Cell-to-cell heterogeneity among T. brucei parasites in vivo

Trypanosoma brucei, the causative agent of human and animal African trypanosomiasis, exhibits great adaptability within its mammalian host. One of the biggest reservoirs is the gonadal white adipose tissue (gWAT), while the mechanisms of tissue invasion and preference for the adipose tissue during early infection remain poorly understood. Endothelial cells (ECs) play a role in AT invasion, and their response varies by organ, influencing disease progression and drug sensitivity.

This Ph.D. research explores T. brucei biology, focusing on its heterogeneity within the host and interactions with gWAT and vasculature. Single-cell RNA sequencing (scRNA-seq) was employed to dissect host-parasite interactions, aiming to uncover novel insights and potential therapeutic targets. We performed some methodological changes involved adapting the scRNA-seq protocol for in vivo parasites. This enabled analysis of parasite heterogeneity across the bloodstream and gWAT, linking it to infection progression and identifying genes associated with tissue invasion and adaptation.

Using 10x Genomics scRNA-seq, we implemented rapid cooling of parasites before encapsulation, in order to decrease their motility while preserving transcriptomic integrity. Samples were sequenced at 5, 6, and 7 days post-infection (p.i.), forming an atlas of early infection. Parasites exhibited organotypic profiles, being highly similar within the same tissue but distinct across different tissues. Replicas were also analysed, to confirm the transcriptomic differences do not come from batch effect. The study found that parasites transitioning from blood to gWAT are primarily in early G1 phase, undergo metabolic shifts, and cease glucose utilisation. Within gWAT, parasites display greater heterogeneity, including adaptations to the environment and the different cell cycle stages. Additionally, increased Variant Surface Glycoprotein (VSG) diversity was observed in gWAT parasites, supporting the idea that tissue-resident parasites drive higher antigenic variation than in the blood.

To complete this comprehensive view of host-parasite interactions, scRNA-seq was also performed on ECs from gWAT and brain vasculature in T. brucei and T. congolense infections in mice. Brain tissue, where few parasites are found early, served as a comparison, while T. congolense, which remains in the bloodstream, acted as a control. This approach revealed that gWAT ECs undergo a robust immune and inflammatory response to T. brucei, absent in brain ECs and in T. congolense infections. Notably, T. congolense triggered a distinct brain EC response linked to their early neurological effects.

This research highlights the dynamic interplay between T. brucei and gWAT ECs, identifying host and parasite factors that may facilitate invasion. The findings provide a list of potential drug targets and shed light on tissue-specific adaptations of the parasite. By leveraging refined scRNA-seq methodologies and in vivo models, this work advances the understanding of African trypanosomiasis and sets the stage for future studies to validate key genes involved in invasion and adaptation.