Hijacking of the HAUSP/p53 pathway by the Toxoplasma protein GRA16: a mechanism regulating p53-dependent metabolism, cell fate and immune signaling during toxoplasmosis? – ToxoP53

A global analysis of host cell gene expression comparing wild type parasites with parasites mutated for the parasite effector determines the impact of parasite effectors on infected cells.
The mechanism of interaction of GRA16 with its target protein HAUSP is investigated using a variety of biochemical and genetic methods. Metabolomics methods utilizing HRMAS-NMR and real-time metabolic analysis using Seahorse are used to evaluate the influence of GRA16 on HAUSP, MDM2, and p53 dependent metabolic signaling pathways.

Major Project Outcomes:
Using a biochemical method, we established the minimal requirements for GRA16 to interact with HAUSP, as well as the HAUSP binding region that GRA16 recognizes.

This project has multiple prospects. The continuation of this effort on other parasite effectors should enhance our understanding of parasite biology and mechanisms of interference with the host cell's different signaling pathways from a fundamental scientific standpoint. In terms of applications, identifying host functions targeted by the parasite as well as the export processes of the effectors in the infected cell should allow for the development of new therapeutic strategies.

The ANR ToxoP53 project has led to the following scientific publications. The members involved in the project are underlined.

Valeria Bellini, Christopher Swale, Marie-Pierre Brenier-Pinchart, Tiffany Pezier, Sonia Georgeault, Fabrice Laurent, Mohamed-Ali Hakimi, Alexandre Bougdour. Target Identification of an Antimalarial Oxaborole Identifies AN13762 as an Alternative Chemotype for Targeting CPSF3 in Apicomplexan Parasites. iScience 2020 Nov 27;23(12):101871.

Toxoplasma gondii (T. gondii) belongs to the phylum Apicomplexa, which includes numerous important human and animal pathogens such as Plasmodium species, Cryptosporidium, and Neospora caninum. T. gondii is the etiologic agent of toxoplasmosis, one of the most widespread protozoan parasites of domestic, wild, and companion animals. While T. gondii infects about a third of the human population worldwide and persists usually as a quasi-cryptic population and accordingly with little clinical signs, it can turn life threatening when the immune balance is transiently or more sustainably ruptured, or still immature, as during embryonic development.
T. gondii is an obligate intracellular parasite that has the unique ability to establish a lifelong chronic infection in virtually any warm-blooded animal. The strategy of T. gondii as a parasite is based on a quest for avirulence, a capacity to attenuate but not to fully counteract the immune defense of the host, thus securing the permanent residence required to await transmission. How the parasite achieves this subtle balance in thousands of potential intermediate hosts, with strikingly different immune systems is one of the major unknown in T. gondii biology. We have recently discovered that T. gondii uses a repertoire of products stored in a large heterogeneous family of secretory dense granules (DGs) as effectors (GRA effectors) to subvert key functions of the host cells, primarily by taking over host cell gene expression during infection. One of these effectors, GRA16, forms a complex with HAUSP, the main ubiquitin-protease regulating the p53-Mdm2 pathway to alter the expression of host cell genes involved in metabolism, in cell-cycle regulatory processes, and the p53 tumor suppressor pathway. TP53 is a transcription factor with broad functions in cell stress responses through cell cycle arrest, apoptosis, DNA repair, metabolism, and immune response. In response to multiple forms of stress, p53 is activated through control of Mdm2, a ubiquitin ligase regulating p53 stability. Many infectious agents, as oncogenic viruses, have developed mechanisms to interfere with the p53-Mdm2 pathway, impeding p53-dependent cell responses. Hence, the main objective of this proposal is to generate targeted breakthrough information on the mechanisms by which the protozoan T. gondii hijacks the HAUSP-Mdm2-p53 pathway to interfere with host immune signaling and cell metabolism. We propose that obligate intracellular parasites such as T. gondii may also possess their own proprietary mechanisms to regulate p53 functions by deploying subversive proteins and modulating p53 post-transcriptional status. Elucidating how the DG-resident effector protein GRA16 subverts HAUSP activity to co-opt downstream p53-dependent and independent metabolic reprogramming of cells infected by parasites should provide novel strategies for pharmacological intervention against toxoplasmosis and other related parasites.