Profiling the bloodstream form and procyclic form Trypanosoma brucei cell cycle using single-cell transcriptomics.

Area of Science:

• Molecular Biology
• Parasitology
• Genomics

Background:

• African trypanosomes exist as bloodstream forms (BSFs) in mammals and procyclic forms in tsetse flies, crucial for host colonization and life cycle progression.
• Mechanisms governing the cell replication cycle of these distinct trypanosome forms remain largely unelucidated.

Purpose of the Study:

• To characterize the cell cycle-regulated (CCR) transcriptomes of both procyclic and slender BSF *Trypanosoma brucei* using single-cell transcriptomics.
• To develop and validate an efficient protocol for single-cell transcriptomic analysis of cryopreserved *T. brucei*.
• To computationally reconstruct cell cycle dynamics and compare gene expression patterns between life cycle forms.

Main Methods:

• Single-cell RNA sequencing (scRNA-seq) applied to unsynchronized *T. brucei* populations.
• Development of a freeze-thawing protocol for cryopreserved trypanosomes compatible with scRNA-seq.
• Computational analysis using periodic pseudotime inference to reconstruct cell cycle dynamics.
• Comparative transcriptomics and proteomic analysis.

Main Results:

• High-resolution CCR transcriptomes were obtained for both procyclic and slender BSF *T. brucei* without cell synchronization.
• A core cycling transcriptome conserved across both life cycle forms was identified, alongside form-specific dynamic gene expression.
• A delay between peak transcript and protein expression was observed for most cycling genes.

Conclusions:

• This study provides novel insights into the CCR transcriptomes of *T. brucei* life cycle forms.
• The findings highlight conserved and divergent gene regulation strategies between mammalian and insect stages.
• An interactive webtool is available for further exploration of the generated transcriptomic data.