Unveiling the Unique Structure and Singular Function of the Histone Deacetylase 2 (TgHDAC2) of Toxoplasma gondii

Histone deacetylases (HDACs) are enzymes traditionally recognized for their role in removing acetyl groups from lysines on histones. However, recent findings have revealed that many HDACs also target non-histone proteins. In Toxoplasma gondii, although we identified TgHDAC2, an enzyme annotated as a class I HDAC, we found that its substrates are non-histone proteins. Notably, TgHDAC2 possesses two unique peptide insertions within its HDAC domain, whose structural and functional roles were previously unknown. Using cross-linking mass spectrometry (XLMS), we resolved the three-dimensional structure of TgHDAC2, while biophysical analyses demonstrated that these insertions do not compromise the proteins stability but play an important part in its function. Localization studies revealed differential expression of TgHDAC2 throughout the cell cycle, with prominent enrichment around daughter cells during mitosis and cytokinesis. Its deletion severely disrupts parasite replication, suggesting a critical role in cell cycle regulation. RNA sequencing of TgHDAC2 knockout parasites highlighted significant downregulation of genes involved in membrane composition, cytoskeletal organization, and cell signaling pathways, further supporting its role in modifying non-histone proteins. Collectively, our results suggest that TgHDAC2 acts as a deacetylase for non-histone proteins, modulating cytoskeletal and membrane proteins critical for T. gondii cell cycle progression and replication. IMPORTANCEToxoplasma gondii is an obligate intracellular parasite and a significant global public health concern. It is estimated that up to one-third of the worlds population may be infected, depending on the region, with even higher prevalence rates in South America due to the circulation of atypical and more virulent strains. Understanding the biology of this parasite and identifying novel therapeutic targets is therefore critical, as current treatments are outdated and ineffective against the chronic phase of toxoplasmosis. In this study, we identified a novel lysine deacetylase that plays an essential role in T. gondii replication, highlighting its potential as a promising therapeutic target.