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Single cell transcriptomic analysis of bloodstream form Trypanosoma brucei reconstructs cell cycle progression and differentiation via quorum sensing

Emma M. Briggs, Richard McCulloch, Keith R. Matthews, Thomas D. Otto

Preprint posted on 11 December 2020 https://www.biorxiv.org/content/10.1101/2020.12.11.420976v1

Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-021-25607-2

The hidden steps of the life of T. brucei.

Selected by Mariana De Niz

Categories: cell biology

Background

During their life cycle, Trypanosoma brucei parasites undergo various developmental transitions. These transitions involve changes in nutrient-specific metabolism, morphology, organelle organization and structure, and stage-specific surface protein expression, which facilitates survival and transmission. In the mammalian host, these forms include long slender bloodstream forms, which can differentiate into stumpy bloodstream forms through a quorum sensing process. Although these 2 extremes are well identified, there are possibly multiple intermediate stages between both forms which have not been well defined. Stumpy forms remain arrested in the cell cycle until ingested by a tsetse fly. In the fly midgut the stumpy forms undergo a further differentiation event and re-enter the cell cycle as tsetse-midgut procyclic forms. Procyclic, slender, and stumpy forms, differ at the transcript and protein level. However, understanding the detailed progression between slender and stumpy cells has been hampered due to the asynchrony of this differentiation step. To address this, single-cell RNA sequencing offers the opportunity to study individual cells in a heterogeneous population, to decipher in detail this developmental process. In their current work, Briggs et al (1) applied single cell transcriptomics (scRNA-seq) to dissect the asynchronous differentiation of slender to stumpy forms, deriving a temporal map of the transition between these forms, based on individual cells.

Figure 1. Single cell transcriptomic analysis of BSF T. brucei reconstructs cell cycle progression and differentiation via quorum sensing (From Ref 1).

Key findings and developments

scRNA-seq identifies transcriptionally distinct long slender and short stumpy form T. brucei.  To model stumpy differentiation in vitro the authors used a pleomorphic line, and began by treating parasites with oligopeptide-rich BHI broth, which can induce T. brucei bloodstream form differentiation in a titratable manner. To capture the transcriptomes of slender, intermediate and stumpy forms, they combined parasites after 0, 24, 48, or 72h after 10% BHI treatment in equal numbers. scRNA-Seq was then performed using the Chromium Single Cell 3’ workflow (10x genomics) and Illumina Sequencing, of two independent biological replicates with a total of 9344 cells examined. Medians of 1051 and 1439 genes were detected per cell. Cells from both experiments were integrated and visualized using UMAP. The authors identified four distinct groups containing transcriptionally similar cells, with two of those groups being clear slender and stumpy-like cells. The four groups were termed slender A, slender B, stumpy A and stumpy B. Differential expression analysis of the transcripts between the 4 groups showed significant overlap between the genes of group slender A and B, and between stumpy A and B. However, the study showed 183 markers unique to the slender A group, 95 to slender B, 55 to stumpy A, and 9 to stumpy B. Gene ontology enrichment analysis revealed the association of each cluster’s marker genes with distinct biological processes. Altogether, the authors emphasize that a distinct cluster representative of ‘the intermediate’ stage transcriptome between slender and stumpy forms was not evident.

Trajectory analysis of long slender to short stumpy differentiation. Given the overlap detected in the clustering analysis, the authors conducted trajectory interference and pseudotime analysis to study gene expression changes during stumpy development in detail. Here, individual cells were re-plotted as a PHATE (potential of heat-diffusion for affinity-based transition embedding) map, which allows preservation of the continual progression of developmental processes. They found that slender A and B clusters remained separate, while stumpy A and stumpy B showed significant overlap. This allowed identification of 2001 genes differentially expressed as a function of pseudotime, which were grouped into 9 modules of co-expressed genes showing similar patterns of expression throughout differentiation. 66% of those genes had been previously shown to be significantly differentially expressed between slender and stumpy populations isolated from low and peak parasitemias in vivo. Altogether, the authors highlight the advantage of scRNA-Seq to reveal transient events in an asynchronous developmental trajectory. Moreover, GO term enrichment for biological processes associated with each gene module revealed a potential order of biological events during slender-to-stumpy development. Besides of the annotated genes, 635 hypothetical genes were identified as differentially expressed during slender to stumpy differentiation. Altogether, pseudotime analysis allowed identification of novel genes differentially expressed during bloodstream form differentiation, as well as each gene’s detailed expression pattern.

Transcript abundance during the bloodstream slender cell cycle. Given that replicating slender bloodstream form cells were captured in the experiments described above, the authors went on to explore if the scRNA-Seq data could reveal greater detail than what is known, on gene expression changes during the cell cycle. Each cell was assigned to a cell cycle phase using marker genes previously identified in bulk RNA-Seq analyses. Slender A and B cells were grouped closer to cells of the same phase, with parasites most distal to Stumpy A and B labelled as late G1, followed by S and G2/M phase cells. Slender B cells most proximal to stumpy A contained all 4 cycle phases, although early G1 cells were enriched here. Interestingly, stumpy A and B cells were marked in a variety of cell cycle phases. An important finding of this section of the work was a) the identification of genes driving the cell cycle, and b) the identification of 3 genes previously shown to be involved in stumpy development with differential expression patterns in slender cells- namely, RBP7B (increased in late G1 cells through to G2/M), PPC2 (decreased in late G1/S phase parasites), and ZC3H20 (dropped in expression in late G1/S phase).

ZC3H20 null parasites fail to differentiate in response to BHI. ZC3H20 peaks in expression at the slender B to stumpy transition in pseudotime, and it has been previously shown to be required for differentiation in vivo and in vitro. Based on this, the authors used a ZC3H20 null T. brucei line to investigate where parasites fail in their development to stumpy forms with respect to transcriptome changes, and aimed to identify mRNA targets of ZC3H20 itself. Incubation of ZC3H20 KO in 10% BHI broth showed that these parasites continued to replicate beyond time points where WT cells had arrested, and after 72h of culture, failed to express PAD1. Moreover, consistent with their inability to produce stumpy forms, ZC3H20 KO failed to differentiate into procyclic cells. scRNA-Seq was then performed on ZC3H20 KO cells at 0, 24, 48, or 72h after 10% BHI treatment- as done for WT cells. Clustering the ZC3H20 KO and WT integrated cells resulted in 6 distinct clusters: stumpy A and B, and 4 slender clusters, called slender A.1, A.2, B.1, and B.2. While 77.3% of WT cells were found in clusters stumpy A or B, only 0.3% of ZC3H20 KO cells were in either, consistent with the near complete ablation of stumpy formation in the mutant parasites. The B.2 cohort was comprised almost entirely of ZC3H20 KO cells. Marker gene analysis between clusters identified 94 marker genes upregulated in slender B.2 cells, 18 of which were unique to this cluster.

Trajectory comparison between WT and ZC3H20 KO cells reveals functional separation of downregulation and upregulation of transcripts during differentiation. The authors then compared transcriptomic changes in ZC3H20 KO and WT parasites after BHI treatment, by inferring a trajectory from the WT and ZC3H20 KO integrated parasites. This identified a branched trajectory – while early in pseudotime WT and ZC3H20 KO parasites are transcriptionally similar and arrange on the same lineage, later there was a clear branching in their development, whereby WT cells ended in stumpy forms, and ZC3H20 KO in slender B.2. 587 genes of the 2001 identified as differentially expressed during stumpy development in WT cells, significantly changed in expression in ZC3H20 KO cells across the truncated trajectory. ZC3H20 KO cells failed to upregulate transcripts later in development that are required for stumpy formation, and this point of dysregulation coincided with the peak of ZC3H20 expression during normal WT differentiation.

The authors then aimed to identify regulators of early stumpy development, and so looked for genes which changed significantly in abundance at the start of the trajectory to a point downstream of the ZC3H20 branch. 234 genes changed in transcript abundance between these points, and were associated with trajectory progression. RDK2 and PAD2 were associated with both trajectories, but had different patterns of expression. 83 genes were differentially expressed only in the trajectory of WT parasites. Conversely, 35 genes with early altered expression were associated with the truncated ZC3H20 KO development only. Altogether, comparison of the differentiation of WT and ZC3H20 KO cells through scRNA-Seq allowed the identification of direct and indirect targets of ZC3H20 altered specifically during differentiation; the failure point of ZC3H20 KO cells during differentiation; and putative immediate early regulators of differentiation.

What I like about this preprint

I think the question addressed in this work is one that remained outstanding in the field of T. brucei, namely, little is known about the intermediate stages of development of the parasite from slender to stumpy forms. I think the use of scRNA-Seq in T. brucei research, and in this work allowed opening various avenues of research, relevant to the whole field.

 

References

  1. Briggs et al, Single cell transcriptomic analysis of bloodstream form T. brucei reconstructs cell cycle progression and differentiation via quorum sensing. bioRxiv, 2020.

 

Posted on: 23 December 2020 , updated on: 8 January 2021

doi: https://doi.org/10.1242/prelights.26638

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Author's response

Emma Briggs shared

Open questions

1. This is very exciting work! You mention in your results that it was surprising that an intermediate stage per se, between the slender and stumpy forms was not so clear as expected. Infection with other parasites, such as Plasmodium falciparum, or Toxoplasma gondii has shown that for them, not all stages of development were straight-forward reproducible in vitro. And some stages were missed also in vivo until detailed work was performed (as is the case of all the intermediate stages of gametocytes of P. falciparum, which reside in the bone marrow only). Do you think for T. brucei, we might be missing intermediate stages that a) only reside in specific tissues (as is the case of Plasmodium), and b) the right conditions for their development in culture have not been considered nor identified?

Interesting idea! The “intermediate” stage has previously been defined by morphology, so we were interested to see if we could find robust marker genes for this stage. As you say, we couldn’t find a distinct intermediate group of parasites based on changes in transcript abundance alone. We observed a clear change from slender to stumpy transcriptomes, rather than slender, to intermediate, and then to stumpy.  However, we did note some transiently expressed genes that increase and then decrease again during differentiation (or vice versus). It will be interesting to observe protein levels for these genes during differentiation and see if the proteins are expressed exclusively in T. brucei with intermediate morphology. Without a marker it’s pretty tricky to define intermediates, but if we assume the stumpy forms must develop via an intermediate stage then I think we will have captured them, as we took multiple time points after inducing differentiation.  But that is an assumption! So repeating experiments in vivo would help to clarify. I think taking this further and comparing T. brucei parasites isolated from different tissues with scRNA-seq analysis, as you say, will be very interesting. It should even be possible to integrate different data sets and assemble the parasites by transcriptome changes to see if tissue resident parasites take a specific place in the life cycle.

2.Previous work has identified morphologically different parasites in, for instance, the brain, as compared to those in blood. Is there an idea of what is the range of parasites that can be seen in culture, compared to the ones that exist altogether?

We compared slender vs stumpy gene expression change identified by scRNA-seq to bulk RNA-seq analysis of T. brucei isolated during low and peak parasitaemia from the blood (Silvester, Ivens and Matthews PLoS Negl. Trop. Dis. 2018) and found most genes had similar expression changes in both studies. There are studies where authors use bulk RNA-seq to compare T. brucei from the blood, adipose tissue or cerebrospinal fluid to those grown in culture (Trindade et al Cell Host & Microbe 2016 and Mulindwa et al PLoS Negl. Trop. Dis. 2018, for example). These find cultured strains are closely related to those in the blood, and in the most part the CSF, but there are differences to those from the adipose tissue. The comparison is tricky with bulk analysis as the parasites are normally in a heterogenous pool, so scRNA-seq could be used for more detailed comparison of the morphologically different cell types.

3.A naïve question: You performed incubation of the parasites with 10% BHI broth. Do you think this influences the results in any way? For instance, would any other method for inducing stumpy formation yield different results?

Rojas et al. (Cell 2019) first demonstrated the BHI broth could be used to model slender to stumpy differentiation and demonstrated that oligopeptides can act as the “stumpy induction factor” via TbGPR89, which works at a peptide transporter. I think the important comparison initially is with differentiating parasites in vivo. As discussed above, we did this with bulk RNA-seq data, and it will be interesting to perform scRNA-seq with parasites isolated during infection. However, the analysis of the ZC3H20 null mutant already shows that genes that influence differentiation in BHI also influence differentiation in vivo (Cayla et al., 2020, eLife), indicating the underlying molecular controls are equivalent. Hence, I think these in vitro experiments give us an accurate “reference” of slender to stumpy differentiation and then other methods of inducing stumpy development could be compared without too much difficultly. For instance, forcing expression of a second variant surface glycoprotein can induce differentiation (Zimmerman et al. PLOS Pathogens 2017) and scRNA-seq could be used to investigate this independent pathway in more detail.

4.Another naïve question: Your findings on the branching difference between WT and ZC3H20 KO cells are very interesting. How much is known regarding the differences between T. brucei pleomorphic and monomorphic strains in terms of RNA-Seq? What differences would you expect between the WT pleomorphic line you used, the ZC3H20 KO and a monomorphic line?

Federico Rojas and others (Cell 2019) tested BHI treatment on monomorphic T. brucei and didn’t see a significant difference in growth, which is distinct from the response of pleomorphs and consistent with their developmental incompetence. However, you could perform BHI treatment and scRNA-seq with a monomorphic line to assess if the parasites move towards a stumpy-like transcriptome at all, in the same way we analysed ZC3H20 null parasites in this paper. I would be completely guessing to compare monomorphic and ZC3H20 null results! Presumably, monomorphic T. brucei would fail to express the majority of stumpy-associated transcripts, but how far along the trajectory of differentiation they proceed I’m not sure. It is also not certain that all monomorphic strains are equivalent, since there are many steps at which parasites could fail to differentiate in response to the stumpy inducing signal.

5.You mention a reference work that identified genes differentially expressed between slender and stumpy populations, and that these were isolated from low and peak parasitemias in vivo. Again a naïve question: is it certain that T. brucei populations are homogeneous slenders or stumpies at various parasitemia levels?

Yes, Silvester, Ivens and Matthews (PLoS Negl. Trop. Dis. 2018). We know that the parasites are definitely not homogeneous at different parasitaemia levels, which is why we wanted to use scRNA-seq for this project to follow the trajectory of differentiation. However, at the very highest point of parasitaemia in the first wave of infection we know that the majority of parasites (around 99% in that project) have 1 nucleus and 1 kinetoplast, which is indicative of the G1/G0 cell cycle phase. As we know stumpy parasites are arrested in G1/G0 (most likely G0 based on scRNA-seq!) this indicates most cells are stumpy at the peak and this is supported by their expression of molecular markers for that stage (e.g. PAD proteins) on most parasites (Silvester Nature micro. 2017; Cayla et al. elife 2020 and others).

6.I found your finding regarding the branching point very interesting. Is it known in T. brucei, when does the commitment to stumpy (with no return) occurs, and what regulates it? My question comes from the equivalent in P. falciparum, whereby parasite exposure to specific conditions can regulate commitment to gametocytemia but only until a certain point in the life of the parasite-after the commitment to a certain path of development is irreversible.

Excellent question! We were interested to see if we could narrow down this commitment point by comparing differentiating wild-types to the ZC3H20 null parasites. The single cell analysis has given us some clues I think; we can see a clear switch from slender to stumpy transcriptomes and cell cycle exit specifically in early G1. We also found that, although, ZC3H20 KO parasites have a slight growth defect and down regulate some slender-associated transcripts, they continue to replicate, appear slender in morphology, and don’t express stumpy-associated factors. In short, these parasites aren’t able to irreversibly “commit” to cell cycle exit, and so either ZC3H20 itself or a downstream (or parallel) factor(s) (Ling et al J. Biol. Chem. 2011; Liu et al Mol. Micro. 2020 and Cayla et al Elife 2020) are likely to regulate this. This phenotype is consistent with modelling studies that suggested that parasites can begin some events of differentiation before their irreversible arrest and development to stumpy forms (MacGregor et al. Cell Host and Microbe 2011). More targeted wet lab experiments I think are needed to see if any of the genes with interesting expression patterns in this analysis control an irreversible commitment point.

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