Principal Supervisor: Prof Eva FrickelLink opens in a new window

Co-supervisor: 

PhD project title: The role of Human Guanylate Binding Proteins in Toxoplasma gondii infection

University of Registration: University of Warwick

Project outline:

Toxoplasma gondii is a successful intracellular protozoan parasite that can invade any nucleated cell, a trait that has allowed it to have a broad range of animal hosts, including humans. Global human Toxoplasma infections are estimated at 30% of the population and once chronically infected, the host cannot clear the parasite. Infections are often asymptomatic, with serious complications and death mainly restricted to immunocompromised and congenitally infected humans. It is unclear how normal immunocompetent individuals control Toxoplasma during acute infection.

During its life cycle, the fast replicative form of the parasite known as the tachyzoite causes systemic acute infection and replicates within a membrane bound replicative compartment known as the parasitophorous vacuole (PV). Toxoplasma infection leads to the production of the inflammatory cytokine interferon gamma (IFNg), which stimulates host cells to upregulate defence mechanisms to control the parasite. Macrophages are front-line innate immune cells important for the control of pathogens and/or eliciting an immune response via undergoing programmed host cell death.

One protein family produced by IFNg stimulation are the guanylate binding proteins (GBPs). During infection, GBPs traffic to microbial replicative compartments resulting in the control pathogen replication and the induction of host cell death (1). We have shown that GBP1 targets the Toxoplasma PV and the parasite plasma membrane, disrupts these releasing parasite DNA and triggers macrophage apoptosis (2, 3). However, the mechanisms for human GBP targeting to microbial compartments and its function in conjunction with other cellular defence proteins to control microbial replication remain poorly understood.

The Frickel lab routinely analyses Toxoplasma growth and host cell death in human macrophages employing both cell lines (THP-1) or primary cells (MDMs, monocyte-derived macrophages). We have set up high-content image analysis that measures infection parameters such as parasite numbers and size, host protein recruitment to the PV, as well as PV and parasite integrity using artificial intelligence algorithms (HRMAn, Host Response to Microbe Analysis) (4, 5). We perform siRNA downregulation, CRISPR KO and mutational analysis of host proteins to define their mechanistic contribution in host innate defence to pathogens. This enables us to quantitatively and reproducibly assess the effect of GBP mutants and novel proteins throughout timepoints of infection.

We have recently conducted a full genome siRNA screen in IFNg-stimulated macrophages infected with Toxoplasma and identified a range of potential new host proteins that curb parasite replication. We have also identified point mutants that abolish post-translational modification of GBP1 that abrogate its anti-Toxoplasma function.

In this project, we will pursue the following aims:

• Define novel players at the Toxoplasma PV that regulate parasite growth in IFNg-stimulated macrophages.

This aim will rely of sequential siRNA downregulation of the previously identified candidate proteins and the characterisation of their contribution to Toxoplasma control as well as GBP1 recruitment to the PV. Mutational analysis of confirmed hits will define their functional contribution to parasite control at the PV.

• Analyse the spacio-temporal requirement for ubiqitination and phosphorylation of GBP1 at the Toxoplasma

This aim will analyse two mutants of GBP1 that cannot be ubiquitinated or phosphorylated respectively. Both mutants fail to control Toxoplasma, and we will define their capacity to be recruited to the PV and where the post-translational modification takes place. If time allows we will identify the E3 ubiquitin ligase or the kinase responsible for the post-translational modification.

In summary, this project will define novel players and mechanisms of IFNg-driven control of Toxoplasma in human macrophages.

References:

1. Tretina K, Park ES, Maminska A, MacMicking JD. Interferon-induced guanylate-binding proteins: Guardians of host defense in health and disease. J Exp Med. 2019;216(3):482-500.
2. Fisch D, Bando H, Clough B, Hornung V, Yamamoto M, Shenoy AR, et al. Human GBP1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis. EMBO J. 2019;38(13):e100926.
3. Fisch D, Clough B, Domart MC, Encheva V, Bando H, Snijders AP, et al. Human GBP1 Differentially Targets Salmonella and Toxoplasma to License Recognition of Microbial Ligands and Caspase-Mediated Death. Cell Rep. 2020;32(6):108008.
4. Fisch D, Yakimovich A, Clough B, Wright J, Bunyan M, Howell M, et al. Defining host-pathogen interactions employing an artificial intelligence workflow. Elife. 2019;8.
5. Fisch D, Evans R, Clough B, Byrne SK, Channell WM, Dockterman J, et al. HRMAn 2.0: Next-generation artificial intelligence-driven analysis for broad host-pathogen interactions. Cell Microbiol. 2021;23(7):e13349.

 

BBSRC Strategic Research Priority: Understanding the rules of life – Immunology.

 

Techniques that will be undertaken during the project:

Culturing of human cells and Toxoplasma gondii parasite lines

Fluorescent microscopy: superresolution, confocal, widefield and high content imaging

Artificial intelligence driven image analysis

Genetic manipulation of tissue culture cells: siRNA, CRISPR

Immunoblotting

Immunoprecipitation from cellular lysates

ELISA and cell death assays

Preparation of cellular lysates for mass spectrometry analysis

 

Contact: Prof Eva FrickelLink opens in a new window