Redox regulation of protein functions in the plastid of Toxoplasma gondii

All humans and animals are made of billions of microscopic units named cells. Some organisms, like the parasites we study in this project, comprise only a single cell. Cells are themselves divided into compartments. This division allows proteins, the cell's "workers", to perform their functions in parts of the cell where the conditions are the most suitable for their task. Thus, cell partitioning into compartments with specialized conditions is necessary for proper cell function.

An important factor affecting the conditions within each sub-cellular compartment is the balance between chemicals that "oxidize" proteins, namely that take away electrons from them, and chemicals that "reduce" proteins, namely that give proteins electrons. This balance is collectively named "redox". The adjustment of protein function to the redox conditions in the compartment where they work is a crucial cellular control mechanism and if it goes wrong, the cell or the whole animal may die.

The key factors that mediate between the redox conditions in a compartment and the activity of the proteins in that compartment are specialized molecules called thioredoxins (Trxs). Trxs react to the compartment's redox conditions by altering the activity of proteins that work in that compartment.

The single-celled parasites that are the focus on this study are named apicomplexan parasites. Apicomplexans are harmful parasites causing diseases such as malaria and toxoplasmosis, which kill or cause disabilities to millions of people in the UK and worldwide annually. The ability of these parasites to cause disease depends on a unique structure found only in these parasites - called the apicoplast - without which the parasites cannot survive.

The apicoplast comprises four compartments in the parasite cell. We hypothesize that special Apicoplast Trxs (ATrxs) in each of these compartments control the activity of other proteins in them; and that these ATrxs will be essential for the apicoplast functions that are critical to the parasites' survival.

We started testing this hypothesis in an apicomplexan parasite named Toxoplasma gondii. We discovered two apicoplast activities that are regulated by ATrxs. Our work further found that one of the ATrxs has unique features that are not found in human Trxs, so it is now being studied as a new drug target for malaria. However, with drug-discovery being a particularly unpredictable process, we believe that to maximise the chances of success we should identify as many potential drug target candidates, operating in the same pathway, as possible. There is every reason to believe that other players of the redox regulatory network of apicoplast functions will also be essential, unique to these parasites and promising candidate for drug targets.

We have identified total of seven ATrxs which we expect to regulate many proteins in the apicoplast. We propose to identify the proteins in the apicoplast that are regulated by these new ATrxs and to characterize the roles of each of the ATrxs in this critical regulatory mechanism. This work will enhance the understanding of how this unique and essential parasite structure works. The knowledge that will be generated will likely continue to seed drug discovery for apicomplexans.

You can read more about the importance of this project and about the work that we did leading to this project and already published in "the conversation":
http://theconversation.com/finding-the-achilles-heel-of-the-cat-parasite-could-mean-more-effective-treatment-for-toxoplasmosis-and-malaria-92232

Compartmentalization is fundamental for eukaryotic cells, allowing proteins to function in optimal biochemical environments. Redox control of protein function plays a critical role in defining compartmental functions, and thioredoxins (Trxs) are key players in this regulatory mechanism.
Apicomplexan parasites are global killers and their ability to sustain infection depends on the functions of their plastid, the apicoplast. The apicoplast contains four compartments and is predicted to have seven Trxs, two of which control essential apicoplast housekeeping functions. Through functional study of all apicoplast Trxs (ATrxs) and their substrates we will create a comprehensive understanding of the redox regulatory network controlling apicoplast functions. This work will improve our understanding of how apicomplexans sustain infection, and will seed drug discovery.

Aims
1. Trx control of protein function occurs via disulfide exchange. We will use the biochemical nature of this interaction to trap ATrxs with their substrates through genetic modification and to isolate and identify the substrates via pull-downs and proteomics analyses.
Substrate identity will unravel the apicoplast pathways regulated by ATrxs and will point out new essential players in this redox control network beyond the ATrxs.
2. We will perform a systematic functional analysis of all ATrxs and of selected substrates, using our inducible knock-down system to deplete each of them, and will use our array of functional assays to analyze the phenotypes resulting from their depletion. This will provide an alternative and complementary approach to aim 1 for identifying the cellular pathways controlled by the ATrx redox network.
We will also analyze the biochemical and cellular properties of substrates after depletion of their respective ATrxs. This will clarify the molecular mechanism of how the disulfide exchange between ATrxs and substrates achieves functional control of essential apicoplast pathway

Our project addresses fundamental cell biology questions, so its primary output will be relevant to a broad range of academics in the UK and globally, providing significant advances in our understanding of how redox controls cellular pathways, and bridging a critical gap in our knowledge of parasite biology. Our findings will also inform drug discovery by dissecting an apicomplexan cellular regulatory network that is essential for parasite survival in the host, and through identifying new drug targets. Impact will also be in training of early career professionals and academics and through public engagement.

Academic impact
The academic beneficiaries include researchers of apicomplexan cell biology, of cell evolution and endosymbiosis, and of redox biology. Our findings will be published in open access journals, presented at national and international conferences and in invited seminars and further disseminated via the lab and institute websites and social media feeds.

Impact on the pharma industry
The knowledge and reagents generated by the proposed work will inform and facilitate drug development. The direct beneficiaries are pharmaceutical companies and public-private partnerships invested in the discovery and development of anti-malaria and anti-toxoplasmosis drugs. The work leading to this proposal already identified a new drug target, which would kill the parasites via a novel mode of action compared to the drugs hitherto being developed, thus meeting one of the high priority criteria defined by organizations like the Medicines for Malaria Venture (MMV). Any new targets identified in the proposed work would kill through this same new mechanism, thus maximizing the chance for this new strategy to lead to successful drug development. We are already collaborating with GSK to screen for inhibitors for our recently identified target, and any new targets discovered in the proposed work will follow the translational pathway that we already have in place with GSK.

Impact to patients
Malaria infects 200 million people and kills almost 0.5 million annually. The increase in resistance to current drugs highlights the need for more drugs and particularly for drugs with new modes of action. Thus, malaria patients are potentially very important future beneficiaries, if new targets are discovered and if suitable inhibitors are found that could be developed into clinical leads.

Impact via training
Training in an academic research setting provides continuous impact to the growth and progress of science and technology in the UK. This project will employ a technician and an RA both of whom will be mentored by the PI. Likewise, throughout the funded period the PI will continue to train students at all levels. Training will include cutting edge technical skills (e.g. advanced molecular biology, genetic manipulation techniques, microscopy, proteomics and analysis of large datasets) and essential transferable skills (e.g. leadership, project management, study design, problem solving, interpretation of data, creative thinking, efficient time management, effective communication and networking). The PI is highly committed to enhancing trainee employability and to help early career researchers in their career progress, and she will provide the guidance for acquisition of these skills.

Impact via public engagement
The PI is enthusiastic about informing broad audiences about the most recent academic advances and is highly active in various engagement initiatives. For example, she is the scientific editor for the Toxoplasma chapter of the Wellcome Centre for Molecular Parasitology popular comic series. In other examples, the PI wrote an article in The Conversation and was interviewed for local media about the work leading to this proposal. The PI, RA and technician will take part in public engagement activities throughout the duration of the grant.