Abstract
Abstract
Histone H2A.X is a conserved variant classically associated with the DNA damage response (DDR), where its phosphorylation (γH2A.X) marks double-strand breaks and recruits repair factors. H2A.X is absent in many parasitic protozoans such as
Plasmodium
,
Trypanosoma
and
Leishmania
. In
Toxoplasma gondii
, the obligate intracellular parasite, causative agent of toxoplasmosis, however, its presence raises the question of whether it contributes to genome stability. To tackle this, we generated a
T. gondii
knockout of H2A.X (RHΔH2A.X) and evaluated its phenotype. Although H2A.X is phosphorylated in response to DNA damage in
T. gondii
as in other systems, RHΔH2A.X parasites showed no increased sensitivity to genotoxic agents, indicating that H2A.X is dispensable for canonical DDR. Instead, loss of H2A.X caused reduced gliding motility, increased host cell invasion and clustering of extracellular parasites after egress. Despite no defects in replication rate, RHΔH2A.X showed delayed mitotic progression with abnormal vacuole morphology. Transcriptomic profiling revealed a moderate yet coordinated reprogramming of protein-coding genes. Specifically, H2A.X loss altered the expression of key apical secretory and virulence factors, especially ROP18, while inducing the transcriptional activation of mitotic and DNA replication machinery. Differential enrichment analysis further revealed that upregulated genes were preferentially associated with S phase, whereas downregulated genes were enriched during mitosis and cytokinesis. This is consistent with the delayed mitotic progression and spindle abnormalities observed by microscopy and flow cytometry and suggests a cell cycle phase-specific mechanism of H2A.X-dependent transcriptional control. Rather than a driver of DDR, these findings suggest that
T. gondii
H2A.X operates as a chromatin organizer that coordinates cell division fidelity and virulence gene expression.
Author Summary
Cells package DNA into chromatin, composed of histone proteins, whose variants fine-tune gene activity in response to cellular needs. One such variant, H2A.X, is best known across eukaryotes for its canonical role in signaling DNA damage: upon double-strand breaks, H2A.X is phosphorylated and acts as a molecular beacon to recruit repair machinery. Intriguingly, H2A.X is absent in many parasites closely related to
Toxoplasma gondii
, the opportunistic pathogen responsible for toxoplasmosis. We therefore explored the functional role of H2A.X in
Toxoplasma
. By generating parasites completely lacking this histone variant, we found that H2A.X is dispensable for the canonical DNA damage response. Instead, H2A.X operates as a global chromatin organizer that controls the timing of cell-cycle progression and regulates the expression of genes essential for parasite motility, host cell invasion, and virulence. Our findings reveal that
Toxoplasma
has repurposed a conserved histone variant for functions beyond DNA repair, with potential consequences for how this parasite establishes and maintains infection.