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

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

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.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011178/1 01/10/2015 30/09/2023
1949152 Studentship BB/M011178/1 30/09/2017 30/09/2021 Henry James Benns
 
Description Using an established chemoproteomic approach, cysteine reactivity has been successfully profiled in Toxoplasma gondii, a protozoan pathogen of medical and veterinary importance. This has revealed a number of attractive sites in parasite proteins with potential to be targeted with cysteine-directed covalent small molecules, a drug class that has with well-established therapeutic value but has had little application for the treatment of infectious disease.

To systematically assess which sites are therapeutically-tractable, a novel technology platform has been developed that uses CRISPR/Cas9-based gene editing to identify functional amino acids in proteins. This screen is system-agnostic and addresses a fundamental bottleneck in drug target identification, enabling the efficient prioritisation of protein targets for ligand discovery.
Exploitation Route Parasite proteins identified to feature functional and reactive cysteines may be pursued as viable targets for the discovery and development of novel covalent inhibitors. Given the close evolutionary relationship of T. gondii to other clinically-important pathogens (such as the malaria parasite), any drug-like lead molecules could be applied to other parasitic diseases and/or explored as broad-spectrum antimicrobials.

While the developed CRISPR/Cas9-based genetic screen has been applied to reactive cysteines in the model eukaryotic pathogen T. gondii, it is system- and amino acid-agnostic in nature. Future work could involve applying this technology to functionally profile other types of chemically-targetable amino acids and/or in other disease scenarios, allowing identification of novel targets in diverse areas of biomedical research.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Title CRISPR-TAPE: a protein-centric CRISPR guide design tool for targeted proteome engineering 
Description CRISPR-TAPE is a Python-based bioinformatics tool for protein-centric design of CRISPR gRNAs. The program enables users to quickly identify gRNAs that target specific residues or amino acid types within proteins. 
Type Of Material Improvements to research infrastructure 
Year Produced 2020 
Provided To Others? Yes  
Impact Rational molecular engineering of proteins with CRISPR-based approaches is challenged by the gene-centric nature of gRNA design tools. CRISPR-TAPE is a protein-centric gRNA design algorithm that overcomes this bottleneck by that allowing users to target specific residues, or amino acid types within proteins. gRNA outputs can be customized to support maximal efficacy of homology-directed repair for engineering purposes, removing time consuming post-hoc curation, simplifying gRNA outputs, and reducing CPU times. 
URL http://www.laboratorychild.com/crispr-tape
 
Title CRISPR-TAPE: a protein-centric CRISPR guide design tool for targeted proteome engineering 
Description CRISPR-TAPE is a Python-based bioinformatics tool for protein-centric design of CRISPR gRNAs. The program enables users to quickly identify gRNAs that target specific residues or amino acid types within proteins. 
Type Of Technology Webtool/Application 
Year Produced 2020 
Open Source License? Yes  
Impact Rational molecular engineering of proteins with CRISPR-based approaches is challenged by the gene-centric nature of gRNA design tools. CRISPR-TAPE is a protein-centric gRNA design algorithm that overcomes this bottleneck by that allowing users to target specific residues, or amino acid types within proteins. gRNA outputs can be customized to support maximal efficacy of homology-directed repair for engineering purposes, removing time consuming post-hoc curation, simplifying gRNA outputs, and reducing CPU times. 
URL http://www.laboratorychild.com/crispr-tape