Human GBPs are a family of seven ~65kDa GTPases whose expression is upregulated by IFNg, for example during an infection. All mammalian GBPs contain a GTPase domain and a C-terminal helical region. GBP1, GBP2 and GBP5 have a C-terminal CXXX motif that is modified by lipidation which then can anchor them to membranes. GBP1 is a unique enzyme with GTPase and GDPase activities that convert GTP to GMP in two steps. GBPs can traffic to microbial compartments destroying these membranes, enhance inflammation by activating the inflammasome and limit pathogen replication. All of these pathways happen inside the infected cell with the goal of controlling the infection and are thus called cell-intrinsic host defence. There are several gaps in our understanding of GBP-regulated pathways of cell-intrinsic defence. For example, how GBP trafficking to pathogens is regulated, how they open microbial compartments, and how they ultimately limit microbial replication are important unanswered questions in the field. GBPs play a role in pathogen defence for viruses, bacteria and protozoa.
For many of these pathogens, the prerequisite for GBP-mediated control is targeting of the GBP to the pathogen-containing compartment or the pathogen directly, with the consequence being the disruption of the compartment or the co-recruitment of signalling molecules. Toxoplasma gondii is an apicomplexan parasite that resides in an intracellular compartment, the parasitophorous vacuole (PV). The PV is derived from the host’s plasma membrane and modified by Toxoplasma to sustain its growth. However, IFNg-stimulated host cells can control Toxoplasma burden and replication in the PV by upregulating GBPs. GBP deficient cells fail to control Toxoplasma replication. Surprisingly, we have shown that for some GBPs or their mutants, Toxoplasma growth control happens without targeting of the vacuole and without inducing host cell death ( and unpublished). Hence, we hypothesise that Toxoplasma growth control by some GBPs is uncoupled from the direct attack on the PV membrane that usually leads to Toxoplasma killing and host cell death.
This project will discover the pathways and mechanisms of how GBPs accomplish Toxoplasma control without direct targeting of the PV. We can analyse Toxoplasma burden and growth and host cell death in human macrophages employing both cell lines (THP-1) or primary cells (MDMs, monocyte-derived macrophages) [2, 3]. We downregulate the GBP under study using siRNA in both systems or employ CRISPR technologies in THP-1s to analyse the behaviour of GBP mutants reconstituted in the corresponding GBPKO background. We have set up high-content image analysis that measures infection parameters such as parasite number/cell, parasite number/vacuole, host protein recruitment percentages and intensities and PV and parasite integrity using artificial intelligence algorithms .
This enables us to assess quantitatively and reproducibly the effect of many GBP mutants throughout many timepoints of infection. In this project, we will define the cellular localisation and dynamics of GBPs (and their mutants) that do not target the PV, yet control Toxoplasma replication. We will employ our high content technologies, as well as previously set-up live cell imaging and confocal microscopy. In addition to the GTPase and lipidation mutants described above, we will analyse mutants of GBPs that fail to be ubiquitinated or phosphorylated to define the post-translational regulatory mechanisms active in Toxoplasma control. Using these GBP mutants and the knowledge acquired in Toxoplasma infection control dynamics, we will employ proximity-labelling and mass spectrometry to discover novel cellular host factors participating in this pathway. These novel players will be characterised for their ability to control Toxoplasma and their cellular localisation dynamics. In summary, this project will define novel regulatory mechanisms of GBP-driven Toxoplasma control that are distinct from direct parasite targeting, elimination and subsequent host cell death.
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1. Johnston, A.C., et al., Human GBP1 does not localize to pathogen vacuoles but restricts Toxoplasma gondii. Cell Microbiol, 2016. 18(8): p. 1056-64.
2. Fisch, D., et al., Human GBP1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis. EMBO J, 2019. 38(13): p. e100926.
3. Fisch, D., et al., Human GBP1 Differentially Targets Salmonella and Toxoplasma to License Recognition of Microbial Ligands and Caspase-Mediated Death. Cell Rep, 2020. 32(6): p. 108008.
4. Fisch, D., et al., Defining host-pathogen interactions employing an artificial intelligence workflow. Elife, 2019. 8.