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

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African trypanosomes proliferate as bloodstream forms and procyclic forms in the mammal and tsetse fly midgut, respectively. This allows them to colonise the host environment upon infection and ensure life cycle progression. Yet, understanding of the mechanisms that regulate and drive the cell replication cycle of these forms is limited. Using single cell transcriptomics on unsynchronised cell populations, we have obtained high resolution cell cycle regulated transcriptomes of both procyclic and slender bloodstream form Trypanosoma brucei without prior cell sorting or synchronisation. Additionally, we describe an efficient freeze-thawing protocol that allows single cell transcriptomic analysis of cryopreserved T. brucei . Computational reconstruction of the cell cycle using periodic pseudotime inference allowed the dynamic expression patterns of cycling genes to be profiled for both life cycle forms. Comparative analyses identify a core cycling transcriptome highly conserved between forms, as well as several genes where transcript levels dynamics are form-specific. Comparing transcript expression patterns with protein abundance revealed that the majority of genes with periodic cycling transcript and protein levels exhibit a relative delay between peak transcript and protein expression. This work reveals novel detail of the cell cycle regulated transcriptomes of both forms, which are available for further interrogation via an interactive webtool.

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  1. eLife assessment

    This important study identifies more than 1500 genes that are differentially transcribed over the cell cycle of the single-celled eukaryotic pathogen, Trypanosoma brucei. Analysis of the two major developmental stages of these pathogens suggests that a core set of genes are similarly regulated in both stages, while many cell cycle-related changes in gene expression were unique to one stage. Intriguingly, the levels of far fewer proteins are differentially regulated over the trypanosome cell cycle, indicating that protein levels are primarily regulated by post-transcriptional processes. The study represents a significant technical advance in analyzing gene expression at the single-cell level in unfractionated trypanosome cultures.

  2. Reviewer #1 (Public Review):

    This study utilizes scRNA-seq to generate a detailed map of transcriptional changes that occur in asynchronously replicating the Trypanosoma brucei insect (PCF) and mammalian (BSF) stages. The analyses were performed on both fresh and cryo-preserved parasites, and transcriptional changes in PCF compared to existing proteomic datasets at the same stage. This is the first study to comprehensively map cell cycle-related transcriptional changes in T. brucei BSF and to undertake a side-by-side analysis of the two major parasite developmental stages. The study identified >1,500 transcripts that exhibit dynamic changes during the cell cycle across the two stages, substantially increasing the number of cell cycle-regulated (CCR) genes compared to previous analyses. Analysis of the data revealed common as well as stage-specific CCR transcripts and identified transcripts with known/suspected functions in cell cycle regulation as well as hypothetical proteins. The findings also support and quantify previous observations suggesting that most transcript changes (83-86% of CCR transcripts) are not reflected by similar changes in corresponding proteins, and where there is a correlation, protein expression levels expectedly lag behind transcripts. Overall, the study provides the most comprehensive transcriptome atlas of the T. brucei cell cycle undertaken to date, highlighting a large number of genes and cellular processes that are linked to cell cycle progression, while further confirming the importance of post-transcriptional regulatory processes in these divergent eukaryotes. The work represents a significant technical advance, particularly in the validation of the use of cryo-preserved parasites for single-cell RNS-seq, and nicely integrates results from previous proteomics and gene-knockout studies.

  3. Reviewer #2 (Public Review):

    Parasitic African trypanosomes are agents of devastating diseases in humans and animals. Currently, no vaccines exist, with control of human disease being realized thru vector suppression and elimination of infected hosts while animal diseases remain rampant on the continent. The molecular aspects of the multiple developmental stages the parasite undergoes thru its mammal and tsetse hosts, and the unique aspects of parasite gene expression regulation and host evasion mechanisms have been extensively investigated. Recent applications of single-cell transcriptomics (scRNA) to these approaches have expanded knowledge gained from total RNA and revealed new insights.

    In this paper, Briggs et al., set out to determine the cell cycle-related genes (CCR) of T. brucei, which follows the typical eukaryotic progression through G1, S, G2, and M phases followed by cytokinesis, although trypanosomes are unusual in that both nuclear and mitochondrial genome replication and segregation are orchestrated during cell division. while many regulators remain unidentified, are absent, or have been replaced by trypanosomatid-specific factors. For these studies, they apply scRNA methodology using asynchronous mixed populations of cultured 'monomorphic' slender mammalian (BSF) and insect stage (PCF) cells and then determine their cell cycle phases computationally. Of interest, performing similar analysis with fresh and cryopreserved cells made minimal difference to the outcome, thus enabling future investigations with preserved cells.

    The study identified 1,550 genes with dynamic transcript level changes reflective of the cell cycle, 1,151 of which had not been previously identified by bulk analysis. These revealed a common set of highly conserved CCR genes as well as unique gene transcript levels expressed thru the cell cycle for BSF and PCF cells. Expression patterns of the G1 and S phase genes are highly comparable between BSF and PCF forms, whereas, after the S phase, the timing of gene expression for the S-G2 transition is far less synchronized. Comparison between transcript expression patterns and previously published protein abundance changes identified a relative delay in peak levels for transcript and protein for at least 50% of the genes that could be compared. Collectively, this foundational analysis generates transcript atlases for BSF and PCF cell cycles, which can be further mined for downstream functional investigations.

  4. Reviewer #3 (Public Review):

    In this article, Briggs et al. used scRNAseq to get high-resolution cell cycle-regulated transcriptomes of both replicative forms of Trypanosoma brucei (PCF and BSF) without prior synchronization. Briggs et al. also demonstrated that performing the scRNAseq library immediately after thawing cryopreserved samples did not show significant differences. The authors used computational reconstruction of the cell cycle to get the dynamic expression patterns of cycling genes in both life cycle forms. They identified a core cycling transcriptome highly conserved between forms. However, some slight differences were found between them, e.g.: a switch in gene expression associated with the S-G2 transition is much more discrete in PCFs than BSFs. Moreover, as proteomics data across the cell cycle is not available for BSFs, the authors tagged the top most significant genes with transcripts peaking in G1, S, and G2/M phases with a fluorescent epitope. After comparing the transcript expression patterns with protein abundance, the authors found that the majority of genes with periodic cycling transcript and protein levels exhibited a relative delay between peak transcript and protein expression, which was expected. In summary, this work provides a valuable public tool for further investigation into gene expression dynamics throughout the cell cycle in T. brucei.