Single-cell transcriptomic analysis of bloodstream Trypanosoma brucei reconstructs cell cycle progression and developmental quorum sensing.
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ABSTRACT:
SUBMITTER: Briggs EM
PROVIDER: S-EPMC8421343 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mitochondrial organelles need to be replicated during cell division. Many aspects of this process have been studied in great detail, however the actual size increase and the position of organelle growth are less well understood. We use the protozoan parasite Trypanosoma brucei that contains a single mitochondrion to study organelle biogenesis by fluorescence microscopy. From the analysis of more than 1000 T. brucei bloodstream form cells of a nonsynchronous population we conclude that the mitochondrial network mostly grows from two areas along the main organelle axis, posterior and anterior of the nucleus. Loops and branches from these two areas eventually fuse to build a complex network. Together with the appearance of the division fold in the posterior part of the cell, pruning of the mitochondrial network and finally separation into the two daughter cells occurs. Overall organelle biogenesis is not continuous during cell growth and occurs mostly in the last part of the cell cycle. Furthermore, using 3D STED super resolution microscopy we reconstruct the volume of the organelle and characterize the region where the mitochondrial genome is positioned by serial block face scanning electron microscopy.
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Project description:Early in an infection the bloodstream forms of the African trypanosome Trypanosoma brucei brucei are long, slender and rapidly dividing. Later, non-dividing, short, stumpy forms may be found. In this report we described biochemical differences between the two parasite populations in the phosphorylation of their proteins in vitro. Compared with the slender populations, the non-dividing stumpy forms of the parasites exhibit decreased phosphorylation of an 80 kDa protein and enhanced phosphorylation of 37 kDa and 42 kDa proteins (pp37 and pp42). These changes occurred regardless of whether the stumpy trypanosomes were generated naturally during the course of the infection or induced by difluoromethylornithine treatment. The phosphorylation of pp37 and pp42 occurs on serine and threonine residues and is totally dependent upon the presence of Mn2+ or Mg2+. However, excess Mn2+ or Mg2+ inhibits phosphorylation. Maximal phosphorylation of pp42 occurs with 1 mm-Mn2+ or 10 mm-Mg2+, whereas that of pp37 occurs with 50 mM-Mn2+ or greater than 100 mm-Mg2+. The phosphorylation of pp37 is greatly enhanced by KCl, whereas that of pp42 is only slightly increased by this salt. Ca2+, calmodulin, phospholipids and cyclic AMP have no discernible effect upon the phosphorylation of pp42 or pp37 in vitro, whereas heparin, suramin, polylysine, polyarginine and polyamines all inhibit phosphorylation. Thus the enzymes that phosphorylate pp42 and pp37 have properties similar to, but distinct from, those of mammalian casein kinase II. Since the casein-kinase-like activity is higher in the slender than in the stumpy forms, the enhanced phosphorylation of pp42 and pp37 in the non-dividing parasites is probably a result of the enhanced synthesis of these acidic proteins.
| S-EPMC1150005 | biostudies-other
Project description:For persistent infections of the mammalian host, African trypanosomes limit their population size by quorum sensing of the parasite-excreted stumpy induction factor (SIF), which induces development to the tsetse-infective stumpy stage. We found that besides this cell density-dependent mechanism, there exists a second path to the stumpy stage that is linked to antigenic variation, the main instrument of parasite virulence. The expression of a second variant surface glycoprotein (VSG) leads to transcriptional attenuation of the VSG expression site (ES) and immediate development to tsetse fly infective stumpy parasites. This path is independent of SIF and solely controlled by the transcriptional status of the ES. In pleomorphic trypanosomes varying degrees of ES-attenuation result in phenotypic plasticity. While full ES-attenuation causes irreversible stumpy development, milder attenuation may open a time window for rescuing an unsuccessful antigenic switch, a scenario that so far has not been considered as important for parasite survival.
| S-EPMC5398725 | biostudies-literature
Project description:African trypanosomes proliferate as bloodstream forms (BSFs) 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 (CCR) transcriptomes of both procyclic and slender BSF 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 CCR transcriptomes of both forms, which are available for further interrogation via an interactive webtool.
| S-EPMC10212563 | biostudies-literature
Project description:The protozoan parasites Trypanosoma brucei spp. cause important human and livestock diseases in sub-Saharan Africa. In mammalian blood, two developmental forms of the parasite exist: proliferative 'slender' forms and arrested 'stumpy' forms that are responsible for transmission to tsetse flies. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission. This response is triggered by an elusive 'stumpy induction factor' (SIF) whose intracellular signalling pathway is also uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains respond inefficiently to SIF but can generate forms with stumpy characteristics when exposed to cell-permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNA interference library screen to identify the signalling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) or 8-pCPT-2'-O-methyl-5'-AMP to select cells that were unresponsive to these signals and hence remained proliferative. Genome-wide Ion Torrent based RNAi target sequencing identified cohorts of genes implicated in each step of the signalling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. Genes at each step were independently validated in cells naturally capable of stumpy formation, confirming their role in density sensing in vivo. The putative RNA-binding protein, RBP7, was required for normal quorum sensing and promoted cell-cycle arrest and transmission competence when overexpressed. This study reveals that quorum sensing signalling in trypanosomes shares similarities to fundamental quiescence pathways in eukaryotic cells, its components providing targets for quorum-sensing interference-based therapeutics.
| S-EPMC3908871 | biostudies-literature
Project description:The protozoan parasites Trypanosoma brucei spp. are responsible for important human and livestock diseases in sub Saharan Africa. In the mammalian blood, two developmental forms of the parasite exist: proliferative ?slender? forms and transmissible ?stumpy? forms that are quiescent, awaiting uptake in a tsetse fly bloodmeal. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission. The response is triggered by an elusive ?stumpy induction factor? (SIF) whose intracellular signaling pathway is also completely uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains cannot respond to SIF, but can generate forms with stumpy characteristics when exposed to cell permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNAi library screen to identify the signaling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to cell permeable cAMP or AMP analogues to select cells that remained proliferative and so were unresponsive to these signals. Genome-wide ion torrent-based RNA interference Target sequencing (RIT-seq) identified a cohort of genes implicated in all steps of the signaling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. The identified genes at each step have been validated in cells naturally capable of stumpy formation, confirming their role in SIF-induced density sensing and cellular quiescence.
2013-12-16 | GSE46501 | GEO
Project description:The sleeping sickness parasite, Trypanosoma brucei, uses quorum sensing (QS) to balance proliferation and transmission potential in the mammal bloodstream. A signal transduction cascade regulates this process, a component of which is a divergent member of the DYRK family of protein kinases, TbDYRK. Phylogenetic and mutational analysis in combination with activity and phenotypic assays revealed that TbDYRK exhibits a pre-activated conformation and an atypical HxY activation loop motif, unlike DYRK kinases in other eukaryotes. Phosphoproteomic comparison of TbDYRK null mutants with wild-type parasites identified molecules that operate on both the inhibitory 'slender retainer' and activatory 'stumpy inducer' arms of the QS control pathway. One of these molecules, the RNA-regulator TbZC3H20, regulates parasite QS, this being dependent on the integrity of its TbDYRK phosphorylation site. This analysis reveals fundamental differences to conventional DYRK family regulation and links trypanosome environmental sensing, signal transduction and developmental gene expression in a coherent pathway.
| S-EPMC7136023 | biostudies-literature
Project description:Progression of the eukaryotic cell cycle requires the regulation of hundreds of genes to ensure that they are expressed at the required times. Integral to cell cycle progression in yeast and animal cells are temporally controlled, progressive waves of transcription mediated by cell cycle-regulated transcription factors. However, in the kinetoplastids, a group of early-branching eukaryotes including many important pathogens, transcriptional regulation is almost completely absent, raising questions about the extent of cell-cycle regulation in these organisms and the mechanisms whereby regulation is achieved. Here, we analyse gene expression over the Trypanosoma brucei cell cycle, measuring changes in mRNA abundance on a transcriptome-wide scale. We developed a "double-cut" elutriation procedure to select unperturbed, highly synchronous cell populations from log-phase cultures, and compared this to synchronization by starvation. Transcriptome profiling over the cell cycle revealed the regulation of at least 430 genes. While only a minority were homologous to known cell cycle regulated transcripts in yeast or human, their functions correlated with the cellular processes occurring at the time of peak expression. We searched for potential target sites of RNA-binding proteins in these transcripts, which might earmark them for selective degradation or stabilization. Over-represented sequence motifs were found in several co-regulated transcript groups and were conserved in other kinetoplastids. Furthermore, we found evidence for cell-cycle regulation of a flagellar protein regulon with a highly conserved sequence motif, bearing similarity to consensus PUF-protein binding motifs. RNA sequence motifs that are functional in cell-cycle regulation were more widespread than previously expected and conserved within kinetoplastids. These findings highlight the central importance of post-transcriptional regulation in the proliferation of parasitic kinetoplastids.
| S-EPMC3069104 | biostudies-literature
Project description:The protozoan parasites Trypanosoma brucei spp. are responsible for important human and livestock diseases in sub Saharan Africa. In the mammalian blood, two developmental forms of the parasite exist: proliferative ?slender? forms and transmissible ?stumpy? forms that are quiescent, awaiting uptake in a tsetse fly bloodmeal. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission. The response is triggered by an elusive ?stumpy induction factor? (SIF) whose intracellular signaling pathway is also completely uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains cannot respond to SIF, but can generate forms with stumpy characteristics when exposed to cell permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNAi library screen to identify the signaling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to cell permeable cAMP or AMP analogues to select cells that remained proliferative and so were unresponsive to these signals. Genome-wide ion torrent-based RNA interference Target sequencing (RIT-seq) identified a cohort of genes implicated in all steps of the signaling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. The identified genes at each step have been validated in cells naturally capable of stumpy formation, confirming their role in SIF-induced density sensing and cellular quiescence. RNA was isolated from AnTat1.1 cells grown in mice with or without doxycycline. RBP7 (Tb927.10.12100) RNAi lines were grown in mice with or without doxycycline. Since the RBP7 RNAi is leaky (six independent cell lines were analysed and all were significantly leaky), transcripts that showed elevated or reduced abundance in both RNAi induced and uninduced populations were identified in comparison to the AnTat1.1 control. A similar approach was adopted for the RBP7 over-expressing lines.
2013-12-16 | E-GEOD-46501 | biostudies-arrayexpress
Project description:Trypanosoma brucei EATRO 1125 parasites were grown in adult MF1 mice with parasites harvested on day 3 post-infection ('ascend/slender') or on day 6 post-infection ('peak/stumpy').
2018-10-01 | GSE114810 | GEO