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

Lead Research Organisation: University of Aberdeen
Department Name: School of Medical Sciences


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.

Technical Summary

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.

Planned Impact

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.


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Derevnina L (2016) Emerging oomycete threats to plants and animals. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

Description Saprolegnia parasitica secretes proteins that have a function inside the fish host cells.
The route of entry into the fish cells of two of these proteins was discovered.
Furthermore, we have discovered the function of both proteins inside the fish cells.
Exploitation Route The aquaculture industry will benefit from our research findings.
Our results show how we can translocate proteins into cells this can be exploited for vaccine and drug delivery.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology,Other

Description The results led to a new (successful) application to exploit the translocation mechanism for delivery of vaccines
First Year Of Impact 2016
Sector Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology
Description Development of an immersion vaccine for salmonids
Amount £700,000 (GBP)
Funding ID NE/P010873/1 
Organisation Natural Environment Research Council 
Department NERC Postdoctoral Fellowship
Sector Charity/Non Profit
Country United Kingdom
Start 01/2017 
End 01/2019
Title immersion vaccination 
Description We have been developing an immersion vaccine method for salmonid fish 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2018 
Provided To Others? No  
Impact The method of immersion vaccination needs to be further optimised before we can report here in detail. Also a patent will be filed and this prevents us from further reporting here at this stage. 
Description A composition comprising a translocation sequence derived from a fish pathogen and a heterologous payload coupled to the translocation sequence is provided. The composition is able to translocate across the plasma membrane of a eukaryotic cell, for example a fish cell, and thus stimulate an immune response. Accordingly, nucleic acids, vectors, host cells, compositions and vaccines based thereon are also provided. 
IP Reference WO2014191759 
Protection Patent application published
Year Protection Granted 2014
Licensed Commercial In Confidence
Impact Patent gave us the opportunity to submit a new grant to perform proof of concept studies
Description Cafe Scientifique 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I presented our work on Saprolegnia infections in salmon farms and how we are looking into new methods too control saprolegniosis and other fish diseases.
Year(s) Of Engagement Activity 2018
Description Organising Microbiology School lectures at University of Aberdeen 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Every year I am organising a school lecture for secondary school children to learn about microbiology and show them a career option in sciences and in particular microbiology. Usually we get 100-250 children attending between the ages of 16-18 from several regional schools (Aberdeenshire)
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012,2013,2014,2015,2016,2017
Description Press release 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release about our paper: Route parasite takes to infect fish uncovered
Scientists in Aberdeen have discovered one strategy a fungus-like organism - which is a big problem for the aquaculture industry - uses to infect salmon and trout.

The disease Saprolegniosis, caused by the water mould Saprolegnia parasitica, is a serious problem for the fish farming industry in countries such as Scotland, Norway, Chile, Japan and Canada.

It is highly pathogenic to fish, extremely difficult to control and is estimated to kill about 10% of all farmed salmon and trout.

Now researchers at the University of Aberdeen have discovered one trick this pathogen uses to infect freshwater fish.

Their findings - published in the Proceedings of the National Academy of Sciences - could help to find new ways of controlling Saprolegnia infections in fish.

It might also yield new clues on how to control similar diseases in plants as Saprolegnia is closely related to for example, Phytophthora infestans, the Irish potato famine fungus or late blight, which seems to use a similar trick to infect potatoes.

Dr Pieter van West, a Reader in Mycology at the University, said: "Saprolegnia, which is found in rivers, lochs and lakes, produces swimming spores that attach to the fish and penetrate its underlying tissue".

"We have discovered that Saprolegnia secretes proteins that are able to enter fish cells by binding to a receptor of the fish cell that must be sulphated"

"We don't really know what the proteins are doing once inside the fish cell, but it is possible that they interfere with the fish's ability to defend itself against infection."

The discovery of this trick paves the way for scientists to try to develop new ways of blocking the protein's actions and possibly controlling Saprolegniosis.

Dr Stephan Wawra, research fellow at the University added: " The trick these proteins use to enter host cells may not only be important for infections caused by water moulds, but may also be used by other pathogens. If this turns out to be the case, we would have a good understanding as to how we can block the protein entry process."

"In addition, we might be able to use this trick ourselves to deliver possible cures for other diseases. Obviously this is something that would need further investigation".


Notes to Editors:
Dr Pieter van West is available for interview. To arrange contact Jennifer Phillips on 01224 273174.

Issued by the Communications Team, Office of External Affairs, University of Aberdeen, King's College, Regent Walk. Tel: 01224 273174.
Year(s) Of Engagement Activity 2012