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Regulation of gene expression and parasite-parasite interactions in African trypanosomes

English title Regulation of gene expression and parasite-parasite interactions in African trypanosomes
Applicant Roditi Isabel
Number 184669
Funding scheme Project funding (Div. I-III)
Research institution Institut für Zellbiologie Departement Biologie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Molecular Biology
Start/End 01.09.2019 - 31.01.2022
Approved amount 533'514.00
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Keywords (10)

ChIP-seq; Social motility; Stage-specific; Gene expression; RNA-seq; Nuclear architecture; Differentiation; Trypanosoma; Host-parasite interactions; GRO-seq

Lay Summary (German)

Lead
Afrikanische Trypanosomen, die Erreger der menschlichen Schlafkrankheit und der Naganaseuche im Tier, haben zwei Wirte - Tsetsefliege und Säugetier. Ein Ziel unserer Forschung ist es, die molekulare Grundlage zu verstehen, wie sich die Parasiten durch Veränderung der Genexpression an diese sehr unterschiedlichen Umgebungen anpassen können. Zudem untersuchen wir, wie Trypanosomen untereinander kommunizieren und ihr Verhalten gegenseitig beeinflussen.
Lay summary

Afrikanische Trypanosomen sind die Erreger der menschlichen Schlafkrankheit und der Naganaseuche im Tier. Viel Forschung an dem einzelligen Parasiten wird aus medizinischem Interesse an der Krankheit betrieben. Zusätzlich kam es in den letzten Jahrzehnten auch zu zahlreichen grundlegenden Entdeckungen der Zellbiologie durch die Forschung an den Trypanosomen als Modellorganismus. Trypanosomen haben zwei Wirte, die Tsetsefliege und das Säugetier, wobei die Übertragung durch einen Stich einer infizierten Fliege stattfindet. Die Trypanosomen können sich zwar auf den Wechsel zwischen den Wirten vorbereiten, sie wissen jedoch nie, wann genau das passieren wird. Die beiden Wirte bieten ein komplett unterschiedliches Milieu. Vor allem in der Fliege wird der Parasit mit Änderungen der Temperatur, der Nährstoffe und der Gegenwart anderer Mikroorganismen konfrontiert. Ein Ziel unserer Forschung ist es, die molekulare Grundlage zu verstehen, wie sich die Parasiten durch Veränderung der Genexpression an unterschiedliche Umgebungen anpassen können.

Ein weiterer Aspekt unserer Forschung ist die Frage, wie Trypanosomen untereinander kommunizieren und ihr Verhalten gegenseitig beeinflussen können. Eine Form der Kommunikation ist unter dem Begriff "Social Motility" bekannt und passiert, wenn Trypanosomen auf weichen feuchten Oberflächen kultiviert werden. In dieser Situation formen die Parasiten grosse Verbände, welche sich koordiniert in eine Richtung bewegen können. Interessanterweise spüren sich verschiedene Verbände und vermeiden, mit einander in Kontakt zu kommen. Wir haben vor kurzem gezeigt, dass ein bestimmtes Gen sowohl für Social Motility als auch für die Migration der Parasiten in der Fliege benötigt wird. 

Neueste Ergebnisse deuten darauf hin, dass die Parasiten spezifische Moleküle ausscheiden, die ihr Migrationsverhalten bestimmen. Wir vertreten die Hypothese, dass dieses Verhalten, zusammen mit Signalen von der Fliege, entscheidet, ob die Trypanosomen sich erfolgreich entwickeln und übertragen werden können. Um diese Hypothese zu belegen, werden wir eine Serie von Mutanten auf deren Migrationsverhalten untersuchen und versuchen, verantwortlichen Moleküle zu identifizieren, die für Social Motility wichtig sind.

Direct link to Lay Summary Last update: 25.07.2019

Responsible applicant and co-applicants

Employees

Datasets

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.

Collaboration

Group / person Country
Types of collaboration
Dr Manfred Heller, University of Bern Switzerland (Europe)
- Publication
Professor Shulamit Michaeli, Bar Ilan University Israel (Asia)
- in-depth/constructive exchanges on approaches, methods or results
Prof. Peter Bütikofer, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Jan van den Abbeele, Tropical Institute Antwerp Belgium (Europe)
- Research Infrastructure
Dr Laurie Read, University of Buffalo United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
Prof. Doris Rentsch, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Dr Pamela Nicholson, NGS Platform, University of Bern Switzerland (Europe)
- Publication
- Research Infrastructure

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Swiss Trypanosomatid Meeting 2022 Talk given at a conference A Split-Cre-Lox system permanently labels trypanosomes that undergo flagellar fusion 28.01.2022 Bern (virtual), Switzerland Benninger Mattias;
Swiss Trypanosomatid Meeting 2022 Talk given at a conference The export factor Mex67 sets boundaries for polymerase I transcription in Trypanosoma brucei 28.01.2022 Bern (virtual), Switzerland Pozzi Maria Berta;
Jacobs School of Medicine and Biomedical Sciences Seminar Series Individual talk The sweet and sour sides of cyclic AMP signalling in trypanosomes 07.12.2021 Buffalo (virtual), United States of America Roditi Isabel;
Seminar Series Pasteur Institute, Paris Individual talk The sweet and sour sides of cyclic AMP signalling in trypanosomes 18.11.2021 Pasteur Institute, Paris, France Roditi Isabel;
Molecular Parasitology Meeting 2021 Poster The export factor Mex67 sets boundaries for polymerase I transcription in Trypanosoma bruce 05.10.2021 Woods Hole (virtual), United States of America Pozzi Maria Berta;
Molecular Parasitology Meeting 2021 Talk given at a conference A Split-Cre-Lox system to permanently label and visualise trypanosomes that have undergone flagellar fusion in the tsetse fly 05.10.2021 Woods Hole (virtual), United States of America Benninger Mattias;
Middle Eastern Biology of Parasitism Summer School Talk given at a conference The sweet and sour sides of cyclic AMP signalling in trypanosomes 11.08.2021 Bern (virtual), Switzerland Roditi Isabel;
Biology of Parasitism Summer School Talk given at a conference The sweet and sour sides of cyclic AMP signalling in trypanosomes 01.08.2021 Woods Hole (virtual), United States of America Roditi Isabel;
Molecular Parasitology Meeting 2020 Talk given at a conference Trypanosomes having pHun: cyclic AMP signalling in perception and response to pH gradients 21.09.2020 Woods Hole (virtual), United States of America Shaw Sebastian;
Molecular Paraasitology Meeting 2020 Poster An Alba-domain protein required for proteome remodelling during trypanosome differentiation and host transition 21.09.2020 Woods Hole (virtual), United States of America Bevkal Subramanyaswamy Shubha;
Department of Chemical Biology Seminar Series Individual talk The sweet and sour sides of cyclic AMP signalling in trypanosomes 04.09.2020 Buenos Aires (virtual), Argentina Roditi Isabel;
Swiss Trypanosomatid Meeting 2020 Poster Exploring the mechanisms of transcription and export in Trypanosoma brucei 15.01.2020 Leysin, Switzerland Pozzi Maria Berta;
Swiss Trypanosopmatid Meeting 2020 Talk given at a conference Modulation of differentiation by Alba3 in Trypanosoma bruce 15.01.2020 Leysin, Switzerland Bevkal Subramanyaswamy Shubha;


Awards

Title Year
Dr Lutz and Celia Zwillenberg Prize 2021

Associated projects

Number Title Start Funding scheme
166427 Regulation of gene expression and parasite - parasite interactions in African trypanosomes 01.09.2016 Project funding (Div. I-III)

Abstract

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.
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