Multidimensional profiling of the Toxoplasma gondii proteome

Abstract

Universally, external signals are transduced and propagated in cells by secondary messengers. In the asexual and replicating stages of apicomplexan parasites, these pathways initiate and sustain transitions within the lytic cycle responsible for parasite spread and pathogenesis. Among these early-branching parasitic protists are the etiologic agents of the widespread, persistent, and deadly human diseases malaria (Plasmodium spp.) and toxoplasmosis (Toxoplasma gondii), making the understanding of these parasite signaling pathways of global importance. Although components of secondary messenger signaling pathways are conserved among apicomplexans and higher eukaryotes, 800 million years of divergence from existing model organisms precludes identification of parasite-specific secondary messenger responses or a priori reconstruction of their signaling pathways.

This thesis addresses that gap. I have adapted state-of-the-art proteomics methods to study the proteome of the model apicomplexan T. gondii across multiple dimensions: abundance, stability, time, and space. Chapter 2 describes how I employed thermal proteome profiling to identify the target of an antiparasitic compound, thereby enhancing our understanding of parasite calcium signaling pathways. In a conceptual leap, I applied this method to systematically identify calcium-responsive proteins on the basis of biochemical interactions with this second messenger in Chapter 3. From this analysis, the protein phosphatase PP1 emerged as an unanticipated calcium-responsive phosphatase along with dozens of novel proteins belonging to this critical signaling network.

Signaling pathways communicate to orchestrate complex cellular processes, yet in apicomplexan parasites they are often studied in isolation. In Chapter 4, I identify a node linking three key second messenger pathways in T. gondii: calcium, cyclic GMP, and cyclic AMP. The apicomplexan-specific kinase SPARK regulates the AGC kinases PKG, PKA C1, and PKA C3, which together control transitions within the asexual cycle of this important family of parasites.