Unraveling Transcriptional Regulatory Networks in Toxoplasma gondii

Abstract

Toxoplasma gondii, an obligate intracellular parasite, infects nearly one-third of the global population, causing the disease toxoplasmosis. Despite its significant health impact, the molecular mechanisms governing its lytic cycle and stress-induced adaptation remain incompletely understood. The unique asexual cell division mechanism, endodyogeny, used by T. gondii to expand its parasitic biomass in intermediate hosts, including humans, leads to severe pathological consequences through repeated rounds of the lytic cycle, resulting in acute toxoplasmosis. The parasite’s cell cycle is characterized by a prolonged G1 phase, with centrosome duplication marking the onset of the S phase, followed by a transient G2 phase and a near-simultaneous onset of mitosis and cytokinesis. These overlapping division processes, coupled with the challenges of synchronizing T. gondii, obscure the precise molecular mechanisms of its transcriptional programs. To address these challenges, we employed single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq), combined with advanced machine learning tools, to reveal ‘transition points’ in gene expression and chromatin accessibility that correspond to shifts in biological activity during the lytic cycle. RNA velocity and time-course clustering analyses uncovered a significant G1a transcriptional burst and identified specific AP2 family transcription factors (TFs) that peak during the C-to-G1a transition, likely driving this burst to regulate G1 progression. Further, we conducted an in-depth functional characterization of G1-specific TFs, focusing on AP2XII-8, which plays a critical role in activating a ribosome regulon to promote G1 progression. The study identified combinatorial binding motifs and suggested the existence of a large AP2XII-8 protein complex, involving other TFs and epigenetic factors, that reuglates the intricate processes of T. gondii cell cycle replication. Additionally, we examined stress-responsive AP2 TFs associated with enhanced virulence during in vitro evolution, providing insights into adaptive mechanisms that enable T. gondii to thrive under extracellular stress conditions. Collectively, these findings enhance our understanding of T. gondii’s complex regulatory networks, offering potential targets for therapeutic intervention against acute toxoplasmosis. This dissertation provides the time-resolved transcriptional and chromatin accessibility landscapes of T. gondii’s lytic cycle, resolves transcriptional programs to DNA motifs, and identifies key regulatory elements involved in its cell cycle progression and stress response.