ChIP-seq; Social motility; Stage-specific; Gene expression; RNA-seq; Nuclear architecture; Differentiation; Trypanosoma; Host-parasite interactions; GRO-seq
An Alba-domain protein required for proteome remodeling during differentiation of Trypanosoma brucei
| Author |
RODITI, ISABEL |
| Publication date |
16.12.2020 |
| Persistent Identifier (PID) |
PRJEB38690 |
| Repository |
European Nucleotide Archive
|
| Abstract |
arasites. Over the past decade, it has been shown that Alba-domain proteins contribute to developmental regulation in protozoan parasites. Trypanosoma brucei, responsible for human sleeping sickness, progresses through distinct life cycle stages as it cycles between its mammalian and tsetse fly host. T. brucei has 4 Alba-domain proteins that have been shown to interact with each other and with proteins of the translation machinery. Here we describe deletion mutants of two Alba-domain proteins, Alba3 and Alba4, and the requirements for them in bloodstream forms and during the transition to procyclic forms. We show that deletion of Alba3 or Alba4 does not have an effect on parasite’s survival as bloodstream forms and that Alba3 and Alba4 are functionally redundant in bloodstream forms. In contrast, only Alba3 can support successful differentiation from the stumpy to the procyclic form. Comparative proteomic and transcriptomic analysis of the Alba3 deletion mutant and wild-type parasites during differentiation, together with polysome profile analysis shows that differentiation regulation by Alba3 is achieved by extensive remodeling of the proteome. In summary, our studies show that Alba3 plays an important role in remodeling the proteome at the level of translation during differentiation of stumpy forms to procyclic forms.
pH taxis in trypanosomes
| Author |
Roditi, Isabel |
| Publication date |
24.07.2021 |
| Persistent Identifier (PID) |
PRJEB41935 |
| Repository |
European Nucleotide Archive
|
| Abstract |
More than a decade ago it was proposed that the collective migration of African trypanosomes on semi-solidsurfaces could be explained by a combination of migration factors and repellents released by the parasites, but the identity of these molecules was unknown. Here we show that procyclic (insect midgut) forms acidify their environment as a consequence of glucose metabolism, generating pH gradients by diffusion. Early and late procyclic forms exhibit self-organising propertieson surfaces. Both forms are attracted to alkali, but while early procyclic forms are repelled by acid and migrate outwards, late procyclic forms remain at the inoculation site. pH taxis relieson cyclic AMP signalling. Acid sensing requires a flagellar adenylate cyclase, ACP5, and a cyclic AMP response protein, CARP3, that interacts with ACP5. Deletion of the flagellar phosphodiesterase PDEB1 abolishes pH taxis completely. Trypanosomes can also respond to exogenously formed gradients. pH sensing is likely to be biologically relevant as trypanosomes experience large differences in pH as they progress through their tsetse fly host. In addition, self-generated gradients may help reinforce directionality. Moreover, since trypanosomes encode a large family of adenylate cyclases, these may govern other chemotactic responses and tissue tropisms in both the mammal and the fly.
pH taxis in African trypanosomes
| Author |
Roditi, Isabel |
| Publication date |
17.02.2022 |
| Persistent Identifier (PID) |
PXD030766 |
| Repository |
ProteomeXchange
|
| Abstract |
The collective movement of African trypanosomes on semi-solid surfaces, known as social motility, is presumed to be due to migration factors and repellents released by the parasites. Here we show that procyclic (insect midgut) forms acidify their environment as a consequence of glucose metabolism, generating pH gradients by diffusion. Early and late procyclic forms exhibit self-organising properties on agarose plates. While early procyclic forms are repelled by acid and migrate outwards, late procyclic forms remain at the inoculation site. Furthermore, trypanosomes respond to exogenously formed pH gradients, with both early and late procyclic forms being attracted to alkali. pH taxis is mediated by multiple cyclic AMP effectors: deletion of one copy of adenylate cyclase ACP5, or both copies of the cyclic AMP response protein CARP3, abrogates the response to acid, while deletion of phosphodiesterase PDEB1 completely abolishes pH taxis. The ability to sense pH is biologically relevant as trypanosomes experience large changes as they migrate through their tsetse host. Supporting this, a CARP3 null mutant is severely compromised in its ability to establish infections in flies. Based on these findings, we propose that the expanded family of adenylate cyclases in trypanosomes might govern other chemotactic responses in their two hosts.
African trypanosomes are unicellular eukaryotes that cause fatal diseases in humans and animals. This proposal consists of three parts - I aim to tie up two strands of my current research and to explore a new topic, nuclear architecture and transcriptional control, which arose from our analysis of nascent RNA. i) Social motility (SoMo) describes the coordinated group movement of early procyclic forms on a semi-solid surface and may also be involved in migration in the host. We have previously taken two approaches to identify genes involved in SoMo, genome-wide RNAi screens and knockdown of genes that are differentially expressed between early and late procyclic forms. Despite efficient knockdown by RNAi, analysis of candidates has revealed only mild phenotypes. With CRISPR/Cas9 now established in my laboratory, we will perform knockouts of candidate genes from these two categories and test the ability of mutants to perform SoMo on plates. Mutants showing a defect will be tested for their ability to infect their insect host, the tsetse fly.ii) The multigene retrotransposon hotspot (RHS) family is found only in the trypanosome clade. In African trypanosomes there are 7 RHS subfamilies, 6 of which are expressed in both bloodstream and procyclic forms. Our analysis of 3 RHS subfamilies (RHS2, 4 and 6) has revealed that they play a role in transcription elongation by RNA polymerase II and export of mRNAs. To complete this analysis, we will perform targeted experiments (principally RNAi, chromatin immunoprecipitation and proteomics) on the 3 remaining subfamilies and on selected orthologues of transcription elongation factors found in other eukaryotes. iii) While analysing RHS proteins we established procedures for labelling nascent RNAs with 5-ethynyl uridine (5-EU) and performed global run-on sequencing (GRO-Seq). This revealed that transcripts from different chromosomes and, even more surprisingly, from genes within the same polycistronic transcription unit, showed vastly different levels of incorporation of 5-EU. One reason for these results might be that different regions of the trypanosome nucleus are not equally accessible to exogenous nucleotides. We will perform fluorescence in situ hybridisation to determine whether there are chromosome territories in the nucleus and/or foci where highly transcribed genes are localised. Global run-on analysis (GRO-Seq) will also be performed on mRNA export mutants in order to determine whether the differences are due to selective export or retention of transcripts, rather than differences in transcription rates, and on nuclear exosome mutants to obtain information about whether stage-specific transcripts might be degraded co-transcriptionally or shortly before export.