Biology subjects

Ganuza, A.

Publications and source records attributed to Ganuza, A..

5 recordsLinked to original sources

bioRxiv2026

Histone H2A.X contributes to Gene Expression Regulation and Cell-Cycle Progression in Toxoplasma gondii Tachyzoites

Histone H2A.X is a conserved variant classically associated with the DNA damage response (DDR), where its phosphorylation ({gamma}H2A.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 (RH{Delta}H2A.X) and evaluated its phenotype. Although H2A.X is phosphorylated in response to DNA damage in T. gondii as in other systems, RH{Delta}H2A.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, RH{Delta}H2A.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.

cell biology↗

bioRxiv2025

Toxoplasma gondii RAD51 recombinase is required to overcome DNA replication stress and its inactivation leads to bradyzoite differentiation

Toxoplasma gondii is an obligate intracellular parasite with a high replication rate that can lead to DNA replicative stress, in turn associated with the generation of DNA double-strand breaks (DSBs). Cells have two main pathways to repair DSBs: non-homologous end joining and homologous recombination repair (NHEJ and HRR respectively). RAD51 is the key recombinase in the HRR pathway. In this work, we achieved endogenous tagging of the RAD51 gene using the Auxin Inducible Degron (AID) system, to generate the clonal line RH RAD51HA-AID. Here we demonstrate that RAD51 is expressed in replicative tachyzoites and establishes damage foci. Auxin-induced knock-down (KD) affects the correct replication of tachyzoites which show loss of synchronization. The use of the RAD51 inhibitor B02 also affects parasite growth, with an IC50 of 4.8 {micro}M. B02 produced alterations in tachyzoite replication and arrest in the S phase of the cell cycle. Additionally, B02 induced tachyzoite to bradyzoite differentiation showing small cyst-like structures. In conclusion, the HRR pathway is necessary for maintaining proper tachyzoite replication under normal growth conditions, supporting that replicative stress occurs during the cell cycle. Our findings also suggest that DNA replication stress can induce bradyzoite differentiation.

molecular biology↗

bioRxiv2024

A bioinformatic approach for the prediction andfunctional classification of Toxoplasma gondii longnon-coding RNAs

Long non-coding RNAs (lncRNAs) have emerged as significant players in diverse cellular processes, including cell differentiation. Advancements in computational methodologies have facilitated the prediction of lncRNA functions, enabling insights even in non-model organisms like pathogenic parasites, in roles such as parasite development, antigenic variation, and epigenetics. In this work, we focus on the apicomplexan Toxoplasma gondii differentiation process, where the infective stage, tachyzoite, can develop into the cysted stage, bradyzoite, under stress conditions. Using a publicly available transcriptome dataset, we predicted lncRNA sequences associated with this differentiation process. Notably, a substantial proportion of these predicted lncRNAs exhibited stage-specific expression, particularly at the bradyzoite stage. Furthermore, co-expression patterns between coding transcripts and lncRNAs suggest their involvement in shared processes, such as bradyzoite development. TglncRNA loci analysis revealed their potential influence on the expression of nearby coding genes, including subtelomeric genes unique to the T. gondii genome. Finally, with a k-mer analysis approach, we identified functional relationships between characterized lncRNAs from model organisms like Homo sapiens and predicted T. gondii lncRNAs. Our perspective led to the identification of a T. gondii lncRNA potentially mediating DNA damage repair pathways, shedding light on the adaptive mechanisms of T. gondii in response to stress conditions.

bioinformatics↗

bioRxiv2023

Histone variant H2B.Z acetylation is necessary for maintenance of Toxoplasma gondii biological fitness

Through regulation of DNA packaging, histone proteins are fundamental to a wide array of biological processes. A variety of post-translational modifications (PTMs), including acetylation, constitute a proposed histone code that is interpreted by "reader" proteins to modulate chromatin structure. Canonical histones can be replaced with variant versions that add an additional layer of regulatory complexity. The protozoan parasite Toxoplasma gondii is unique among eukaryotes in possessing a novel variant of H2B designated H2B.Z. The combination of PTMs and the use of histone variants is important for gene regulation in T. gondii, offering new targets for drug development. In this work, T. gondii parasites were generated in which the 5 N-terminal acetylatable lysines in H2B.Z were mutated to either alanine (c-Myc-A) or arginine (c-Myc-R). c-Myc-A mutant only displayed a mild effect in its ability to kill mice. c-Myc-R mutant presented an impaired ability to grow and an increase in differentiation to latent bradyzoites. This mutant line was also more sensitive to DNA damage, displayed no virulence in mice, and provided protective immunity against future infection. While nucleosome composition was unaltered, key genes were abnormally expressed during in vitro bradyzoite differentiation. Our results show that the N-terminal positive charge patch of H2B.Z is important for these procceses. Pull down assays with acetylated N-terminal H2B.Z peptide and unacetylated one retrieved common and differential interactors. Acetylated peptide pulled down proteins associated with chromosome maintenance/segregation and cell cycle, opening the question of a possible link between H2B.Z acetylation status and mitosis.

cell biology↗

bioRxiv2020

Resveratrol induces H2A.X phosphorylation and H4K16 de-acetylation in Toxoplasma gondii

Resveratrol (RSV) is a multi-target drug that demonstrated activity against Toxoplasma gondii in macrophage and HFF cell line infection models. In addition to modulate redox homeostasis, RSV is also an activator of Sir2, a type III HDAC. RSV inhibited intracellular T. gondii tachyzoite growth at concentrations below the toxic effect on host cells. The IC50 value in a 24-hours treatment was 53 M. After 96 hours of treatment the maximum non-toxic concentration for host cell, 20 M, only inhibited T. gondii growth a 50%. RSV induced a reduction in H4K16 acetylation (H4K16ac), a mark associated to transcription, DNA replication and homologous recombination repair, without any effect on H3 acetylation (H3ac). RSV also enhanced the SQE motif phosphorylation on T. gondii H2A.X (termed {gamma}H2A.X), a DNA damage associated PTM. Sirtinol, a specific Sir2 inhibitor also inhibited T. gondii but did not altered the acetylation status of H3 and H4K16 as well as H2A.X phosphorylation. Our findings suggest a possible link between RSV and DNA damage or DNA repair process maybe due to DNA replication stress and/or another undetermined mechanism.

microbiology↗