Toxoplasma: Sphingolipids in host-parasite interactions syntheses versus scavenging

Lead Research Organisation: Durham University
Department Name: Wolfson Research Institute

Abstract

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 animals causing a disease known as toxoplasmosis. In recent times Toxoplasma has come to prominence as causing serious diseases in patients whose defences (immune response) against such invading organisms have been damaged, such as those suffering from acquired immunodeficiency syndrome ¿ AIDS. In addition, infection of unborn lambs with Toxoplasma is a major cause of miscarriage, leading to annual multi-million pound losses to UK sheep farmers. Humans and other animals usually become infected with Toxoplasma following ingestion of contaminated food or faeces. Subsequently, the parasites can invade almost any of the cells that make up the body of an animal. However, in most cases this infection does not cause serious disease, instead Toxoplasma is controlled by the immune response of the animal and retreats into cysts in the brain or in muscle tissue. These cysts can remain throughout the animal¿s life without causing any problems. However, a damaged (in an AIDS patient) or undeveloped (in an unborn animal) immune response is unable to control the parasite in this way leading to disease. Within the animal cell Toxoplasma manufactures many substances it needs for growth, however, it is also able to scavenge various materials that it uses for its own purposes. This research proposal aims to investigate how the parasite manufactures or acquires an important component of its plasma membrane (a fatty barrier that separates the inside of the parasite cell from the external environment) ¿ sphingolipid. This will help us understand how Toxoplasma grows within animal cells and how it subsequently causes disease. In addition, the observation that some of the machinery needed to make sphingolipids in Toxoplasmand in other apicomplexans) is very unusual may eventually allow the development of new drugs against these organisms.

Technical Summary

The important human and agricultural apicomplexan, protozoan parasite Toxoplasma gondii can infect the nucleated cells of almost any warm blooded animal, where it subsequently proliferates within a parasitophorous vacuole (PV). Although the PV is known to exhibit an intimate relationship with the host cell and Toxoplasma is able to scavenge many essential, particularly lipid, macromolecules from it, the mechanisms of this interaction and the balance between parasite de novo biosynthesis and salvage pathways remains unclear. Unlike the vacuoles harbouring many intracellular pathogens, the non-fusagenic Toxoplasma PV is not believed to interact directly with either the exo- or endocytic pathways of the host cell, and therefore is thought to be unable to scavenge essential molecules via vesicular traffic from either of these routes. Sphingolipids perform a diverse array of functions, from acting as secondary signalling molecules to forming part of micro-domains, termed lipid rafts, which have been proposed to function in processes from cell signalling to polarised trafficking. Within the context of this research proposal the roles of de novo synthesised and host acquired sphingolipid in Toxoplasma invasion and proliferation will be investigated. In the first instance this will be achieved by characterising the parasite serine palmitoyltransferase (SPT), an enzyme which mediates the first, rate-limiting step in sphingolipid biosynthesis. Unlike in all other eukaryotes studied to date, the putative SPT from Toxoplasma and other apicomplexans resembles the homodimeric enzyme from the bacterium Sphingomonas paucimobilis and is thought to be targeted to the apicoplast, a vestigal plastid which like all other plastid and mitochondrial organelles is derived from an ancient bacterial endosymbiont. Functional analyses and localisation of this unusual putative enzyme will shed new light on the evolution of sphingolipids in the Eukaryota. However, it will also allow the role of de novo sphingolipid biosynthesis in infection and proliferation to be delineated by the creation of parasites in which SPT function is ablated. Similar investigations assaying for the ability of Toxoplasma to proliferate within host cells deficient in SPT activity themselves will address whether host derived sphingolipid is required for the establishment of an infection. As such the research outlined here will establish the roles of de novo synthesised and host derived sphingolipid in Toxoplasma invasion and proliferation, thereby greatly adding to our understanding of how this parasite interacts with its host.

Publications

10 25 50
 
Description Toxplasma gondii is a model apicomplexan and as such a representative of a group of important human and animal parasites. Work in this project showed that host (mammalian) processes were not vital to Toxoplasma proliferation (and perhaps pathogenicity). In addition key enzymes in the parasite sphingolipid biosynthetic pathway were identified and characterized . Given that sphingolipids are key components of membranes, as well as mediators of vital cellular functions, these may represent novel drug targets. The fact that the enzymes identified differ significantly from their equivalents in mammals indicated that that specific, non-toxic inhibitors (drugs) could be identified.
Exploitation Route The use of assay system developed (and patent protected) in the search for inhibitors could lead to the subsequent development for drugs for a range of human and animal parasitic infections. If successful the impact of this would be substantial. The identification of these enzymes has lead to a drug discovery initiative which could impact on both human and animal health. Specifically, an assay system for analyses of one the the enzymes identified (International Publication Number WO 2007/083116 and International Application Number PCT/GB2007/000142) was developed and has been employed in a drug discovery initiative funded by the Open Lab Foundation.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description The screening technology developed during the execution of this award has, following IP protection, been deployed in collaborative projects with GSK and MRC Technology Ltd towards anti-protozoal drug discovery.
First Year Of Impact 2010
Sector Pharmaceuticals and Medical Biotechnology
 
Description British Council
Amount £2,300 (GBP)
Funding ID 1283 
Organisation British Council 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Description MRC CASE Award with MRC Technology to exploit potential of the kinetoplast IPC synthase as a drug target
Amount £108,438 (GBP)
Funding ID G1100182 
Organisation MRC-Technology 
Sector Private
Country United Kingdom
Start 10/2011 
End 09/2015
 
Description To develop IPC synthase screening platform and screen the GSK 2M compound collection against the Leishmania orthologue at GSK Tres Cantos, Spain
Amount £52,000 (GBP)
Organisation Tres Cantos Open Lab Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2011 
End 01/2012
 
Description To develop IPC synthase screening platform and screen the GSK 2M compound collection against the Leishmania orthologue at GSK Tres Cantos, Spain
Amount £52,000 (GBP)
Organisation Tres Cantos Open Lab Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Description Collaboration with Prof Ralph Schwarz, Marburg 
Organisation Philipp University of Marburg
Country Germany 
Sector Academic/University 
PI Contribution Input into study of sphingolipid biosynthetic enzymes in the protozoa (and plants). Central to three of outputs listed.
Start Year 2005
 
Title Target 
Description Screening assay and proof of concept for a novel protozoan and herbicide target, inositol phosphorylceramide synthase. 
IP Reference WO2007083116 
Protection Patent granted
Year Protection Granted
Licensed Yes
Impact Facilitation of secondary screening of hits generated through HTS of GSK compound library (1.8 million compounds).
 
Description Biosecurity 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Published/broadcast comments regarding biosecurity and Foot and Mouth disease to local and national newspapers, including the Guardian and the Daily Mail, and local radio stations

See above
Year(s) Of Engagement Activity 2007
 
Description Celebrating Science in the North East 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Focussed interactive discussion on malaria with an audience of year 10 pupils

Demonstrable pupil engagement during sessions, particularly when looking at infected blood smears
Year(s) Of Engagement Activity 2009
 
Description NGS - Next Generation Science 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Schools
Results and Impact Approximately 20 students and 5 teachers (UK and Dutch) attended and engaged with a discussion about tropical parasitology and disease.

Further involvement with schools outreach through e.g. Celebrating Science in the North East.
Year(s) Of Engagement Activity 2008
 
Description Perilous Pets? Interviewed by the Northern Echo 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact Clarify link between Toxoplasma infection, cats and mental health

Engagement with local cat charity
Year(s) Of Engagement Activity 2012