# AP2X-8 Is Important for Tachyzoite Growth and Bradyzoite Differentiation of Toxoplasma gondii

## TL;DR

This study shows that AP2X-8 is a key regulator of Toxoplasma gondii's tachyzoite growth and bradyzoite differentiation.

## Contribution

The study identifies AP2X-8 as a novel AP2 transcription factor regulating T. gondii development.

## Key findings

- AP2X-8 promotes tachyzoite growth and suppresses bradyzoite differentiation in vitro.
- Deletion of ap2X-8 impairs plaque formation and intracellular replication but does not affect virulence in mice.
- AP2X-8 is constitutively expressed in both tachyzoite and bradyzoite stages.

## Abstract

Toxoplasma gondii has a complex life cycle involving three main stages: oocysts (containing sporozoites), tachyzoites, and tissue cysts (containing bradyzoites). Understanding the mechanisms that regulate the transition between tachyzoites and bradyzoites has been a major focus of T. gondii research. Previous studies have identified several AP2 transcription factors, such as AP2IV-3, AP2IX-9 and AP2IX-4, as important regulators of this process. In this study, we investigated the role of another AP2 factor, AP2X-8. Using C-terminal endogenous tagging combined with immunofluorescence analysis, we determined that AP2X-8 is a nuclear protein constitutively expressed in both tachyzoite and bradyzoite stages. We then constructed an ap2X-8 knockout strain using CRISPR-Cas9-mediated homologous recombination. Through a series of in vitro phenotypic assays, including plaque formation, invasion, replication, egress, and bradyzoite differentiation, we assessed the impact of ap2X-8 deletion. Our results demonstrated that AP2X-8 promotes tachyzoite growth and suppresses the bradyzoite differentiation in vitro. These findings provide new insights into the regulatory role of AP2X-8 in T. gondii development and lay the groundwork for future investigations into parasite differentiation mechanisms.

Toxoplasma gondii is a protozoan parasite capable of establishing chronic infections, with potential reactivation in immunocompromised individuals. However, the molecular mechanisms governing tachyzoite-to-bradyzoite differentiation remain incompletely understood. Previous studies have identified AP2 transcription factors as key regulators of this developmental switch. In this study, we investigated the role of the AP2 factor AP2X-8. Immunofluorescence analysis revealed that AP2X-8 is constitutively expressed in the nucleus of both tachyzoite and bradyzoite stages. Using CRISPR-Cas9-mediated homologous recombination, we successfully generated an ap2X-8 knockout strain. Phenotypic assays including plaque formation, invasion, replication, and egress, and bradyzoite differentiation assays, were then performed to assess the impact of ap2X-8 deletion. Our analyses showed that the loss of ap2X-8 significantly impaired plaque formation and intracellular replication, while invasion and egress were unaffected. Furthermore, ap2X-8 knockout enhanced bradyzoite differentiation in vitro. Despite these changes, deletion of ap2X-8 did not alter parasite virulence in a mouse infection model. These findings demonstrate that AP2X-8 is an important regulator of T. gondii tachyzoite growth and bradyzoite differentiation, offering new insights into the parasite’s developmental regulation.