Regulation of latent stage differentiation through central carbon metabolism and ubiquitination

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Project summary Toxoplasma latently infects approximately 30% of the human population and is refractory to acute stage therapies. Latent Toxoplasma can act as a reservoir for acute stage reactivation in immunocompromised individuals and can cause progressive blindness in otherwise healthy individuals. Thus, latent stages are clinically important, but currently untreatable. Drug resistance is likely due to changes in the metabolism of latent stages, but what these are remain a complete mystery. Furthermore, latent Toxoplasma is exposed to different nutritional environments when infecting its target tissue types – muscle and the central nervous system (CNS) – and the impact this has on Toxoplasma latency is not understood. We have found that changes in carbon sources, that mimic muscle and CNS trigger Toxoplasma differentiation into latent forms. We then performed a whole-genome CRISPR screen and identified select Toxoplasma carbon catabolic enzymes that are required for differentiation and furthermore, identified a role for ubiquitination in metabolic adaptation and differentiation into latent forms. These background studies form the basis for developing a comprehensive understanding of Toxoplasma metabolism during latency and understanding how metabolic rewiring is linked to differentiation through regulation by ubiquitination. The specific aims of this program are to: Aim 1: Define the metabolic pathways required for differentiation and persistence of latent Toxoplasma. We will determine the role of select enzymes (identified in CRISPR screen) required for utilization of carbon sources that mimic brain and muscle and their impact on differentiation in vitro and in vivo. This will determine how metabolism of latent and acute stages differs and how this is linked to environmental sensing and differentiation. Aim 2: Determine the role of ubiquitination in the regulation of differentiation into latent forms. Define the composition, biochemical activity and structure of an E3 ubiquitin ligase complex that is important for differentiation into latent forms, thus determining how ubiquitination regulates differentiation. Aim 3: Define how ubiquitination regulates metabolism and differentiation into latent forms. We have identified likely substrates of the E3 ubiquitination machinery and will define their function in regulation of both metabolism and differentiation.