The Bloomsbury Colleges | PhD Studentships | Studentships 2021 | Role of thrombospondin type1 repeat (TSR) domain proteins in motility and virulence of Babesia parasites
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Role of thrombospondin type1 repeat (TSR) domain proteins in motility and virulence of Babesia parasites

Principal Supervisor: Dr Ellen Knuepfer (RVC)

Co-Supervisor: Dr Robert W Moon (LSHTM)

Prof Dirk Werling (RVC)

Award includes tuition fees and a stipend of £17,285 (non-vet) or 23,590 (vet) including London Weighting (at 2020/21 rates, so slightly higher for 2021 entry).

Full time for 3 years, from October 2021.

Project description

Babesiosis is a disease symptomatically very similar to malaria, caused by Babesia parasites infecting and multiplying in erythrocytes. Babesia and Plasmodium are closely-related protozoan parasites. Both are transmitted by insect vectors. Babesiosis affects a large range of vertebrates including livestock, companion animals, and also humans. In animals Babesia infections are frequently fatal if left untreated and cause a significant economic burden. Babesiosis in humans is a zoonotic disease. The disease is mostly asymptomatic and only fatal in immune-compromised or elderly individuals. However, numbers of human babesiosis cases are rising, especially in the USA where this parasite is the most common transfusion-transmitted pathogen. In the UK, several Babesia species are endemic and babesiosis cases have been reported in cattle, sheep, dogs and recently also in humans.

All symptoms of this disease, and onward transmission result from the cyclical invasion of host erythrocytes, and thus a better understanding of this process could yield vital new drug or vaccine candidates. Whereas this process is well-studied for malaria parasites, little is known about this process in Babesia. We know that proteins in micronemal organelles are required for host cell recognition and parasite motility. Thrombospondin type 1 repeat domain (TSR) proteins form an important group of micronemal proteins in Plasmodium and have critical roles in motility, invasion of salivary glands, hepatocytes and erythrocytes1. Some TSR-containing proteins recognise glycosaminoglycans on the host cell surface and at the same time connect to the actin-myosin motor through their cytoplasmic tail, generating the motility required for host cell invasion. One such example is the thrombospondin-related anonymous protein (TRAP).  Four TSR-domain containing proteins can be identified in the B. divergens genome. Based on limited transcription profiling all four genes appear expressed in blood stages of Babesia. Three of these hypothetical proteins are secreted TSR-proteins, one of which is showing domain structures reminiscent of TRAP. We hypothesise that similar to Plasmodium, TSR-proteins are involved in and required for erythrocyte invasion of B.divergens.

This project will study the role of B.divergens TSR-proteins in erythrocyte invasion to understand the steps leading to invasion.

The projects main objectives are

1)      Confirm gene models and transcription profiles of all 4 genes encoding TSR-domain containing proteins during the blood cycle stage of B. divergens.

2)      Determine the essentiality/redundancy of each of these proteins during host cell invasion and their vaccine potential.

3)      Visualise the localisation of these potential parasite adhesins during invasion using real-time fluorescence microscopy.

4)      Identify host cell receptors on human and bovine erythrocytes.

The project will utilise in vitro tissue culture systems established for B.divergens elsewhere in the world as well as genome editing methodology using CRSIPR/Cas92 followed by real-time fluorescence imaging techniques3. We will adapt existing CRISPR/Cas9 vectors to generate inducible gene knockouts, domain swaps or deletions and point mutations of essential TSR-proteins. Generating specific antibodies, we will test these in invasion inhibition experiments. Imaging of transgenic B.divergens parasites will pinpoint at which step of the invasion process these ligands act. Furthermore, the motility of free transgenic B.divergens parasites will be quantitated and potential host cell receptors characterised.

We are looking for a person who is self-motivated, has recently successfully completed a degree in a biological sciences or similar subject or a related veterinary degree and has a good understanding of molecular biology. An interest in infectious disease, cell and molecular biology is essential. Pratical experience in tissue culture/sterile working techniques and molecular biology is desirable. This studentship will be held jointly between the labs of Ellen Knuepfer (RVC), who has strong background in reverse genetics using CRISPR/Cas9 tools including the development of inducible gene knockout technologies in Plasmodium4 and Robert Moon (LSHTM) who has recently established CRISPR-Cas9 in a zoonotic Plasmodium parasite and specialises in studying parasite invasion and motility using live-cell imaging approaches5 with further support by Prof Dirk Werling (RVC) who has a strong background in immunology.

This project offers an exciting opportunity to work in two world-class research institutions with leading labs in the field of malaria, branching out newly into the field of babesiosis. Strong international collaborations exist with Oxford University, ITM Antwerp, Francis Crick Institute, the NY Blood Centre and University of Florida which will be able to provide further support.

Subject Areas/Keywords

Infection biology; molecular biology; cell biology; real-time microscopy; CRISPR/Cas9; parasitology; vaccine development.

Key References:

1. Morahan, B. J., Wang, L. & Coppel, R. L. No TRAP, no invasion. Trends in Parasitology 25, 77–84 (2009).

2. Hakimi, H. et al. Genome Editing of Babesia bovis using the CRISPR/Cas9 System. mSphere 4, (2019).

3. Sevilla, E. et al. Kinetics of the invasion and egress processes of Babesia divergens, observed by time-lapse video microscopy. Scientific Reports 8, 14116 (2018).

4. Knuepfer, E., Napiorkowska, M., van Ooij, C. & Holder, A. A. Generating conditional gene knockouts in Plasmodium – a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9. Scientific Reports 7, 3881 (2017).

5. Yahata, K. et al. Gliding motility of Plasmodium merozoites. http://biorxiv.org/lookup/doi/10.1101/2020.05.01.072637 (2020).

Further details about the project may be obtained from:

Principal Supervisor: Dr Ellen Knuepfer (eknuepfer@rvc.ac.uk)

Co-Supervisor: Dr Robert Moon (Rob.Moon@lshtm.ac.uk)

Prof Dirk Werling (dwerling@rvc.ac.uk)

Further information about PhDs at RVC is available from:

https://www.rvc.ac.uk/study/postgraduate/phd

Application forms and details about how to apply are available from:

https://www.rvc.ac.uk/study/postgraduate/phd/how-to-apply

researchdegrees@rvc.ac.uk

 

Closing date for applications is: 
7th February 2021