Understanding chromatin dynamics during antigenic variation in trypanosoma brucei

Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2016African trypanosomiasis is a disease restricted to sub-Saharian Africa and it assumes two forms: Human African trypanosomiasis (HAT or sleeping sickness) or Animal African trypanosomiasis (AAT or n’gana), both being fatal if untreated. While HAT causes an estimated number of 15000 new cases each year and 70 million people are at risk, AAT causes an enormous economic burden due to livestock elimination [1]. HAT is caused by the unicellular Trypanosoma brucei parasite, which is transmitted by the tsetse transmitting vector. Upon a tsetse blood meal, parasites are delivered into a mammalian host where they proliferate in the bloodstream, lymphatic system and inside preferred tissues, such as the adipose tissue [2]. The parasites can progressively migrate to the central nervous system, leading to neurological disorders, coma and death [3]. Each Trypanosoma brucei parasite expresses a single type of Variant Surface Glycoprotein (VSG) at its cell surface. To avoid being eliminated by the host immune system, parasites use antigenic variation, which consists in periodically changing to a new VSG coat, obligating the immune system to reinitiate a new adaptive response and generate new antibodies. VSG genes are transcribed by RNA polymerase I (Pol I) from specialized subtelomeric loci termed Bloodstream Expression Sites (BESs) [4]. Although there are around 15 BESs in the genome, only one is active at any given time, thus ensuring VSG monoallelic expression. Replacement of the VSG coat starts in the nucleus by changing the actively transcribed VSG. This process takes place through: i) switching by recombination, which involves DNA recombination between the active and a silent BES encompassing the VSG gene or by copying a VSG sequence from archival copies present in the genome into the active BES [5, 6]; ii) in situ switching, where the active BES becomes silenced with the concomitant activation of a silent BES [7, 8]. VSG in situ switching is very poorly understood. We do not know the key players or the sequence of events that regulate it. As the actively transcribed BES possesses a very open chromatin conformation relative to transcriptionally silent BESs [9, 10], we proposed to understand how chromatin and transcription interweave during an in situ switching. Another event in which the active BES becomes silenced is during differentiation from bloodstream to procyclic (present in the tsetse) forms [11]. In this situation, we observed that transcriptional silencing precedes chromatin changes. Thus, we postulated that during an in situ switching event transcription is probably also halted prior to closing chromatin structure. We chose to use a tetracycline-inducible BES transcriptional silencing system from the Horn lab that induces VSG silencing [12], resulting in an increase of the switching frequency to 8%. Blockage of transcription was confirmed by the absence of Pol I in the active BES and led to a rapid decrease in transcript levels of this locus within the first 8 hr after BES silencing. Despite such significant changes at the transcriptional level, chromatin of the previously active BES remained in an open conformation. We hypothesized that the cell-cycle was necessary to induce chromatin remodeling as in Pol I-transcribed ribosomal DNA (rDNA) genes in yeast, but this revealed unfruitful. We observed that TDP1 (Trypanosome DNA-binding protein 1), an essential high mobility group box protein [13, 14], remained in the active BES even after BES silencing had been triggered. Depletion of TDP1 in these conditions led to closure of chromatin indicating that TDP1 is a key factor in stabilizing open chromatin conformation under transcriptional switching. We showed that during in situ switching, cells probe more than one silent BES, inclusively expressing other VSGs. We determined that after 24 hr of BES silencing, roughly half of the cells were committed to switch while the remaining could revert to transcribe the initial BES. Interestingly, cells that at 24 hr of BES silencing were not probing a specific BES (and VSG) did not switch to that BES. Overall, these results led us to propose a model in which under an in situ switching, transcription of the active BES is halted but its chromatin is maintained in an open conformation by TDP1. This allows parasites to probe silent BES to make a decision of switching or returning to the initial BES. The importance of TDP1 in Pol I transcription and in the switching process led us to ask if TDP1 overexpression could interfere with VSG monoallelic expression. Using a TDP1-overexpressing cell-line, we observed that genome-wide chromatin becomes more open (at different extent) except in the active BES and rDNA genes, suggesting that these two loci already have the chromatin fully open. Furthermore, the mRNA transcript levelsonly increased in silent BES, MES (Metacyclic Expression Sites) and procyclin loci confirming the role of TDP1 as a Pol I transcription facilitator [14]. Importantly, we observed the expression of a silent VSG upon TDP1 overexpression confirming the disruption of monoallelic expression. Overall, this dissertation enlightens the relevance of chromatin during an in situ switching and elucidates the roles of TDP1 as key factor for antigenic variation

Caracterização biofísica da membrana plasmática da levedura

Tese de mestrado, Bioquímica (Bioquímica Médica), Universidade de Lisboa, Faculdade de Ciências, 2009A membrana plasmática de Saccharomyces cerevisiae foi estudada por espectroscopia de fluorescência, utilizando as sondas de membrana ácido transparinárico e difenil-hexatrieno, de modo a compreender os princípios biofisicos subjacentes à formação e função de compartimentos membranares, cuja importância foi reconhecida recentemente. O estudo foi realizado em (i) células wt; (ii) esferoblastos (celulas wt com remoção completa da parede celular); (iii) lipossomas preparados a partir de extractos lipídicos totais de células wt; (iv) células erg6, que acumulam zimosterol em vez de ergosterol; (v) células scs7, que não sintetizam esfingolípidos com ácidos gordos C26:0 -hidroxilados. As principais observações foram: (a) a membrana plasmática da levedura possui domínios de gel ricos em esfingolípidos com conteúdo baixo ou nulo de esteróis; (b) os lípidos de levedura possuem a capacidade de formar domínios mais rígidos na ausência de proteínas do que os detectados na membrana plasmática; (c) os sistemas erg6e esferoblastos possuem uma maior ordem global de membrana; (d) modificações na biossíntese do ergosterol e a remoção da parede celular não alteram significativamente a rigidez dos domínios ricos em esfingolípidos, mas em ambos os casos há uma diminuição da sua abundância relativa. Os resultados obtidos sugerem que a parede celular desempenha um papel na estabilização dos domínios ricos em esfingolípidos que poderá ocorrer por interacção da mesma com proteínas ancoradas a glucosilfosfatidilinositol. A partir dos resultados também foi proposto um modelo em que nas células onde os domínios ricos em esfingolípidos sao menos abundantes ocorre uma distribuição mais homogénea de esfingolípidos por toda a membrana, responsável pelo aumento de ordem global da mesma. Concluindo, este trabalho comprova a existência de domínios ordenados tipo gel na membrana plasmática de organismos vivos, reforçando a ideia de que os esfingolípidos são componentes essenciais na constituição da membrana e na resposta a alterações fisiológicas que ponham em causa a integridade da célula.The plasma membrane of Saccharomyces cerevisiae was studied by fluorescence spectroscopy with the membrane probes trans-parinaric acid and diphenylhexatriene to understand biophysical principles underlying the formation and role of membrane compartments, with relevance recently recognized. The study included: (i) wt cells; (ii) spheroplasts (wt cells with complete cell wall removal); (iii) liposomes from total lipid extracts of wt cells; (iv) erg6Δ cells, which accumulate zymosterol instead of ergosterol; (v) scs7Δ cells, which lack synthesis of α- hydroxylated sphingolipid-associated C26:0 fatty acids. The main observations were: (a) the yeast plasma membrane contains sphingolipid-enriched gel domains with low or none sterol content; (b) yeast lipids have the ability to form, in the absence of proteins, domains more rigid than those detected in the plasma membrane; (c) erg6 cells and spheropasts have a higher membrane order; (d) alterations in sterol biosynthesis and cell wall removal do not significantly affect sphingolipid-enriched domains rigidity, although significantly reducing their relative abundance. The results obtained suggest that the cell wall has an important role in the stabilization of sphingolipid-enriched domains which might occur through its interaction with glucosylphosphatidylinositol anchored proteins. Furthermore, a model is proposed that explains the lower abundance of sphingolipid-enriched domains by a more homogeneous distribution of those lipids throughout the whole plasma membrane, thereby increasing the membrane global order. In conclusion, this work shows the existence of gel-like ordered domains in the plasma membrane of living cells, supporting the idea that sphingolipids are essential components for the constitution of biomembranes and for the response to physiological changes that are dangerous for the cell integrity

Trypanosoma brucei parasites occupy and functionally adapt to the adipose tissue in mice

This work was supported by 55007419 (HHMI) and 2151 (EMBO) to L.M.F., D.P.-N., F.B., and F.G.; FCT fellowships to S.T., F.R.-F., and F.A.-B. (SFRH/BPD/89833/2012, SFRH/BD/51286/2010, and SFRH/BD/80718/2011, respectively); Wellcome Trust grant (093228), MRC MR/M020118/1, and European Community Seventh Framework Programme under grant agreement No. 602773 (Project KINDRED) to S.A.Y. and T.K.S.; and PAI 7/41 (Belspo) and ERC-NANOSYM to J.V.D.A.Trypanosoma brucei is an extracellular parasite that causes sleeping sickness. In mammalian hosts, trypanosomes are thought to exist in two major niches: early in infection, they populate the blood; later, they breach the blood-brain barrier. Working with a well-established mouse model, we discovered that adipose tissue constitutes a third major reservoir for T. brucei. Parasites from adipose tissue, here termed adipose tissue forms (ATFs), can replicate and were capable of infecting a naive animal. ATFs were transcriptionally distinct from bloodstream forms, and the genes upregulated included putative fatty acid β-oxidation enzymes. Consistent with this, ATFs were able to utilize exogenous myristate and form β-oxidation intermediates, suggesting that ATF parasites can use fatty acids as an external carbon source. These findings identify the adipose tissue as a niche for T. brucei during its mammalian life cycle and could potentially explain the weight loss associated with sleeping sickness.Publisher PDFPeer reviewe

N6-methyladenosine in poly(A) tails stabilize VSG transcripts

© The Author(s), under exclusive licence to Springer Nature Limited 2022RNA modifications are important regulators of gene expression1. In Trypanosoma brucei, transcription is polycistronic and thus most regulation happens post-transcriptionally2. N6-methyladenosine (m6A) has been detected in this parasite, but its function remains unknown3. Here we found that m6A is enriched in 342 transcripts using RNA immunoprecipitation, with an enrichment in transcripts encoding variant surface glycoproteins (VSGs). Approximately 50% of the m6A is located in the poly(A) tail of the actively expressed VSG transcripts. m6A residues are removed from the VSG poly(A) tail before deadenylation and mRNA degradation. Computational analysis revealed an association between m6A in the poly(A) tail and a 16-mer motif in the 3' untranslated region of VSG genes. Using genetic tools, we show that the 16-mer motif acts as a cis-acting motif that is required for inclusion of m6A in the poly(A) tail. Removal of this motif from the 3' untranslated region of VSG genes results in poly(A) tails lacking m6A, rapid deadenylation and mRNA degradation. To our knowledge, this is the first identification of an RNA modification in the poly(A) tail of any eukaryote, uncovering a post-transcriptional mechanism of gene regulation.We are grateful to support from the Howard Hughes Medical Institute International Early Career Scientist Program (55007419), a European Molecular Biology Organization Installation grant (2151) and La Caixa Foundation (HR20-00361). This work was also partially supported by the ONEIDA project (LISBOA-01-0145-FEDER-016417) co-funded by Fundos Europeus Estruturais e de Investimento (FEEI) from ‘Programa Operacional Regional Lisboa 2020’ and by national funds from Fundação para a Ciência e a Tecnologia (FCT). S.R.J. was supported by NIH (R35 NS111631). Researchers were funded by individual fellowships from FCT (PD/BD/105838/2014 to I.J.V., 2020.06827.BD to L.S., SFRH/BD/80718/2011 to F.A.-B., PD/BD/138891/2018 to A.T. and CEECIND/03322/2018 to L.M.F.); a Novartis Foundation for Biomedical-Biological research to J.P.d.M.; a Human Frontier Science Programme long-term postdoctoral fellowship to M.D.N. (LT000047/2019); a Marie Skłodowska-Curie Individual Standard European Fellowship to S.S.P. (grant no. 839960); the GlycoPar Marie Curie Initial Training Network (GA 608295) to J.A.R. We thank J. Thomas-Oates (University of York, Centre of Excellence in Mass Spectrometry, Department of Chemistry) for the mass spectrometry analysis. The York Centre of Excellence in Mass Spectrometry was created thanks to a major capital investment through Science City York, supported by Yorkshire Forward with funds from the Northern Way Initiative, and subsequent support from EPSRC (EP/K039660/1 and EP/M028127/1).info:eu-repo/semantics/publishedVersio