Sphingolipid biosynthesis in the parasitic apicomplexan protozoa: divergent enzymes in key host:pathogen interactions

Lead Research Organisation: Durham University
Department Name: Biosciences

Abstract

Introduction
The apicomplexans are a large group of related, single-celled microscopic organisms that cause a range of diseases (including malaria) in both humans and economically important domestic animals. One of these, Toxoplasma gondii, can infect most species of warm-blooded animal causing a disease known as toxoplasmosis. In recent times toxoplasmosis has come to prominence as a serious disease in patients whose defences (immune response) against infectious microbes have been damaged, such as those suffering from Acquired ImmunoDeficiency Syndrome (AIDS) and those undergoing anti-cancer chemotherapy. Furthermore, during normal pregnancy infection of the unborn child with Toxoplasma is a major cause of miscarriage and congenital defects in humans. Similarly, Toxoplasma causes the spontaneous abortion of unborn lambs, leading to annual multi-million pound losses to UK sheep farmers.
The Problem
Humans and other animals usually become infected with Toxoplasma following ingestion of faeces from the infected cats, or via contaminated food. Subsequently, the parasites can invade almost any of the cells that make up the body. However, in most cases this infection does not cause serious disease, instead Toxoplasma is controlled by the immune response of the human or animal and retreats into cysts in muscle tissues or the brain. These cysts can remain throughout the host's life without causing any physical problems, although some studies have linked the cysts to behavioral changes and mental illness in humans. However, a damaged (in an AIDS or cancer patient) or undeveloped (in an unborn human or animal) immune response is unable to control the parasite in this way and serious disease occurs, commonly leading to severe brain damage. Unfortunately, the few available drugs to treat the diseases caused by Toxoplasma and other apicomplexan parasites show severe problems with effectiveness and major side-effects, meaning that there is an urgent need to discover new therapies for both human and animal health.
The Background
Within the human or animal cell Toxoplasma manufactures many molecules it needs for growth, however it is also able to scavenge various materials that it uses for its own purposes. Sphingolipids are essential components of the Toxoplasma plasma membrane (the fatty barrier that separates the inside of the parasite cell from the external environment). Our previous work has shown that although the parasite can scavenge sphingolipid from the host animal cell, this process is not essential for growth and the spread of infection. This indicated that the machinery that Toxoplasma possesses to synthesize the essential sphingolipids is likely to be crucial for its survival and ability to cause disease.
The Aims
We have identified key enzyme components of this machinery and this research proposal aims to exploit these findings by: [i] Demonstrating that Toxoplasma sphingolipid synthesis is essential for the parasite; and [ii] Characterizing the identified enzymes, the components of the sphingolipid machinery. The data generated will facilitate future work to exploit the Toxoplasma (and other apicomplexan parasites') sphingolipid synthesis machinery as a target for new, much needed, drugs.

Technical Summary

The apicomplexan protozoan parasite Toxoplasma gondii can infect the nucleated cells of almost any warm-blooded animal. In addition to being an important cause of human and animal disease, due to its genetic tractability and host promiscuity Toxoplasma has also been established as the model apicomplexan. In previous work we have established that despite the ready acquisition of host biomolecules, Toxoplasma are not dependent on host sphingolipid biosynthesis. Sphingolipids are essential lipids that perform a diverse array of functions, from participating in the formation of membrane micro-domains to acting as secondary signaling molecules, e.g. in apoptosis. This, and the non-reliance on host biosynthesis, indicated the importance of de novo sphingolipid synthesis in parasitism. We have now identified and partially characterized three pivotal enzymes in Toxoplasma sphingolipid biosynthesis that are conserved across the Apicomplexa: serine palmitoyltransferase (SPT), catalyzing the first and rate-limiting step in sphingolipid biosynthesis, the condensation of serine and palmitoyl-CoA to form keto-dihydrosphingosine; ceramide synthase (CerS), which acylates the downstream product of SPT to form ceramide; and sphingolipid synthase (SLS), which catalyses the addition of a phospho head group to ceramide to form a complex sphingolipid. Whilst the unstudied CerS appears similar to all other eukaryotic orthologues, SPT has a unique bacterial origin and SLS, at least in Toxoplasma, has functionality reminiscent of plants and fungi rather than mammals. The proposed research will fully analyse these enzymes, and the de novo pathway per se, firstly by sequential ablation of the encoding genes in Toxoplasma to establish their role in parasite proliferation. Secondly, by establishing protein production, purification and assay development for biochemical and biophysical analyses. Together this will facilitate the triage and exploration of these enzymes as novel drug targets.

Planned Impact

The fundamental science within this proposal underpins an aim to validate the pathway and its key enzymes as potential drug targets in the Apicomplexa, parasites of humans (e.g. toxoplasmosis and malaria) and economically important animals (e.g. toxoplasmosis and coccidiosis). Problems of effectiveness, toxicity and anti-microbial resistance make the discovery of new anti-apicomplexan therapies a priority. The outcomes will facilitate engagement with industrial partners in human and/or animal health, funded through Industrial Partnership Awards (IPA) or CASE Awards, to exploit findings towards apicomplexan disease control. The outcomes will support UK competitiveness in the pharmaceutical industry (both human and animal health), animal welfare and food security.

To ensure impact non-academic beneficiaries have been identified [i]; how they will benefit described [ii]; the processes to ensure they benefit, and a timeframe, illustrated [iii].

Beneficiary 1
[i] The animal health pharmaceutical sector with programmes for the control of apicomplexan parasitic disease in livestock, and pharmaceutical companies with a commitment to develop equivalent therapies for human health.
[ii] The research outcomes will benefit this commercial sector by [a] validating novel drug targets in Toxoplasma which are conserved across the Apicomplexa; [b] developing enzyme assay platforms for the screening of compound libraries.
[iii] Drawing on our extensive experience of industrial (large and SME) engagement across the human and agricultural sectors (GSK, Bayer Crop Science, Hypha Discovery and Aureogen Inc) we will [a] use the data generated to secure IP on the assays developed, month 18-30; [b] utilize Durham Business and Innovation Services to engage with appropriate partners, month 18-30.

Beneficiary 2
[i] The knowledge economy through skills, training and development
[ii] The PDRA employed (plus research postgraduate students, undergraduate project students and visiting workers who contribute towards project aims) will develop expertise in interdisciplinary research skills in molecular and cellular biology, and biochemistry and biophysics. This skills profile is of great value to UK industry in the knowledge economy, contributing to boosting national economic competitiveness.
[iii] Cross-disciplinary research is embedded in our laboratories and the project workers will [a] benefit directly from this and receive high quality training throughout, month 1-36; [b] the PDRA will receive training in Toxoplasma transgenic manipulation through a collaboration with Prof Markus Meissner (Glasgow), month 3-15.

Beneficiary 3
[i] International development
[ii] In the developing world apicomplexan disease is a major cause of economic loss through their effects on both human health and agricultural production. Therefore the research outcomes will be of direct benefit.
[iii] The research team is already engaged with international partners, e.g. PWD having recently developed a link with the University of Baghdad in apicomplexan disease. This will ensure that developing world postgraduate research students engage in the project, facilitating training and knowledge exchange, month 1-36.

Beneficiary 4
[i] The wider public
[ii] Public understanding of science is a vital function of active research and is central to this proposal. Utilizing existing structures and creating new social media initiatives the research team will ensure that the wider public are engaged.
[iii] The PDRA will be responsible for [a] a researcher blog disseminated as widely as possible, month 6-36; [b] with support, construction of a Wikipedia page on the theme of sphingolipids in the protozoa, month 24-36. The Investigators will [c] engage with local outreach programmes (e.g. Festival of Science and Café Scientifique), month 12-36; and [d] ensure maximal conventional media coverage through engagement with the Durham Communications Office and the BBSRC, month 12-36.

Publications

10 25 50

 
Description 1. The identification and characterisation of a bacterial-derived serine palmitoyltransferase in the apicomplexan protozoa. A key enzyme in lipid metabolism in these pathogens, this work represents a new evolutionary paradigm. See Mina et al, J Biol Chem (2017). 2. Investigation of the mode of action of the antifungal agent auerobasidin A against the apicomplexan protozoan, Toxoplasma gondii. See Alqaisi et al, Parasitology (2018). 3. Training and capacity development in key gene editing and cell biology technologies through secondments to Glasgow and Ruhr (2016-17).
Exploitation Route 1. This work leads onto further analyses regarding the tractability of this unusual enzyme as a possible drug target. Following further validation this may be of interest to the animal and human health sectors. 2. This work definitively shows that this natural compound does not target sphingolipid biosynthesis, thus clearing up some confusion in the research community. However, unusually, the compound also demonstrates activity against the encysted form of the parasite and may represent a new class of antiprotozoal to take forward for animal and/or human application. 3. Currently this is facilitating drug target validation studies which may allow outcomes to be feed into the animal and human health sector discovery pipeline.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

URL https://www.dur.ac.uk/biosciences/about/schoolstaff/academicstaff/?id=2595
 
Description Centre for Doctoral Training in Molecular Sciences for Medicine
Amount £6,916,264 (GBP)
Funding ID EPSRC Centre for Doctoral Training in Molecular Sciences for Medicine 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 05/2019 
End 10/2027
 
Description Deconvoluting the mode-of-action of novel anti-Chagas and anti-leishmanial compounds
Amount £284,068 (GBP)
Funding ID TC279 
Organisation Tres Cantos Open Lab Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2020 
End 03/2023
 
Description Development of platforms to study transmembrane proteins - towards medical, agricultural and biotechnological benefits
Amount £40,000 (GBP)
Organisation São Paulo Research Foundation (FAPESP) 
Sector Public
Country Brazil
Start 12/2018 
End 11/2021
 
Description GCRF Capacity Building
Amount £7,800,000 (GBP)
Funding ID MR/P027989/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 12/2021
 
Description Proof of Concept Award
Amount £86,857 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Department Networks in Industrial Biotechnology and Bioenergy (NIBB)
Sector Academic/University
Country United Kingdom
Start 08/2016 
End 10/2017
 
Description UK:Brazil Joint Centre Partnership in leishmaniasis
Amount £1,416,241 (GBP)
Funding ID MR/S019472/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2019 
End 03/2022
 
Description Lipidomics 
Organisation University of St Andrews
Department School of Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Theoretical, sample preparation and analyses
Collaborator Contribution Data generation and analytics
Impact Lipidomic effects of protozoan sphingolipid dysfunction
Start Year 2016
 
Description Markus Meissner Collaboration 
Organisation University of Glasgow
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of intellectual resource, labour and materials.
Collaborator Contribution Provision of intellectual resource, training and materials.
Impact None yet
Start Year 2016
 
Description Metabolomics 
Organisation University of Glasgow
Department School of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Theoretical input, preparation of samples and analyses
Collaborator Contribution Data generation and analytics
Impact Metabolomic profile of protozoan sphingolipid dysfunction
Start Year 2016
 
Description Toxoplasma peroxisomes 
Organisation Ruhr University Bochum
Department Institute of Biochemistry and Pathobiochemistry
Country Germany 
Sector Academic/University 
PI Contribution Identification and cloning of Toxoplasma PEX open reading frames to facilitate expression and functional analyses in yeast and mammalian systems.
Collaborator Contribution Provision of mutant yeast and mammalian cells, transfection and functional analyses.
Impact Supports a PhD project (Alison Mbekeani)
Start Year 2016
 
Description Article 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Article for the Microbiologist, the magazine of the Society for General Microbiology: A very unpleasant 'kiss'. Microbiologist 18(4): 20-21.
ISSN/ISBN: 1479-2699
Year(s) Of Engagement Activity 2017
 
Description Article for alumni magazine 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Article written for Dunelm Magazine (the Durham Alumni publication).
Year(s) Of Engagement Activity 2018
 
Description Blog 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact BugBitten BMC Blog: Bacteria and protozoa, chemistry and biology discussed
Parasitologists, microbiologists and chemists came together for fruitful discussions at the British Society for Parasitology 2016 Autumn Symposium entitled "Microbial Protein Targets: towards understanding and intervention"
Year(s) Of Engagement Activity 2016
URL http://blogs.biomedcentral.com/bugbitten/2016/10/07/bacteria-and-protozoa-chemistry-and-biology-disc...
 
Description Interview: Chemistry World (magazine of the Royal Society of Chemistry) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Article: Are chemical engineering and biochemistry their own disciplines? Chemistry World by Rachel Brazil
Aim: To highlight interdisciplinary working between physical and biological sciences
Mailed to >49,000 society members and readable on the society website
Year(s) Of Engagement Activity 2019
URL https://www.chemistryworld.com/careers/are-chemical-engineering-and-biochemistry-their-own-disciplin...
 
Description Schools Outreach (Durham) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact >100 Year 11, 12 and 13 school students attended talks on antimicrobial resistance and global health at the British Society for Parasitology Autumn Symposium in Durham (Sept 2016). Subsequently they joined the Symposium poster session and spoke to the presenting researchers (including post and under graduate students). Feedback on engagement and emphasis was universally positive. The type of event will now be embedded in future BSP meetings.
Year(s) Of Engagement Activity 2016
URL http://blogs.biomedcentral.com/blog/author/pauldenny/
 
Description parasitasconectados.com 
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 Public/other audiences
Results and Impact Interview with Brazilian web-based magazine (parasitasconectados.com) designed to increase profile of parasitology in Brazil.
Discussed perceptions on Brazilian science in Europe.
Year(s) Of Engagement Activity 2018
URL https://www.parasitasconectados.com