The role of polyphosphate in Toxoplasma gondii

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PROJECT SUMMARY/ABSTRACT Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases like malaria, toxoplasmosis, and cryptosporidiosis. The phylum member Toxoplasma gondii alone infects approximately one third of the world population. The pathogenesis of T. gondii is reliant on its capacity to replicate within host cells and spread to other host cells. The acute infection is ultimately controlled by the host immune response and the parasite establishes a chronic infection characterized by the presence of tissue cysts containing slow replicating bradyzoites. Tissue cyst rupture releases bradyzoites that convert into tachyzoites that replicate fast and if the host is immunocompromised, could cause severe and even fatal tissue damage. We propose to study how polyphosphate (polyP), a “forgotten molecule”, exerts its role in pathogenicity and virulence of T. gondii. PolyP is a ubiquitous polymer of three to hundreds of phosphate residues linked by high-energy phosphoanhydride bonds and can reach millimolar levels in protozoan parasites, while the concentration in host cells is at the micromolar level. T. gondii stores polyP at molar levels in acidocalcisomes which are hydrolyzed upon alkalinization followed by release of Ca2+ into the cytosol. It is puzzling how little is known about the function of polyP in the T. gondii infection cycle and/or virulence. However, characterizing how polyP functions will impact our knowledge of the mechanism by which T. gondii causes disease. Both in bacteria and eukaryotes polyP is involved in a large number of diverse and apparently unrelated functions. However, these apparently unrelated functions may be the result of polyP’s ability to interact with proteins either electrostatically or covalently and we propose to test this for T. gondii. Our hypothesis is that polyP exerts its essential function in the lytic cycle of T. gondii through its interaction with proteins and/or their covalent modification by polyphosphorylation. We propose two strategies to determine how polyP exerts its role in virulence. To define candidate proteins that interact with polyP we will use Thermal Proteome Profiling and expose intact parasites and/or lysates to heat stress to underscore the role of polyP as a protein-like chaperone. We will also use a bioinformatics approach to discover potential proteins that are covalently modified by polyP. We propose to use polyP mutants (that do not make polyP) already available for the characterization of the role of polyP interaction with target proteins. The synthesis of polyP represents a novel target as it is essential, and the enzymes are absent in the mammalian host. The proteins involved in polyP synthesis are conserved in other apicomplexan parasites. The polyP target proteins will be specific for T. gondii virulence pathways and could have orthologs in other apicomplexans. Additionally, findings from this project could have wider impact for vertebrate biology since the mammalian polyP synthesis machinery is unknown and our findings may uncover novel functions and proteins.