title: Investigating oxidative stress responses in Trypanosoma brucei and Trypanosoma congolense bloodstream forms
Creators: name: Enock Mukibetti, Mararo
Publisher: name: The University of Edinburgh
Year: 2025
Subjects: subject: Oxidative Stress
subject: Redox Balance
subject: Trypanosoma brucei
subject: T. congolense
subject: Metabolomics

description: African trypanosomes are parasites spread by tsetse flies that cause severe diseases in humans and animals. The clinically relevant species include Trypanosoma brucei, upon which the majority of our biological understanding is based, and T. congolense, which is less understood. These parasites are extracellular in all stages of their complex life cycle, as a result, they are continuously exposed to harsh environments in their mammalian and insect hosts. In the mammalian host, the immune system is characterised by key innate immune cells such as macrophages, which release reactive oxygen and nitrogen species, imposing oxidative stress. The parasite response to oxidative stress, as well as redox balance maintenance, have been studied to some degree in T. brucei, but not in T. congolense, which is known to exhibit divergence at the genome, transcriptome and metabolome levels. Understanding these responses and how they differ between closely related species is crucial for uncovering parasite survival strategies and developing therapeutic interventions. This thesis investigates the hypothesis that oxidative stress responses and redox balance mechanisms differ between bloodstream forms (BSFs) of T. brucei and T. congolense, and that these differences underlie phenotypes such as differential sensitivity to oxidative stress and interactions with macrophages. Specifically, it examines the oxidative stress responses, redox balance mechanisms, and interactions with macrophages in these two species to determine their differential parasite sensitivity. Techniques including metabolomics and transcriptomics were used to compare genes and pathways involved in these processes between the two species that may explain any observed phenotypic differences and similarities. Finally, the thesis also explored a gene linked to oxidative stress resistance previously studied in the parasite Leishmania, but not yet explored in African trypanosomes. To facilitate the study of the two trypanosome species under comparable conditions, a novel lipid-rich medium, Morrison’s Universal Medium (MUM) was developed and optimised. This medium enabled the direct comparison of redox balance and oxidative stress response pathways between T. brucei and T. congolense. Experiments were carried out to find suitable candidates for inducing oxidative stress under in vitro conditions, which led to the identification of the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) as a viable candidate. Subsequently, experiments with SNAP showed that T. congolense was more sensitive to nitrosative stress compared to T. brucei. In vitro studies on trypanosome-macrophage interaction further supported these findings, as T. congolense viability was reduced to a greater extent in the presence of macrophages. To investigate potential underlying metabolic differences that could explain the differences in SNAP sensitivity, a comparative analysis of intracellular and extracellular metabolites of T. brucei and T. congolense was carried out using cells derived from the same medium. It was found that both species showed a similar capacity for glucose consumption, in contrast to previous data showing that T. congolense had reduced capacity. Metabolomics data further showed that T. brucei excreted high levels of pyruvate while T. congolense consumed this metabolite, indicating a species-specific difference in pyruvate utilisation. T. brucei culture supernatants consistently showed elevated acetate levels, which was not consistent with previous literature. Taken together, these results are indicative of metabolic adaptation to MUM. T. brucei supernatants also exhibited higher levels of immunomodulatory aromatic ketoacids such as indolepyruvate, hydroxyphenylpyruvate and phenylpyruvate. Conversely, T. congolense exhibited higher levels of antioxidant metabolites, such as glutathione, oxidised glutathione, and ornithine, which help protect cells from damage. Metabolomics data were integrated with previously generated transcriptomics data, highlighting that T. congolense exhibited a higher expression of genes associated with the trypanothione cycle and polyamine biosynthesis – pathways that play a role in handling oxidative stress. Moreover, T. congolense displayed a higher transcript abundance of genes linked to the oxidative branch of the Pentose Phosphate Pathway (PPP), which produces antioxidant components. In contrast, T. brucei showed higher activity of genes from the non-oxidative branch of the PPP, which are involved in energy production. Combined, the data indicated that T. congolense has more activity in oxidative stress response pathways compared to T. brucei, even under steady-state conditions. Studies in the related kinetoplastid parasite Leishmania spp. showed that overexpressing the gene aconitase (ACO) increases resistance to nitrosative stress. The role of this gene in nitric oxide resistance has not been investigated in African trypanosomes. I, therefore, investigated the impact of ACO overexpression in T. brucei and T. congolense BSFs on SNAP sensitivity. Overexpressing ACO in T. congolense caused BSF parasites to differentiate into a form resembling the insect stage procyclic forms (PCFs), suggesting a role for ACO in parasite differentiation. In contrast, overexpressing ACO in the monomorphic T. brucei Lister 427 led to significant mortality without any morphological change, likely because this strain is incapable of differentiation. Analyses, including adaptation to PCF culture media, flow cytometry, and microscopy, confirmed that ACO overexpression caused T. congolense to differentiate into a form closely resembling PCFs. I was unable to evaluate the role of ACO in oxidative stress management across both species, which warrants further investigation. In addition, further work is required to determine the mechanisms by which ACO overexpression is linked to differentiation. In summary, this thesis presents the development of a universal medium enabling the culture of multiple African trypanosome species. This system revealed T. congolense’s increased sensitivity to oxidative stress compared to T. brucei and highlighted metabolic similarities and differences between species. Additionally, this thesis describes the potential role of ACO in promoting differentiation in trypanosomes. These findings offer valuable insights into the adaptive mechanisms of African trypanosomes, and further support a growing body of literature evidencing biological divergence between two closely related species of clinically relevant protozoan parasites.