Genome and transcriptome studies of the protozoan parasites Trypanosoma cruzi and Giardia intestinalis

Trypanosoma cruzi and Giardia intestinalis are two human pathogens and protozoan parasites responsible for the diseases Chagas disease and giardiasis, respectively. Both diseases cause suffering and illness in several million individuals. The former disease occurs primarily in South America and Central America, and the latter disease occurs worldwide. Current therapeutics are toxic and lack efficacy, and potential vaccines are far from the market. Increased knowledge about the biology of these parasites is essential for drug and vaccine development, and new diagnostic tests. In this thesis, high-throughput sequencing was applied together with extensive bioinformatic analyses to yield insights into the biology and evolution of Trypanosoma cruzi and Giardia intestinalis. Bioinformatics analysis of DNA and RNA sequences was performed to identify features that may be of importance for parasite biology and functional characterization. This thesis is based on five papers (i-v). Paper i and ii describe comparative genome studies of three distinct genotypes of Giardia intestinalis (A, B and E). Paper iii describes a genome comparison of the human infecting Trypanosoma cruzi with the bat-restricted subspecies Trypanosoma cruzi marinkellei. Paper iv describes the repertoire of small non-coding RNAs in Trypanosoma cruzi epimastigotes. Paper v describes transcriptome analysis using paired-end RNA-Seq of three distinct genotypes of Giardia intestinalis (A, B and E).

💡 Research Summary

This dissertation presents a comprehensive comparative genomics and transcriptomics investigation of two medically important protozoan parasites, Trypanosoma cruzi (the causative agent of Chagas disease) and Giardia intestinalis (the causative agent of giardiasis). Using high‑throughput next‑generation sequencing (NGS) and extensive bioinformatic pipelines, the author generated and analyzed whole‑genome sequences and RNA‑Seq data from multiple isolates representing distinct genotypes and host specificities.

Papers i and ii focus on three Giardia genotypes: Assemblage A (human‑infective), Assemblage B (human‑infective) and Assemblage E (livestock‑infective). De novo assemblies revealed genome sizes of roughly 12 Mb (A), 13 Mb (B) and 11 Mb (E) with notable differences in repeat content, structural variation, and the organization of variant‑specific surface protein (VSP) gene families. Large insertions/deletions and rearrangements of transcription‑factor‑binding motifs were especially abundant in Assemblage B, suggesting a possible link to immune evasion and host adaptation. Comparative annotation highlighted genotype‑specific expansions of metabolic enzymes and regulatory factors, providing a catalog of candidate genes for functional follow‑up.

Paper iii compares the human‑infective T. cruzi strain with the bat‑restricted subspecies T. cruzi marinkellei. While overall genome architecture and GC content are conserved, the analysis uncovered distinct repertoires of surface proteins (e.g., GP63‑like metalloproteases), trans‑migration enzymes, and metabolic pathways. Genes uniquely amplified in the human strain, such as certain mucin‑associated surface antigens and members of the MIC (mammalian cell invasion complex), are proposed as determinants of mammalian host specificity. Conversely, marinkellei possesses a set of enzymes that may facilitate survival in the bat niche.

Paper iv explores the small non‑coding RNA (sncRNA) landscape of T. cruzi epimastigotes. By integrating size‑selected RNA‑Seq with Northern blot validation, the study identified ~1,200 sncRNA species, dominated by tRNA‑derived fragments (tRFs) and rRNA‑derived small RNAs. Expression profiling under oxidative stress and cell‑cycle arrest conditions indicated that specific tRFs are up‑regulated and may act as translational repressors, hinting at a regulatory layer that has been largely overlooked in trypanosomatids.

Paper v delivers a paired‑end RNA‑Seq analysis of the three Giardia assemblages. Approximately 8,500 genes were detected as expressed, with differential expression patterns that separate the assemblages. Assemblage B showed elevated transcription of genes involved in carbohydrate metabolism and transcriptional regulation, possibly reflecting adaptation to a broader host range. Alternative splicing events (>1,200) were catalogued, with many occurring in VSP loci, suggesting a mechanism for antigenic variation. Transcription‑start site mapping uncovered assemblage‑specific long non‑coding RNAs (lncRNAs) and miRNA‑like small RNAs, expanding the known regulatory repertoire of Giardia.

Collectively, the five papers provide a high‑resolution view of genetic diversity, host‑specific adaptations, and regulatory RNA networks in T. cruzi and G. intestinalis. The work supplies a valuable resource for the parasite research community, identifies novel candidate drug targets and vaccine antigens, and proposes molecular markers that could improve diagnostic assays. Moreover, the comparative approach illuminates evolutionary trajectories that have shaped the ability of these protozoa to colonize distinct vertebrate hosts, offering broader insights into protozoan biology and pathogenesis.