Inchausti, L. | bioRxiv Science

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↗

bioRxiv2025

Single-cell RNA-seq reveals trans-sialidase-like superfamily gene expression heterogeneity in Trypanosoma cruzi populations

Trypanosoma cruzi, the causative agent of Chagas disease, presents a major public health challenge in Central and South America, affecting approximately 8 million people and placing millions more at risk. The T. cruzi life cycle includes transitions between epimastigote, metacyclic trypomastigote, amastigote, and blood trypomastigote stages, each marked by distinct morphological and molecular adaptations to different hosts and environments. Unlike other trypanosomatids such as Trypanosoma brucei, T. cruzi does not employ a monoallelic model of antigenic variation; instead, it relies on a diverse repertoire of cell-surface associated proteins encoded by large multigene families, which are essential for infectivity and immune evasion. This study analyzes cell-specific transcriptomes using single-cell RNA sequencing of amastigote and trypomastigote cells to characterize stage-specific surface protein expression during mammalian infection. Through clustering and identification of cell-specific markers, we assigned cells to distinct parasite developmental forms. Analysis of individual cells revealed that surface protein-coding genes, especially members of the trans-sialidase like superfamily (TcS), are expressed with greater heterogeneity than single-copy genes. Moreover, no recurrent combinations of TcS genes were observed between individual cells in the population. Remarkably, a small subset of TcS mRNAs, encoded by genes preferentially located in the core genomic compartment, are frequently detected across the cell population, whereas the vast majority of TcS mRNAs show low detection frequencies and are mainly encoded in the disruptive compartment. Our findings thus reveal transcriptomic heterogeneity within trypomastigote populations where each cell displays unique TcS expression profiles. Focusing on the diversity of surface protein expression, this research aims to deepen our understanding of T. cruzi cellular biology and infection strategies.

genomics↗