Biology subjects

Corvi, M. M.

Publications and source records attributed to Corvi, M. M..

3 recordsLinked to original sources

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↗

bioRxiv2023

Identification of Toxoplasma Calcium Dependent Protein Kinase 3 as a Stress-Activated Elongation Factor 2 Kinase

Toxoplasma gondii is an obligate intracellular parasite whose tachyzoite form causes disease via a lytic growth cycle. Its metabolic and cellular pathways are primarily designed to ensure parasite survival within a host cell. But during its lytic cycle tachyzoites are exposed to the extracellular milieu and prolonged exposure requires activation of stress response pathways that include reprogramming the parasite proteome. Regulation of protein synthesis is therefore important for extracellular survival. We previously reported that in extracellularly stressed parasites that the elongation phase of protein synthesis is negatively regulated by the Toxoplasma oxygen sensing protein, PhyB. PhyB acts by promoting the activity of elongation factor eEF2, which is a GTPase that catalyzes the transfer of the peptidyl-tRNA from the A site to the P site of the ribosome. In the absence of PhyB, eEF2 is hyper-phosphorylated, which inhibits eEF2 from interacting with the ribosome. eEF2 kinases are atypical calcium-dependent kinases and BLAST analyses revealed the parasite kinase, CDPK3, as the most highly homologous to the Saccharomyces cerevisiae eEF2 kinase, RCK2. In parasites exposed to extracellular stress, loss of CDPK3 leads to decreased eEF2 phosphorylation and enhanced rates of elongation. Furthermore, co-immunoprecipitation studies revealed that CDPK3 and eEF2 interact in stressed parasites. Since CDPK3 and eEF2 normally localize to the plasma membrane and cytosol, respectively, we investigated how the two can interact. We report that under stress conditions that CDPK3 is not N-myristoylated likely leading to its cytoplasmic localization. In summary, we have identified a novel function for CDPK3 as the first protozoan extracellular-stress induced eEF2 kinase. IMPORTANCEHere, we identify the first protozoan kinase that phosphorylate elongation factor 2 and demonstrate that it is part of an integrated stress response.

microbiology↗

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↗