, the cause of potato blight, and the fish pathogen Saprolegnia parasitica. Both pathogens cause serious diseases and are responsible for large economic losses and animal suffering. Potato is the third most important crop consumed by man and P. infestans is estimated to be responsible for 16-20% production losses worldwide. Fish farming is the world fastest growing food sector and recent numbers from the Scottish salmon industry show that S. parasitica causes yield losses of about 10% with increasing tendency. At present there are no adequate control measures in place to reduce Saprolegnia infections.
We recently studied two putative effector proteins SpHtp1 and SpHtp3 from S. parasitica, and found that they are able to enter fish cells by utilising a host transport mechanism.
Our project aims to identify the host receptor molecules for SpHtp1 and SpHtp3 and to characterise the respective interactions, will provide information for the development of inhibitors/strategies to block their translocation activity. On the other hand, our research could also lead to the development of protein-based molecules able to deliver drugs into cells or the development of immersion vaccines. Development of immersion vaccines is of great interest to the aquaculture business (including Pfizer, our industrial partner) since such vaccines provide the ultimate goal for protecting fish from diseases. Immersion vaccines are particularly sought after because oral/immersion vaccine administering will reduce stress and handling of the fish, which often promotes disease itself (especially Saprolegnia). Investigating the receptors and the interaction with the translocating effectors could significantly aid the development of an immersion vaccine based on the SpHtp1-translocation machinery, which could ultimately revolutionise vaccination methods in the aquaculture industry. Efficient pest control will beneficially impact food availability, price and energy requirements for the production process.
Recent studies investigating the oomycete effector protein translocation have been published in high to very high-ranking international journals. Therefore, the results of our proposed research are likely to be reported in influential journals and have a strong scientific impact. The identification and characterisation of the receptors for SpHtp1 and SpHtp3 could reveal novel protein translocation pathways, which will influence the scientific progress in other areas that study host-microbe interactions.
We have a good track record in exploiting our research through collaborations with the industry (including Merck, Marine Harvest, Landcatch Ltd. and Syngenta) and other (international) scientists. Intellectual property will be protected by the UoA and Pfizer.
Dissemination of our work through public available research papers, the media and the internet will increase the knowledge of the general public. This project involves skills that are only available in very few labs in the UK and we will train under- and postgraduate students to increase the local skill base.