Biology subjects

Roson, J. N.

Publications and source records attributed to Roson, J. N..

4 recordsLinked to original sources

bioRxiv2026

NEK kinase TcRDK2 controls differentiation, host-cell infection, and remodeling of the translation initiation machinery in Trypanosoma cruzi

Trypanosoma cruzi, the etiologic agent of Chagas disease, alternates between replicative epimastigotes and amastigotes and non-dividing, mammal-infective metacyclic and bloodstream trypomastigotes. Protein phosphorylation is a major regulatory mechanism in trypanosomatids, whose kinomes reveal an expanded family of NIMA-related kinases (NEKs). Here, we investigated the role of T. cruzi RDK2 (Repressor of Differentiation Kinase 2), a conserved NEK that carries a C-terminal pleckstrin homology (PH) domain. Endogenous gene tagging showed that TcRDK2 is expressed in all major life-cycle stages and displays a cytoplasmic distribution. CRISPR/Cas9-mediated knockout of TcRDK2 did not markedly alter epimastigote growth in rich medium but caused a significant accumulation of cells with abnormal nuclear/kinetoplast configurations, consistent with defects in kinetoplast segregation and cytokinesis; TcRDK2-null parasites also showed reduced in vitro metacyclogenesis and failed to establish efficient infections in human fibroblasts. To probe gain-of-function effects, we generated tetracycline-inducible overexpression lines for full-length TcRDK2 (RDK2WT), a PH-deleted variant (RDK2{Delta}PH), and a catalytic-dead mutant (RDK2K70A). Overexpression of RDK2WT or RDK2{Delta}PH decreased epimastigote growth, enhanced metacyclogenesis, and strongly impaired host-cell invasion and intracellular amastigote proliferation, with more pronounced phenotypes for RDK2{Delta}PH, suggesting that the PH domain normally restrains TcRDK2 activity in vivo. Phosphoproteomic profiling of RDK2WT-overexpressing epimastigotes identified candidate TcRDK2 substrates and pathways, including translation initiation and cytoskeletal regulation. Together, these data identify TcRDK2 as a NEK kinase that coordinates kinetoplast replication/segregation, metacyclogenesis, and host-cell infection in T. cruzi and support TcRDK2 as a promising, kinetoplastid-specific therapeutic target for Chagas disease. IMPORTANCEChagas disease, caused by the parasite Trypanosoma cruzi, remains a major health problem with limited treatment options. To persist in both insect vectors and mammalian hosts, the parasite must precisely coordinate cell division, differentiation into infectious forms, and survival inside host cells. Protein kinases are central regulators of these processes and attractive drug targets, yet many remain poorly understood in T. cruzi. In this study, we investigate RDK2, a member of the NIMA-related kinase family. Using gene knockout, inducible overexpression, and global analysis of phosphorylated proteins, we show that RDK2 is required for accurate segregation of mitochondrial DNA, efficient formation of infective insect-stage forms, and successful infection and replication in human cells. These findings identify RDK2 as a key regulator that links parasite cell division to infectivity and highlight it as a promising, parasite-specific candidate for future drug development against Chagas disease.

microbiology↗

bioRxiv2025

New Histone H4 variant and H2B variant Exhibits Distinct Genomic Locations, Chromatin Affinities, and Dynamics Throughout Life Cycle and Cell Cycle of Trypanosoma cruzi

Histone variants play crucial roles in chromatin organization and transcriptional regulation in eukaryotes. Unusually, trypanosomatids display histone variants for all histones, although a functional homolog of histone H4 variant (H4.V) had not yet been described in Trypanosoma cruzi. In this study, we identified a H4.V in T. cruzi that is encoded by a single-copy gene located apart from the typical tandem arrays of canonical histone H4. Functional characterization using ChIP-seq assays revealed that H4.V is located at telomeric regions, demarcates convergent strand-switch regions (cSSRs), and determines new transcription termination sites at codirectional PTUs interrupted by tDNA loci. Throughout the cell cycle, H4.V transcript levels remain stable, while protein abundance increases in G2/M, as shown by immunofluorescence and image flow cytometry. In contrast, the histone H2B variant (H2B.V) transcripts peak at S-phase, and protein abundance accumulates progressively. H4.V is more abundant in the nuclei of metacyclic trypomastigotes but barely detectable in amastigotes and bloodstream trypomastigotes. In contrast, H2B.V shows a punctate nuclear pattern and is present in all life stages, with the highest levels also observed in metacyclics. During metacyclogenesis, both variants show a progressive increase in expression, particularly H4.V, suggesting a role in parasite differentiation. Ultimately, salt extraction experiments demonstrated differing chromatin binding affinities across life stages for both variants, with H4.V displaying a more permanent chromatin association than H2B.V, indicating that H4.V may be associated with a more compact chromatin state. Our data reveal H4.V as a novel histone variant in T. cruzi, characterized by unique genomic localization, expression profiles, and chromatin-binding dynamics in contrast to H2B.V, underscoring its potential function as an epigenetic marker in transcriptional regulation and adaptation to diverse host environments. Author summaryChromatin is composed of a group of proteins called histones that are involved in compacting DNA and thereby affecting many DNA-associated mechanisms, such as replication and transcription. Histone variants share distinct primary sequences from the canonical ones and display different roles and genomic locations. While variants of histone H3 and H2A are common in all eukaryotes, variants of H2B and H4 are unusual, except in trypanosomatids, a group of parasites that cause human and veterinary diseases. Among them, Trypanosoma cruzi, the causative agent of Chagas disease, exhibits variants for three core histones, but a variant of histone H4 (H4.V) had not been described. Here, we identify H4.V and demonstrate its preferential deposition at telomeres, convergent strand-switch regions (cSSRs), and codirectional polycistronic transcription units (PTUs) interrupted by tDNA loci, likely marking transcription termination sites. Both variants are enriched in the nuclei of metacyclic trypomastigotes; however, H4.V is barely detectable in amastigotes and bloodstream trypomastigotes, while H2B.V is consistently present in all forms. Additionally, each variant shows distinct chromatin-binding affinities across life stages, with H4.V potentially linked to a more compact chromatin state. Our findings reveal a novel histone variant with unique dynamics and epigenetic roles distinct from H2B.V in T. cruzi.

microbiology↗

bioRxiv2021

Open chromatin analysis in Trypanosoma cruzi life forms highlights critical differences in genomic compartments and developmental regulation at tDNA loci

BackgroundGenomic organization and gene expression regulation in trypanosomes are remarkable because protein-coding genes are organized into codirectional gene clusters with unrelated functions. Moreover, there is no dedicated promoter for each gene, resulting in polycistronic gene transcription, with posttranscriptional control playing a major role. Nonetheless, these parasites harbor epigenetic modifications at critical regulatory genome features that dynamically change among parasite stages, which are not fully understood. ResultsHere, we investigated the impact of chromatin changes in a scenario commanded by posttranscriptional control exploring the parasite Trypanosoma cruzi and its differentiation program using genome-wide approaches supported by transmission electron microscopy. The integration of FAIRE and MNase-seq data, two complementary epigenomic approaches, enabled us to identify differences in T. cruzi genome compartments, putative transcriptional start regions and virulence factors. In addition, we also detected developmental chromatin regulation at tRNA loci (tDNA), which seems to be linked to the translation regulatory mechanism required for parasite differentiation. Strikingly, a positive correlation was observed between active chromatin and steady-state transcription levels. ConclusionTaken together, our results indicate that chromatin changes reflect the unusual gene expression regulation of trypanosomes and the differences among parasite developmental stages, even in the context of a lack of canonical transcriptional control of protein-coding genes.

microbiology↗

bioRxiv2021

{-}{-} Histone H2B.V demarcates strategic regions in the Trypanosoma cruzi genome, associates with a bromodomain factor and affects parasite differentiation and host cell invasion

Histone variants play a crucial role in chromatin structure organization and gene expression. Trypanosomatids have an unusual H2B variant (H2B.V) that is known to dimerize with the variant H2A.Z generating unstable nucleosomes. Previously, we found that H2B.V protein is enriched in nonreplicative life forms of Trypanosoma cruzi, suggesting that this variant may contribute to the differences in chromatin structure and global transcription rates observed among parasite life forms. Here, we performed the first genome-wide profiling of histone localization in T. cruzi using replicative and nonreplicative life forms, and we found that H2B.V was preferentially located at the edges of divergent switch regions, which encompass putative transcriptional start regions; at some tDNA loci; and between the conserved and disrupted genome compartments, mainly at trans-sialidase, mucin and MASP genes. Remarkably, the chromatin of nonreplicative forms was depleted of H2B.V-enriched peaks in comparison to replicative forms. Interactome assays indicated that H2B.V associated specifically with H2A.Z, bromodomain factor 2, nucleolar proteins and a histone chaperone, among others. Parasites expressing reduced H2B.V levels were associated with higher rates of parasite differentiation and mammalian cell infectivity. Taken together, H2B.V demarcates critical genomic regions and associates with regulatory chromatin proteins, suggesting a scenario wherein local chromatin structures associated with parasite differentiation and invasion are regulated during the parasite life cycle. Author SummaryTrypanosomatids have to adapt to different environmental conditions, changing their morphology, gene expression and metabolism. These organisms have many unique features in terms of gene expression regulation. The genomic organization includes polycistronic regions with the absence of well-defined transcription start sites. In T. brucei, histone variants mark the start and ending sites of transcription; however, little is known about whether these proteins change their genome location, expression levels and interactors along life forms and what the impact is of these changes on parasite differentiation and infection. In T. cruzi, the causative agent of Chagas disease, we previously found that the histone variant of H2B is enriched in nonreplicative and infective forms, suggesting that this variant may contribute to the differences in chromatin structure and global transcription rates observed among these life forms. Here, we aimed to go one step further and performed the first histone ChIP-seq analysis in T. cruzi, in which we found that H2B.V was enriched at divergent strand switch regions, some tDNA loci and other critical genomic regions associated with T. cruzi genome compartments. We found that H2B.V interacts with a bromodomain factor, suggesting an intricate network involving chromatin acetylation around H2B.V enriched sites. Moreover, parasites expressing reduced H2B.V levels were associated with higher rates of differentiation and mammalian cell infectivity.

microbiology↗