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

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.

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.

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.