Periodic Reporting for period 1 - SMART (Selective Molecular Approaches to Remove Trypanosomiasis)
Reporting period: 2021-10-04 to 2023-10-03
Human African trypanosomiasis (HAT) is a debilitating and deadly disease caused by the protozoan parasite Trypanosoma brucei spp. The disease threatens millions of people in Sub-Saharan Africa and represents a serious environmental, social, and economic burden for populations living in endemic areas. The recent discovery of the oral treatment fexinidazole has significantly improved disease outcomes, however, the risk of drug resistance remains a threat that requires the development of new treatment options. This project aims to characterise DNA structures, called G-quadruplexes (G4s), as novel cellular targets to treat and eradicate HAT. G4s are guanine-rich DNA sequences that self-assemble through G-G base pairing, forming a non-canonical DNA structure that can be found throughout the genome of all eukaryotes. For example, the human G4s have been widely characterised and validated as therapeutic targets, with novel G4-ligands entering clinical studies for cancer therapy. In contrast, parasitic G4s are still poorly characterised, and their potential as therapeutic targets is yet to be validated. In this proposal, we aim to characterise G4s in Trypanosoma brucei cells, using genomic approaches to determine the potential of these DNA structures as novel targets to fight parasitic infections.
To characterise the role of DNA G-quadruplexes (G4s) in trypanosomes, we used G4-selective chromatin immunoprecipitation assays followed by high-throughput sequencing (G4 ChIP-seq, Figure 1A). ChIP-seq is typically used to determine the location of specific proteins within the entire genome of an organism. G4 ChIP-seq has been widely applied in human cells to develop a genomic map of G4s, however, so far there is no evidence of this technique performed in parasitic pathogens. Broadly, this complex technique required the expression of a G4-selective antibody using Escherichia coli cells, and preparation of parasite chromatin extracted from T. brucei cells cultured in vitro, which is segmented into DNA fragments of approx. 500 bp. Then, the optimal ChIP-seq assay conditions (e.g. antibody-to-chromatin ratio) were determined in order to obtain a successful enrichment of G4-structures in ChIP samples compared to the non G4-enriched controls (Input sample). Finally, a newly optimised bioinformatic pipeline was implemented to analyse the data derived from DNA sequencing. Results from preliminary ChIP-seq assays with T. brucei cells ultimately identified thousands of G4-forming sequences (i.e. G4-peaks) within the genome of the parasites. Specifically, 3,144 G4-peaks were identified to be shared between two experimental ChIP-replicates. The genomic distribution of the peaks shows a significant enrichment of G4s in exons (61%) and promoter regions (20%), compared to intergenic regions (5%) and transcription termination sites (TTS, 13%) (Figure 1B), suggesting an important biological role of G4s in transcriptional regulation. Notably, these results are in agreement with previously published bioinformatic predictions. Functional annotation of the G4-peaks revealed genes with important biological implications, including, for example, transcriptional regulation of the Variant Surface Glycoprotein (VSG). VSG represents the most important virulence factor in trypanosomes, helping the parasites to escape host immune responses and therefore causing prolonged infections. Two G4-peaks were identified in the bloodstream expression site (BES) 1, where VSG genes are actively transcribed. Interestingly, these G4-peaks are found in genomic regions nearby putative G4-sequences (PQS) identified by the bioinformatic G4-searching tool, G4Hunter (Figure 1C). These preliminary results highlight the potential role of G4s in controlling antigenic variation in trypanosomes and could be further exploited in the future to understand the link between DNA secondary structures and virulence control in infectious agents.
Work derived from this project was presented at national and international seminars and conferences with poster and oral presentations.
Work derived from this project was presented at national and international seminars and conferences with poster and oral presentations.
This project has made significant progress beyond the state of the art by uncovering intricate details of the parasite's genomic architecture, transcription, and replication mechanisms, using next-generation genomic approaches (e.g. ChIP-seq) and novel bioinformatic analyses. This newfound knowledge holds the promise of unravelling critical aspects of the parasite's life cycle, pathogenicity, and drug resistance. Expected results include a deeper understanding of the genetic factors facilitating virulence and the evolution of drug resistance in Trypanosoma brucei, paving the way for the development of more effective therapeutic interventions. The socio-economic impact is substantial, as these parasites cause devastating diseases in humans and livestock across sub-Saharan Africa. Improved interventions could lead to more targeted and efficient treatments, resolving and reducing the economic burden of disease on affected communities. Furthermore, the wider societal implications of my project includes increased awareness and understanding of neglected tropical diseases, parasite biology, which contributes to broader efforts in advancing infectious diseases research and global health initiatives in low-middle income countries.