Chemical proteomic mapping of redox signalling in the intracellular pathogen, Toxoplasma gondii

Background
Of all protozoan parasitic infections, T. gondii is the most widespread, infecting 30-50% of the human population across 88 countries. Clinical toxoplasmosis is a serious health risk for immunodeficient individuals, and T. gondii is a significant veterinary pathogen, imposing economic burden on agricultural industries. However, current drugs suffer from issues with toxicity, prolonged treatment regimens and the emergence of resistance. Hence, there is a continuing need to discover novel drug targets.

As an obligate intracellular pathogen, T. gondii requires the environment of a host cell to survive and propagate. Pathogenesis is driven by iterative lytic growth of asexual tachyzoites, a process associated with a range of molecular events that occur in both the parasite and host. Signalling molecules such as Ca2, K and cyclic nucleotides are key regulators of tachyzoite invasion, replication and egress. More recently, hydrogen peroxide (H2O2) and nitric oxide (NO) have been recognised as important signalling molecules in eukaryotes. While notorious transducers of stress, at low levels these reactive oxygen species (ROS) can modulate protein function via reversible, discrete and selective oxidation of redox-sensitive cysteine residues. For H2O2, cysteine oxidation can alter the activity and/or localisation of diverse protein classes including kinases, phosphatases, ion channels and metabolic enzymes. Indeed, T. gondii encounters ROS throughout its life cycle and is known to suppress the production of H2O2 during oxidative challenge by host macrophages. Despite this, the full impact of redox signals on the parasite's biology is unclear.

As part of efforts to identify novel druggable nodes in the proteomes of intracellular pathogens such as T. gondii and Plasmodium, the overarching aim of this project is to molecularly map redox signalling in T. gondii. To achieve this, a combination of multidisciplinary techniques spanning biochemistry, molecular, cell and chemical biology will be used.

Objectives
1. Identify protein-associated reactive cysteines using chemical proteomics
Redox signalling is associated with the sensitivity of reactive cysteine thiols to oxidative post-translational modification. To first profile cysteine thiol reactivity in the T. gondii proteome, a quantitative mass-spectrometry-based chemical proteomic workflow will be established based on a published platform. A series of bioinformatics analyses will be performed on identified hits to gain insight into their biological importance and prioritise downstream molecular interrogation.

2. Systematic genetic validation of reactive cysteines
To systematically assess the contribution of the identified reactive cysteines to protein function and parasite fitness, a novel CRISPR/Cas9-based phenotypic screen will be undertaken. Those cysteines considered essential will be prioritised for validation using traditional genetic and biochemical approaches.

3. Identification and characterisation of redox sensors
The proteomic workflow described in Objective 1 will be modified to enable detection of reactive cysteines that are sensitive to H2O2 oxidation and thus have the capacity to transduce redox signals. Redox sensors will be validated biochemically, and reverse genetic approaches will be used to assess their contribution to essential cellular processes including host-cell invasion, replication and egress.

Broad impact
The current resurgence of covalent drugs reflects their track-record of success in the clinic. Examples include the inhibitors Afatinib and Ibrutinib, which form covalent bonds with cancer-associated kinases. With global sales of Ibrutinib expected to reach $9 billion in 2020, there is sustained industrial interest in covalent inhibitors targeting cysteines. Our research has the potential to uncover new druggable hotspots in clinically-important parasites, and thus has broad impact on industry and health.