Prof. Dr. Isabel Roditi
Regulation of gene expression and parasite-parasite interactions in African trypanosomes
African trypanosomes are unicellular parasites that cause two diseases – human sleeping sickness and the animal disease Nagana. Related parasites cause Chagas Disease in South America and various forms of Leishmaniasis worldwide. African trypanosomes cycle between two hosts - tsetse fly and mammal – that provide them with radically different environments. Although trypanosomes can pre-adapt for transmission from the mammal to the tsetse fly, and vice versa, they cannot foresee when this will occur. The fly stages in particular are confronted with shifts in temperature, variation in nutrients and the presence of other microbes. While the importance of post-transcriptional control of gene expression is undisputed (and a long-standing topic of our research) the contribution of transcriptional control may have been underestimated. Another area that is still largely unexplored is how parasites within a community interact with each other and influence behaviour and gene expression. Finally, we are continuing our analysis of genes required for successful transmission and studying the transcriptome of parasites as they progress through the fly host.
Unusual features of gene expression
Trypanosomes branched early in the eukaryotic lineage and several features of gene expression deviate from those in more "mainstream" eukaryotes. The vast majority of protein-coding genes in trypanosomes are organised in polycistronic transcription units; these are transcribed as polycistronic precursor RNAs and co-transcriptionally processed into monocistronic mRNAs by trans-splicing and polyadenylation. Trypanosomes possess basal transcription factors, but lack recognisable orthologues of many factors required for transcription elongation and mRNA export in other eukaryotes.
Retrotransposon hotspot (RHS) proteins constitute 7 sub-families They are found exclusively in the trypanosome clade. Because of their complexity and abundance, RHS have often been discarded from transcriptomic and proteomic analyses and this, together with the absence of homology to any other known proteins, made it challenging to predict their functions. We recently showed that RHS2, RHS4 and RHS6 sub-families are required for transcription (Florini et al., 2019). ChIP analyses demonstrated that these 3 RHS are associated with RNA Polymerase II transcription units, and that their binding is reduced in Pol I transcription units and absent from silent regions of the genome.
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 the architecture of the trypanosome nucleus, such that different regions of the genome are not equally accessible to exogenous nucleotides. Alternatively, the differences might due to selective export or retention of transcripts or to certain transcripts being degraded co-transcriptionally or shortly before export. More controversially, different regions within a polycistronic transcription unit may be transcribed to different extents. We will investigate this in the context of a grant recently funded by the Swiss National Science Foundation.
Link to publication:
How do trypanosomes communicate?
Social motility (SoMo) describes the coordinated group movement of early procyclic (tsetse midgut ) form trypanosomes on a semi-solid surface. When trypanosomes are inoculated onto the surface of an agarose plate, they initially grow at the inoculation site; once they reach a threshold number, they form radial projections that move outwards at a rate of 500 body lengths per day. This behaviour implies that cells are producing migration factors that might be soluble and/or cell-associated. Interestingly, two communities derived from the same inoculum can sense each other and reorient to avoid contact, indicating that they produce and sense repellents. Finally, the cells might also produce surfactants/lubricants that allow them to move as a group.
Does this behaviour reflect an event in the trypanosome life-cycle? In collaboration with Kent Hill’s laboratory at UCLA, we recently showed that cyclic AMP signalling is required both for SoMo and for trypanosomes to cross a barrier, the peritrophic matrix, in the tsetse midgut (Shaw/DeMarco et al., 2019). We are currently performing screens for additional genes involved in SoMo and studying the nature of the migration and repellent factors.
Flagellar fusion and bidirectional protein exchange represent a novel form of cell-cell communication. We discovered this phenomenon serendipitously when we co-cultured procyclic forms tagged with two different fluorescent proteins and observed that a small proportion of them became double-positive. We initially interpreted this as a form of precocious sex (since mating, if it occurs, does so later in the life cycle) or as a possible parasexual cycle, but could not find any evidence of genetic exchange. We now know that trypanosomes can fuse their flagella (Figure 4) and exchange membrane proteins and cytoplasmic proteins within 2-3 minutes (Imhof et al., 2016). Fused cells are able to swim together and are also capable of dividing while joined. Intriguingly, Argonaute, a component of the RNAi pathway, is transferred, suggesting small RNAs might be carried along as cargo. Why do trypanosomes do this? Might it be a mechanism for stressed or damaged cells to obtain help from healthy cells? Are they delivering differentiation signals? Alternatively, might it enable cells to synchronise their behaviour? There is evidence that this can occur in tsetse as well and we are currently working on a system that will allow to permanently mark cells that have undergone flagellar fusion.
Behind the paper: Tackling trypanosome biology takes a lot of guts
pAi1C9 (All in one Cas9)
pAi1C9 (All in one Cas9) was constructed by replacing the tubulin sequence upstream of T7RNAP in pTB011_Cas9_T7RNAP_blast with 366 bp of the EP procyclin promoter and 5’ UTR. This plasmid can be used for transient expression of T7RNAP and Cas9.
pTB011_Cas9_T7RNAP_blast (Shaw et al., 2019) is a derivative of pTB011 (Beneke et al., 2017). We modified tpTB011 by replacing the puromycin acetyltransferase gene with that of T7 RNA polymerase (T7RNAP), giving rise to pTB011_Cas9_T7RNAP_blast. This plasmid allows generation of CRISPR/Cas9-competent cell lines, constitutively expressing T7RNAP and Cas9 from the tubulin locus, in a single round of transfection.
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- Name / Titel
- Dr. Arunasalam Naguleswaran
- Post Doc
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Hulpia, Fabian; Mabille, Dorien; Campagnaro, Gustavo; Schumann, Gabriela; Maes, Louis; Roditi, Isabel; Hofer, Anders; de Koning, Harry; Caljon, Guy; Von Calenbergh, Serge (2019). Combining tubercidin and cordycepin scaffolds results in highly active candidates to treat late-stage sleeping sickness. Nature Communications, 10(1), p. 5564. Springer Nature 10.1038/s41467-019-13522-6
Shaw, Sebastian; DeMarco, Stephanie F; Rehmann, Ruth; Wenzler, Tanja; Florini, Francesca; Roditi, Isabel; Hill, Kent L (2019). Flagellar cAMP signaling controls trypanosome progression through host tissues. Nature communications, 10(1), p. 803. Nature Publishing Group 10.1038/s41467-019-08696-y
Florini, Francesca; Naguleswaran, Arunasalam; Gharib, Walid H; Bringaud, Frédéric; Roditi, Isabel (2019). Unexpected diversity in eukaryotic transcription revealed by the retrotransposon hotspot family of Trypanosoma brucei. Nucleic acids research, 47(4), pp. 1725-1739. Oxford University Press 10.1093/nar/gky1255
Naguleswaran, Arunasalam; Doiron, Nicholas; Roditi, Isabel (2018). RNA-Seq analysis validates the use of culture-derived Trypanosoma brucei and provides new markers for mammalian and insect life-cycle stages. BMC Genomics, 19(1), p. 227. BioMed Central 10.1186/s12864-018-4600-6
Naguleswaran, Arunasalam; Roditi, Isabel (2017). Rodent-free cyclical transmission of Trypanosoma brucei brucei. Molecular and biochemical parasitology, 217, pp. 16-18. Elsevier 10.1016/j.molbiopara.2017.08.005
Poch, Olivier; Frey, Joachim; Roditi, Isabel; Pommerol, Antoine; Jost, Bernhard; Thomas, Nicolas (2017). Remote Sensing of Potential Biosignatures from Rocky, Liquid, or Icy (Exo)Planetary Surfaces. Astrobiology, 17(3), pp. 231-252. Mary Ann Liebert 10.1089/ast.2016.1523
Roditi, Isabel; Schumann-Burkard, Gabriela; Naguleswaran, Arunasalam (2016). Environmental sensing by African trypanosomes. Current opinion in microbiology, 32, pp. 26-30. Current Biology Ltd. 10.1016/j.mib.2016.04.011
Imhof, Simon; Fragoso, Cristina; Hemphill, Andrew; Von Schubert, Conrad; Li, Dong; Legant, Wesley; Betzig, Erik; Roditi, Isabel (2016). Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication? F1000Research, 5(682), p. 682. F1000 Research Ltd 10.12688/f1000research.8249.1
Wenzler, Tanja; Schumann-Burkard, Gabriela; Schmidt, Remo S.; Mäser, Pascal; Bergner, Andreas; Roditi, Isabel; Brun, Reto (2016). A new approach to chemotherapy: drug-induced differentiation kills African trypanosomes. Scientific Reports, 6(22451), p. 22451. Nature Publishing Group 10.1038/srep22451
Vanwalleghem, G; Fontaine, F; Lecordier, L; Tebabi, P; Klewe, K; Nolan, DP; Yamaryo-Botté, Y; Botté, C; Schumann-Burkard, Gabriela; Rassow, J; Roditi, Isabel; Perez-Morga, D; Pays, E (2015). Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1. Nature communications, 6(8078), p. 8078. Nature Publishing Group 10.1038/ncomms9078
de Macêdo, Juan P.; Schumann-Burkard, Gabriela; Niemann, Moritz; Barrett, Michael P.; Vial, Henri; Mäser, Pascal; Roditi, Isabel; Schneider, André; Bütikofer, Peter; Horn, David (2015). An Atypical Mitochondrial Carrier That Mediates Drug Action in Trypanosoma brucei. PLoS pathogens, 11(5), e1004875. Public Library of Science 10.1371/journal.ppat.1004875
Naguleswaran, Arunasalam; Gunasekera, Kapila; Schimanski, Bernd; Heller, Manfred; Hemphill, Andrew; Ochsenreiter, Torsten; Roditi, Isabel (2015). Trypanosoma brucei RRM1 is a nuclear RNA-binding protein and modulator of chromatin structure. mBio, 6(2), e00114. American Society for Microbiology 10.1128/mBio.00114-15
Imhof, Simon; Vu Nguyen, Xuan Lan; Bütikofer, Peter; Roditi, Isabel (2015). A glycosylation mutant of Trypanosoma brucei links social motility defects in vitro to impaired colonisation of tsetse in vivo. Eukaryotic cell, 14(6), pp. 588-592. American Society for Microbiology ASM 10.1128/EC.00023-15
Bühlmann, M; Walrad, Pegine; Rico, E; Ivens, A; Capewell, P; Naguleswaran, Arunasalam; Roditi, Isabel; Matthews, K (2015). NMD3 regulates both mRNA and rRNA nuclear export in African trypanosomes via an XPOI-linked pathway. Nucleic acids research, 43(9), pp. 4491-4504. Oxford University Press 10.1093/nar/gkv330
Cristodero, Marina; Schimanski, Bernd; Heller, Manfred; Roditi, Isabel (2014). Functional characterization of the trypanosome translational repressor SCD6. Biochemical journal, 457(1), pp. 57-67. Portland Press 10.1042/BJ20130747
Lecordier, Laurence; Uzureau, P; Tebabi, P; Perez-Morga, D; Nolan, D; Schumann-Burkard, Gabriela; Roditi, Isabel; Pays, E (2014). Identification of Trypanosoma brucei components involved in trypanolysis by normal human serum. Molecular microbiology, 94(3), pp. 625-636. Blackwell Science 10.1111/mmi.12783
Imhof, Simon; Knüsel, Sebastian; Gunasekera, Kapila; Vu Nguyen, Xuan Lan; Roditi, Isabel (2014). Social motility of African trypanosomes is a property of a distinct life-cycle stage that occurs early in tsetse fly transmission. PLoS pathogens, 10(10), e1004493. Public Library of Science 10.1371/journal.ppat.1004493
Knüsel, Sebastian; Roditi, Isabel (2013). Insights into the regulation of GPEET procyclin during differentiation from early to late procyclic forms of Trypanosoma brucei. Molecular and biochemical parasitology, 191(2), pp. 66-74. Elsevier 10.1016/j.molbiopara.2013.09.004
Schumann Burkard, Gabriela; Käser, Sandro; de Araújo, Patricia; Schimanski, Bernd; Naguleswaran, Arunasalam; Knüsel, Sebastian; Heller, Manfred; Roditi, Isabel (2013). Nucleolar proteins regulate stage-specific gene expression and ribosomal RNA maturation in Trypanosoma brucei. Molecular microbiology, 88(4), pp. 827-840. Blackwell Science 10.1111/mmi.12227
Morand, Sabine; Renggli, Christina Kunz; Roditi, Isabel; Vassella, Erik (2012). MAP kinase kinase 1 (MKK1) is essential for transmission of Trypanosoma brucei by Glossina morsitans. Molecular and biochemical parasitology, 186(1), pp. 73-6. Amsterdam: Elsevier 10.1016/j.molbiopara.2012.09.001
Schumann Burkard, Gabriela; Jutzi, Pascal; Roditi, Isabel (2011). Genome-wide RNAi screens in bloodstream form trypanosomes identify drug transporters. Molecular and biochemical parasitology, 175(1), pp. 91-94. Amsterdam: Elsevier 10.1016/j.molbiopara.2010.09.002
Mani, Jan; Güttinger, Andreas; Schimanski, Bernd; Heller, Manfred; Acosta-Serrano, Alvaro; Pescher, Pascale; Späth, Gerald; Roditi, Isabel (2011). Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery. PLoS ONE, 6(7), e22463. Lawrence, Kans.: Public Library of Science 10.1371/journal.pone.0022463
Aeby, Eric; Ullu, Elisabetta; Yepiskoposyan, Hasmik; Schimanski, Bernd; Roditi, Isabel; Mühlemann, Oliver; Schneider, André (2010). tRNASec is transcribed by RNA polymerase II in Trypanosoma brucei but not in humans. Nucleic acids research, 38(17), pp. 5833-5843. London: Oxford University Press 10.1093/nar/gkq345
Nilsson, Daniel; Gunasekera, Kapila; Mani, Jan; Osteras, Magne; Farinelli, Laurent; Baerlocher, Loic; Roditi, Isabel; Ochsenreiter, Torsten (2010). Spliced leader trapping reveals widespread alternative splicing patterns in the highly dynamic transcriptome of Trypanosoma brucei. PLoS pathogens, 6(8), e1001037. San Francisco, Calif.: Public Library of Science 10.1371/journal.ppat.1001037
Oberle, Michael; Balmer, Oliver; Brun, Reto; Roditi, Isabel (2010). Bottlenecks and the maintenance of minor genotypes during the life cycle of Trypanosoma brucei. PLoS pathogens, 6(7), e1001023. San Francisco, Calif.: Public Library of Science 10.1371/journal.ppat.1001023
Vassella, Erik; Oberle, Michael; Urwyler, Simon; Renggli, Christina Kunz; Studer, Erwin; Hemphill, Andrew; Fragoso, Cristina; Bütikofer, Peter; Brun, Reto; Roditi, Isabel (2009). Major surface glycoproteins of insect forms of Trypanosoma brucei are not essential for cyclical transmission by tsetse. PLoS ONE, 4(2), e4493. Lawrence, Kans.: Public Library of Science 10.1371/journal.pone.0004493
Rufener, Lucien; Mäser, Pascal; Roditi, Isabel; Kaminsky, Ronald (2009). Haemonchus contortus acetylcholine receptors of the DEG-3 subfamily and their role in sensitivity to monepantel. PLoS pathogens, 5(4), e1000380. San Francisco, Calif.: Public Library of Science 10.1371/journal.ppat.1000380
Fragoso, Cristina M.; Schumann Burkard, Gabriela; Oberle, Michael; Kunz Renggli, Christina; Hilzinger, Karen; Roditi, Isabel (2009). PSSA-2, a Membrane-Spanning Phosphoprotein of Trypanosoma brucei, Is Required for Efficient Maturation of Infection. PLoS ONE, 4(9), e7074. Lawrence, Kans.: Public Library of Science 10.1371/journal.pone.0007074
Haenni, Simon; Studer, Erwin; Schumann Burkard, Gabriela; Roditi, Isabel (2009). Bidirectional silencing of RNA polymerase I transcription by a strand switch region in Trypanosoma brucei. Nucleic acids research, 37(15), pp. 5007-5018. London: Oxford University Press 10.1093/nar/gkp513
Goldshmidt, Hanoch; Sheiner, Lilach; Bütikofer, Peter; Roditi, Isabel; Uliel, Shai; Günzel, Mark; Engstler, Markus; Michaeli, Shulamit (2008). Role of protein translocation pathways across the endoplasmic reticulum in Trypanosoma brucei. Journal of biological chemistry, 283(46), pp. 32085-32098. Bethesda, Md.: American Society for Biochemistry and Molecular Biology 10.1074/jbc.M801499200
Roditi, Isabel; Lehane, Michael (2008). Interactions between trypanosomes and tsetse flies. Current opinion in microbiology, 11(4), pp. 345-351. London: Current Biology Ltd. 10.1016/j.mib.2008.06.006
Güttinger, Andreas; Schwab, Claudia; Morand, Sabine; Roditi, Isabel; Vassella, Erik (2007). A mitogen-activated protein kinase of Trypanosoma brucei confers resistance to temperature stress. Molecular and biochemical parasitology, 153(2), pp. 203-206. Amsterdam: Elsevier 10.1016/j.molbiopara.2007.02.001
Burkard, Gabriela; Fragoso, Cristina; Roditi, Isabel (2007). Highly efficient stable transformation of bloodstream forms of Trypanosoma brucei. Molecular and biochemical parasitology, 153(2), pp. 220-223. Amsterdam: Elsevier 10.1016/j.molbiopara.2007.02.008
Spoerri, Iris; Chadwick, Ruth; Renggli, Christina; Matthews, Keith; Roditi, Isabel; Burkard, Gabriela (2007). Role of the stage-regulated nucleoside transporter TbNT10 in differentiation and adenosine uptake in Trypanosoma brucei. Molecular and biochemical parasitology, 154(1), pp. 110-4. Amsterdam: Elsevier 10.1016/j.molbiopara.2007.04.006
Urwyler, Simon; Studer, Erwin; Renggli, Christina; Roditi, Isabel (2007). A family of stage-specific alanine-rich proteins on the surface of epimastigote forms of Trypanosoma brucei. Molecular microbiology, 63(1), pp. 218-28. Oxford: Blackwell Science 10.1111/j.1365-2958.2006.05492.x
Domenicali Pfister, Debora; Burkard, Gabriela; Morand, Sabine; Renggli, Christina Kunz; Roditi, Isabel; Vassella, Erik (2006). A Mitogen-activated protein kinase controls differentiation of bloodstream forms of Trypanosoma brucei. Eukaryotic cell, 5(7), pp. 1126-35. Washington, D.C.: American Society for Microbiology ASM 10.1128/EC.00094-06
Utz, Silvia; Roditi, Isabel; Kunz Renggli, Christina; Almeida, Igor C; Acosta-Serrano, Alvaro; Bütikofer, Peter (2006). Trypanosoma congolense procyclins: unmasking cryptic major surface glycoproteins in procyclic forms. Eukaryotic cell, 5(8), pp. 1430-40. Washington, D.C.: American Society for Microbiology ASM 10.1128/EC.00067-06
Haenni, Simon; Kunz Renggli, Christina; Fragoso, Cristina; Oberle, Michael; Roditi, Isabel (2006). The procyclin-associated genes of Trypanosoma brucei are not essential for cyclical transmission by tsetse. Molecular and biochemical parasitology, 150(2), pp. 144-156. Amsterdam: Elsevier 10.1016/j.molbiopara.2006.07.005
Urwyler, Simon; Vassella, Erik; Abbeele, Jan Van Den; Kunz Renggli, Christina; Blundell, Pat; Barry, J. David; Roditi, Isabel (2005). Expression of Procyclin mRNAs during Cyclical Transmission of Trypanosoma brucei. PLoS pathogens, 1(3), e22. Public Library of Science 10.1371/journal.ppat.0010022
Weynants, V; Gilson, D; Furger, A; Collins, R A; Mertens, P; De Bolle, X; Heussler, Volker; Roditi, Isabel; Howard, C J; Dobbelaere, A E; Letesson, J J (1998). Production and characterisation of monoclonal antibodies specific for bovine interleukin-4. Veterinary immunology and immunopathology, 66(2), pp. 99-112. Elsevier 10.1016/S0165-2427(98)00184-6