Biology subjects

Alonso, G. D.

Publications and source records attributed to Alonso, G. D..

7 recordsLinked to original sources

bioRxiv2026

H3K4me3 exhibits length-dependent deposition patterns at transcription initiation regions in Trypanosoma cruzi and correlates with transcriptional activity

In trypanosmatids genes, transcribed by RNA polymerase II do not have canonical promoters and are organized into directional gene clusters that mature into monocistronic transcripts by a co-transcriptional process known as trans-splicing. Even though gene expression is regulated mainly post-transcriptionally, it is currently understood that chromatin and epigenetics are also involved in this regulation. In eukaryotes, specific signals are normally required for the occurrence of an appropriate transcription initiation. Among them, trimethylation of histone H3 in lysine 4 is the most conserved signal normally detected at transcription start sites of actively transcribed genes. Unlike many model organisms, trypanosomes do not have defined promoters. Instead, transcription initiates in a bidirectional manner from dispersed regions coincident with divergent strand switch regions located between directional gene clusters (DGCs). In T. cruzi, H3K4me3 was observed at the origins of transcription coincident with divergent strand switch regions (dSSRs) in epimastigotes, but it has not been mapped throughout the whole genome at base-pair resolution or in other life stages so far. Here, we set up the CUT&RUN technique for T. cruzi epimastigotes and trypomastigotes. Consistent with a predominant post-transcriptional regulation along the life cycle, we did not find significant differences between life stages. We corroborated that H3K4me3 is enriched at dSSR adjacent to actively expressed DGCs. Moreover, we noticed that this histone mark exhibits different patterns that correlate with the genomic span of the transcription initiation regions and with transcriptional activity. Furthermore, we unveiled that the most actively transcribed DGCs are associated with shorter dSSRs and are located within the core compartment of the genome displaying a more accessible chromatin.

genomics↗

bioRxiv2026

NN-Assisted Image Analysis for Quantifying Intracellular Trypanosoma cruzi Infection

Quantification of intracellular Trypanosoma cruzi infection remains a central, yet methodologically challenging step in Chagas disease research and early-stage drug discovery. Current approaches largely rely on manual microscopy-based counting or on genetically modified parasites, both of which present limitations in scalability, reproducibility, or accessibility. Here, we developed and validated a neural network (NN)-based pipeline for the automated quantification of infection rates and parasite burden in mammalian cells using images stained exclusively with DNA-binding fluorescent dyes. Two independently refined deep-learning models were trained to segment host cell nuclei and intracellular amastigotes, respectively, and subsequently integrated into a unified algorithm that assigns each parasite to its nearest host cell. The pipeline was evaluated using confocal images from six mammalian cell lines infected with two T. cruzi strains and compared against blinded manual quantification. Automated detection of both, host nuclei and parasites, showed high concordance with manual counts, with median deviations around 5% and similar distributions of parasite burden per cell. In contrast to morphology-based image analysis methods, our NN-based approach demonstrated improved robustness across diverse cell types and staining conditions, reduced parameter dependency, and independent segmentation of host and parasite objects, minimizing error propagation. Although minor biases in parasite-to-cell assignment were observed in densely clustered cultures, overall infection indexes and burden estimates closely matched manual analysis. This accessible and scalable AI-assisted workflow provides a reproducible alternative to manual quantification and represents a methodological advance for standardized phenotypic screening of intracellular T. cruzi, supporting more robust and harmonized drug discovery efforts in Chagas disease.

microbiology↗

bioRxiv2026

Histone H3 Ser10 phosphorylation occurs exclusively in replicative stages and peaks during mitosis in Trypanosoma cruzi

Protein phosphorylation is a central post-translational modification that regulates signaling pathways across all living organisms. Through the antagonistic activities of protein kinases and phosphatases, phosphorylation modulates protein function by inducing conformational changes that affect enzyme activity, protein-protein interactions, stability, and subcellular localization. These molecular events regulate diverse cellular processes, including cell cycle progression, differentiation, gene expression, and metabolism. In unicellular parasites such as Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., specialized signaling pathways have evolved to enable adaptation to the fluctuating environments of insect vectors and mammalian hosts. In many eukaryotes, phosphorylation of histone H3 at serine 10 (H3Ser10p) is essential for proper chromosome condensation during mitosis and is catalyzed by Aurora kinase B. Although trypanosomatids possess an Aurora kinase B homolog and a conserved serine residue at position 10 of histone H3, this modification had not been previously detected in these organisms. Here, using a stage-specific approach, we report the first detection of H3Ser10p in T. cruzi and explore its association with cell cycle progression. Western blot analyses using a specific antibody revealed H3Ser10p in exponentially growing epimastigotes, both in total protein extracts and nucleosome-enriched fractions, indicating its incorporation into chromatin. Fluorescence microscopy showed that this histone mark is restricted to the nuclei of dividing cells. Furthermore, H3Ser10p was detected exclusively in replicative stages of the parasite. Analysis of cell cycle-associated structures and flow cytometry demonstrated that H3Ser10 phosphorylation is dynamically regulated, peaking in the G2/M phase. These findings identify H3Ser10p as a novel epigenetic mark in T. cruzi that is tightly regulated during the cell cycle.

cell biology↗

bioRxiv2025

Beyond the transcript: chromatin implications in trans-splicing in Trypanosomatids

Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, usually known as TriTryps, are the causal agents of animal and human sickness, and are characterized by having complex life cycles, alternating between a mammalian host and an insect vector. Their genes are organized in long transcriptional units that give rise to polycistronic transcripts which maturate into mRNA by a process known as trans-splicing. Among those genes, an important subset is composed of multicopy genes, which play crucial roles in host invasion and immune evasion. Here, we predicted the most likely trans-splicing acceptor sites (TASs) for TriTryps and found that the average chromatin organization is very similar among them with a mild nucleosome depletion at the TASs, and the same layout is observed in most of the genome. A detailed examination of the nucleosome landscapes resulting from different levels of chromatin digestion in T. brucei shows that an MNase-sensitive complex is protecting the TASs, and it is at least partly composed of histones. Additionally, comparative analysis for single and multi-copy genes in T. cruzi revealed a differential chromatin structure at the TASs suggesting a novel mechanism to guarantee the fidelity of trans-splicing in trypanosomatids.

genomics↗

bioRxiv2021

Experimental and Bioinformatic upgrade for genome-wide mapping of nucleosomes in Trypanosoma cruzi

1In Trypanosoma cruzi, as in every eukaryotic cell, DNA is packaged into chromatin by octamers of histone proteins that constitute nucleosomes. Besides compacting DNA, nucleosomes control DNA dependent processes by modulating the access of DNA binding proteins to regulatory elements on the DNA; or by providing the platform for additional layers of regulation given by histone variants and histone post-translational modifications. In trypanosomes, protein coding genes are constitutively transcribed as polycistronic units. Therefore, gene expression is controlled mainly post transcriptionally. However, chromatin organization and the histone code influence transcription, cell cycle progression, replication and DNA repair. Hence, determining nucleosome position is of uppermost importance to understand the peculiarities of these processes in trypanosomes. Digestion of chromatin with micrococcal nuclease followed by deep sequencing has been widely applied for genome-wide mapping of nucleosomes in several organisms. Nonetheless, this parasite presents numerous singularities. On one hand, special growth conditions and cell manipulation are required. On the other hand, chromatin organization shows some uniqueness that demands a specially designed analytical approach. An additional entanglement is given by the nature of its genome harboring a large content of repetitive sequences and the poor quality of the genome assembly and annotation of many strains. Here, we adapted this broadly used method to the hybrid reference strain, CL Brener. Particularly, we developed an exhaustive and thorough computational workflow for data analysis, highlighting the relevance of using its whole genome as a reference instead of the commonly used Esmeraldo-like haplotype. Moreover, the performance of two aligners, Bowtie2 and HISAT2 was tested to find the most appropriate tool to map any genomic read to reference genomes bearing this complexity.

genomics↗

bioRxiv2020

AMP-activated protein kinase: A key enzyme to manage nutritional stress responses in parasites with complex life cycles

Trypanosoma cruzi, the etiological agent of Chagas disease, has a digenetic life cycle. In its passage from the insect vector to the mammalian host, and vice versa, it must be prepared to cope with abrupt changes in environmental conditions in order to survive. Sensing and signaling pathways that allow the parasite to adapt, have unique characteristics with respect to their hosts and other free-living organisms. Many of the canonical proteins involved in these transduction pathways have not yet been found in the genomes of these parasites, because they present divergences either at the functional, structural and/or protein sequence level. All of this makes these pathways promising targets for therapeutic drugs. The AMP-activated protein kinase (AMPK) is a serine/threonine kinase activated by environmental stresses that results in reduction of ATP and increase of AMP levels. Thus, AMPK is regarded as a fuel gauge, functioning both as a nutrient and an energy sensor, to maintain energy homeostasis and, eventually, to protect cells from death by nutrient starvation. In the present study, we report the characterization of AMPK complexes for the first time in T. cruzi and describe the function of TcAMPK as a novel regulator of nutritional stress in epimastigote forms. We demonstrate that this complex possesses specific AMPK kinase activity in epimastigotes, which is inhibited by Compound C and is modulated by carbon source availability. In addition, TcAMPK2 subunit has an unprecedented functional substitution (Ser x Thr) at the activation loop and its overexpression in epimastigotes led to higher autophagic activity during prolonged nutritional stress. Moreover, the over-expression of the catalytic subunits resulted in antagonistic phenotypes associated with proliferation. Together, these results point to a role of TcAMPK in autophagy and nutrient sensing, key processes for the survival of trypanosomatids and for its life cycle progression.

cell biology↗

bioRxiv2019

Intracellular cyclic AMP levels modulate differential adaptive responses on epimastigotes and cell culture trypomastigotes of Trypanosoma cruzi

Among the many environmental challenges the parasite Trypanosoma cruzi has to overcome to complete its life cycle through different hosts, oxidative stress plays a central role. Different stages of this parasite encounter distinct sources of oxidative stress, such as the oxidative burst of the immune system, or the Heme released from hemoglobin degradation in the triatomines midgut. Also, the redox status of the surroundings functions as a signal to the parasite, triggering processes coupled to differentiation or proliferation. Intracellular second messengers, like cAMP, are responsible for the transduction of environmental queues and initiating cellular processes accordingly. In trypanosomatids cAMP is involved in a variety of processes, including proliferation, differentiation, osmoregulation and quorum sensing. Trypanosomatid phosphodiesterases (PDE) show atypical pharmacological properties and some have been involved in key processes for the survival of the parasites, which validates them as attractive therapeutic targets. Our work here shows that cAMP modulates different processes according to parasite stage. Epimastigotes become more resistant to oxidative stress when pre-treated with cAMP analogs, while trypomastigotes do not alter their response to oxidative stress under the same treatment. However, cAMP analogs do increase trypomastigotes infectivity in vitro. Also, we show that TcrPDEA1, a functionally enigmatic phosphodiesterase with very high Km, is involved in the epimastigotes response to oxidative stress.

molecular biology↗