65 results found
Davies C, Cher-Pheng O, Sioutas G, et al., 2021, TbSAP is a novel chromatin protein repressing metacyclic variant surface glycoprotein expression sites in bloodstream form Trypanosoma brucei, Nucleic Acids Research, Vol: 49, Pages: 3242-3262, ISSN: 0305-1048
The African trypanosome Trypanosoma brucei is a unicellular eukaryote, which relies on a protective variant surface glycoprotein (VSG) coat for survival in the mammalian host. A single trypanosome has >2000 VSG genes and pseudogenes of which only one is expressed from one of ∼15 telomeric bloodstream form expression sites (BESs). Infectious metacyclic trypanosomes present within the tsetse fly vector also express VSG from a separate set of telomeric metacyclic ESs (MESs). All MESs are silenced in bloodstream form T. brucei. As very little is known about how this is mediated, we performed a whole genome RNAi library screen to identify MES repressors. This allowed us to identify a novel SAP domain containing DNA binding protein which we called TbSAP. TbSAP is enriched at the nuclear periphery and binds both MESs and BESs. Knockdown of TbSAP in bloodstream form trypanosomes did not result in cells becoming more ‘metacyclic-like'. Instead, there was extensive global upregulation of transcripts including MES VSGs, VSGs within the silent VSG arrays as well as genes immediately downstream of BES promoters. TbSAP therefore appears to be a novel chromatin protein playing an important role in silencing the extensive VSG repertoire of bloodstream form T. brucei.
Budzak J, Kerry LE, Aristodemou A, et al., 2019, Dynamic colocalization of 2 simultaneously active VSG expression sites within a single expression-site body in Trypanosoma brucei, Proceedings of the National Academy of Sciences of the United States of America, Vol: 116, Pages: 16561-16570, ISSN: 0027-8424
Monoallelic exclusion ensures that the African trypanosome Trypanosoma brucei exclusively expresses only 1 of thousands of different variant surface glycoprotein (VSG) coat genes. The active VSG is transcribed from 1 of 15 polycistronic bloodstream-form VSG expression sites (ESs), which are controlled in a mutually exclusive fashion. Unusually, T. brucei uses RNA polymerase I (Pol I) to transcribe the active ES, which is unprecedented among eukaryotes. This active ES is located within a unique extranucleolar Pol I body called the expression-site body (ESB). A stringent restriction mechanism prevents T. brucei from expressing multiple ESs at the same time, although how this is mediated is unclear. By using drug-selection pressure, we generated VSG double-expresser T. brucei lines, which have disrupted monoallelic exclusion, and simultaneously express 2 ESs in a dynamic fashion. The 2 unstably active ESs appear epigenetically similar to fully active ESs as determined by using chromatin immunoprecipitation for multiple epigenetic marks (histones H3 and H1, TDP1, and DNA base J). We find that the double-expresser cells, similar to wild-type single-expresser cells, predominantly contain 1 subnuclear ESB, as determined using Pol I or the ESB marker VEX1. Strikingly, simultaneous transcription of the 2 dynamically transcribed ESs is normally observed only when the 2 ESs are both located within this single ESB. This colocalization is reversible in the absence of drug selection. This discovery that simultaneously active ESs dynamically share a single ESB demonstrates the importance of this unique subnuclear body in restricting the monoallelic expression of VSG.
Ooi C-P, Smith TK, Gluenz E, et al., 2018, Blocking variant surface glycoprotein synthesis alters ERES/ Golgi homeostasis in Trypanosoma brucei, Traffic, Vol: 19, Pages: 391-405, ISSN: 1398-9219
The predominant secretory cargo of bloodstream form Trypanosoma brucei is Variant Surface Glycoprotein (VSG), comprising ~10% total protein and forming a dense protective layer. Blocking VSG translation using Morpholino oligonucleotides triggered a precise pre/cytokinesis arrest. We investigated the effect of blocking VSG synthesis on the secretory pathway. The number of Golgi decreased, particularly in post/mitotic cells, from 3.5 ± 0.6 to 2.0 ± 0.04 per cell. Similarly, the number of ER exit sites (ERES) in post/mitotic cells dropped from (3.9 ± 0.6) to (2.7 ± 0.1) eight hours after blocking VSG synthesis. The secretory pathway was still functional in these stalled cells, as monitored using Cathepsin L. Rates of phospholipid and GPI/anchor biosynthesis were relatively unaffected, except for the level of sphingomyelin which increased. However, both ER and Golgi morphology became distorted, with the Golgi cisternae becoming significantly dilated, particularly at the trans/face. Membrane accumulation in these structures is possibly caused by reduced budding of nascent vesicles due to the drastic reduction in the total amount of secretory cargo, i.e. VSG. These data argue that the total flux of secretory cargo impacts upon the biogenesis and maintenance of secretory structures and organelles in T. brucei including the ERES and Golgi.
Ooi C-P, Rudenko G, 2017, How to create coats for all seasons: elucidating antigenic variation in African trypanosomes, Emerging Topics in Life Sciences, Vol: 1, Pages: 593-600, ISSN: 2397-8562
Extracellular parasites of the mammalian bloodstream face considerable challenges including incessant assault by the immune system. African trypanosomes are consummate survivors in this inclement environment and are renowned for their supremely sophisticated strategy of antigenic variation of their protective surface coat during the course of chronic infections. Recent developments are making us realize how complex this antigenic machinery is and are allowing us to tackle previously intractable problems. However, many of the simplest (and arguably the most important) questions still remain unanswered!
Ridewood S, Ooi CP, Hall B, et al., 2017, The role of genomic location and flanking 3’UTR in the generation of functional levels of Variant Surface Glycoprotein in Trypanosoma brucei, Molecular Microbiology, Vol: 106, Pages: 614-634, ISSN: 0950-382X
Trypanosoma brucei faces relentless immune attack in the mammalian bloodstream, where it is protected by an essential coat of Variant Surface Glycoprotein (VSG) comprising ∼10% total protein. The active VSG gene is in a Pol I-transcribed telomeric expression site (ES). We investigated factors mediating these extremely high levels of VSG expression by inserting ectopic VSG117 into VSG221 expressing T. brucei. Mutational analysis of the ectopic VSG 3′UTR demonstrated the essentiality of a conserved 16-mer for mRNA stability. Expressing ectopic VSG117 from different genomic locations showed that functional VSG levels could be produced from a gene 60 kb upstream of its normal telomeric location. High, but very heterogeneous levels of VSG117 were obtained from the Pol I-transcribed rDNA. Blocking VSG synthesis normally triggers a precise precytokinesis cell-cycle checkpoint. VSG117 expression from the rDNA was not adequate for functional complementation, and the stalled cells arrested prior to cytokinesis. However, VSG levels were not consistently low enough to trigger a characteristic ‘VSG synthesis block’ cell-cycle checkpoint, as some cells reinitiated S phase. This demonstrates the essentiality of a Pol I-transcribed ES, as well as conserved VSG 3′UTR 16-mer sequences for the generation of functional levels of VSG expression in bloodstream form T. brucei.
Kerry LE, Pegg EE, Cameron DP, et al., 2017, Selective inhibition of RNA polymerase I transcription as a potential approach to treat African trypanosomiasis., PLOS Neglected Tropical Diseases, Vol: 11, ISSN: 1935-2735
Trypanosoma brucei relies on an essential Variant Surface Glycoprotein (VSG) coat for survival in the mammalian bloodstream. High VSG expression within an expression site body (ESB) is mediated by RNA polymerase I (Pol I), which in other eukaryotes exclusively transcribes ribosomal RNA genes (rDNA). As T. brucei is reliant on Pol I for VSG transcription, we investigated Pol I transcription inhibitors for selective anti-trypanosomal activity. The Pol I inhibitors quarfloxin (CX-3543), CX-5461, and BMH-21 are currently under investigation for treating cancer, as rapidly dividing cancer cells are particularly dependent on high levels of Pol I transcription compared with nontransformed cells. In T. brucei all three Pol I inhibitors have IC50 concentrations for cell proliferation in the nanomolar range: quarfloxin (155 nM), CX-5461 (279 nM) or BMH-21 (134 nM) compared with IC50 concentrations in the MCF10A human breast epithelial cell line (4.44 μM, 6.89 μM or 460 nM, respectively). T. brucei was therefore 29-fold more sensitive to quarfloxin, 25-fold more sensitive to CX-5461 and 3.4-fold more sensitive to BMH-21. Cell death in T. brucei was due to rapid inhibition of Pol I transcription, as within 15 minutes treatment with the inhibitors rRNA precursor transcript was reduced 97-98% and VSG precursor transcript 91-94%. Incubation with Pol I transcription inhibitors also resulted in disintegration of the ESB as well as the nucleolus subnuclear structures, within one hour. Rapid ESB loss following the block in Pol I transcription argues that the ESB is a Pol I transcription nucleated structure, similar to the nucleolus. In addition to providing insight into Pol I transcription and ES control, Pol I transcription inhibitors potentially also provide new approaches to treat trypanosomiasis.
Maree JP, Povelones ML, Clark DJ, et al., 2017, Well-positioned nucleosomes punctuate polycistronic pol II transcription units and flank silent VSG gene arrays in Trypanosoma brucei, EPIGENETICS & CHROMATIN, Vol: 10, ISSN: 1756-8935
Background:The compaction of DNA in chromatin in eukaryotes allowed the expansion of genome size and coincided with significant evolutionary diversification. However, chromatin generally represses DNA function, and mechanisms coevolved to regulate chromatin structure and its impact on DNA. This included the selection of specific nucleosome positions to modulate accessibility to the DNA molecule. Trypanosoma brucei, a member of the Excavates supergroup, falls in an ancient evolutionary branch of eukaryotes and provides valuable insight into the organization of chromatin in early genomes.Results:We have mapped nucleosome positions in T. brucei and identified important differences compared to other eukaryotes: The RNA polymerase II initiation regions in T. brucei do not exhibit pronounced nucleosome depletion, and show little evidence for defined −1 and +1 nucleosomes. In contrast, a well-positioned nucleosome is present directly on the splice acceptor sites within the polycistronic transcription units. The RNA polyadenylation sites were depleted of nucleosomes, with a single well-positioned nucleosome present immediately downstream of the predicted sites. The regions flanking the silent variant surface glycoprotein (VSG) gene cassettes showed extensive arrays of well-positioned nucleosomes, which may repress cryptic transcription initiation. The silent VSG genes themselves exhibited a less regular nucleosomal pattern in both bloodstream and procyclic form trypanosomes. The DNA replication origins, when present within silent VSG gene cassettes, displayed a defined nucleosomal organization compared with replication origins in other chromosomal core regions.Conclusions:Our results indicate that some organizational features of chromatin are evolutionarily ancient, and may already have been present in the last eukaryotic common ancestor.
Cheung JL, Wand NV, Ooi CP, et al., 2016, Blocking synthesis of the Variant Surface Glycoprotein Coat in Trypanosoma brucei leads to an Increase in macrophage phagocytosis due to reduced clearance of surface coat antibodies, Plos Pathogens, Vol: 12, ISSN: 1553-7374
The extracellular bloodstream form parasite Trypanosoma brucei is supremely adapted to escape the host innate and adaptive immune system. Evasion is mediated through an antigenically variable Variant Surface Glycoprotein (VSG) coat, which is recycled at extraordinarily high rates. Blocking VSG synthesis triggers a precytokinesis arrest where stalled cells persist for days in vitro with superficially intact VSG coats, but are rapidly cleared within hours in mice. We therefore investigated the role of VSG synthesis in trypanosome phagocytosis by activated mouse macrophages. T. brucei normally effectively evades macrophages, and induction of VSG RNAi resulted in little change in phagocytosis of the arrested cells. Halting VSG synthesis resulted in stalled cells which swam directionally rather than tumbling, with a significant increase in swim velocity. This is possibly a consequence of increased rigidity of the cells due to a restricted surface coat in the absence of VSG synthesis. However if VSG RNAi was induced in the presence of anti-VSG221 antibodies, phagocytosis increased significantly. Blocking VSG synthesis resulted in reduced clearance of anti-VSG antibodies from the trypanosome surface, possibly as a consequence of the changed motility. This was particularly marked in cells in the G2/ M cell cycle stage, where the half-life of anti-VSG antibody increased from 39.3 ± 4.2 seconds to 99.2 ± 15.9 seconds after induction of VSG RNAi. The rates of internalisation of bulk surface VSG, or endocytic markers like transferrin, tomato lectin or dextran were not significantly affected by the VSG synthesis block. Efficient elimination of anti-VSG-antibody complexes from the trypanosome cell surface is therefore essential for trypanosome evasion of macrophages. These experiments highlight the essentiality of high rates of VSG recycling for the rapid removal of host opsonins from the parasite surface, and identify this process as a key parasite virulence factor
Stanne TM, Narayanan MS, Ridewood S, et al., 2015, Identification of the ISWI chromatin remodeling complex of the early branching eukaryote Trypanosoma brucei (vol 290, pg 26954, 2015), Journal of Biological Chemistry, Vol: 290, Pages: 29754-26967, ISSN: 1083-351X
Background: Eukaryotes typically encode a range of ISWI chromatin remodeling complexes with different functions.Results: We have identified and analyzed three novel ISWI partners in the early branching eukaryote Trypanosoma brucei.Conclusion: T. brucei appears to have a single major ISWI complex.Significance: This unusually simple ISWI configuration could be a consequence of the relative lack of transcriptional regulationin this ancient eukaryote
Stanne T, Narayanan MS, Ridewood S, et al., 2015, Identification of the ISWI chromatin remodeling complex of the early branching eukaryote Trypanosoma brucei, Journal of Biological Chemistry, Vol: 290, Pages: 26954-26967, ISSN: 0021-9258
ISWI chromatin remodelers are highly conserved ineukaryotes and are important for the assembly and spacing ofnucleosomes, thereby controlling transcription initiation andelongation. ISWI is typically associated with different subunits,forming specialized complexes with discrete functions. In the unicellularparasite Trypanosoma brucei, which causes African sleepingsickness, TbISWI down-regulates RNA polymerase I (PolI)-transcribed variant surface glycoprotein (VSG) gene expressionsites (ESs), which are monoallelically expressed. Here, we use tandemaffinity purification to determine the interacting partners ofTbISWI. We identify three proteins that do not show significanthomology with known ISWI-associated partners. Surprisingly, oneof these is nucleoplasmin-like protein (NLP), which we had previouslyshown to play a rolein ES control. In addition, weidentify twonovel ISWI partners, regulator of chromosome condensation1-like protein (RCCP) and phenylalanine/tyrosine-rich protein(FYRP), both containing proteinmotifs typically found on chromatinproteins. Knockdown of RCCP or FYRP in bloodstream formT. brucei results in derepression of silent variant surface glycoproteinESs, as had previously been shown for TbISWI and NLP. Allfour proteins are expressed and interact with each other in bothmajor life cycle stages and show similar distributions at Pol I-transcribedloci. They are also found at Pol II strand switch regions asdetermined with ChIP. ISWI, NLP, RCCP, and FYRP thereforeappear to form a single major ISWI complex in T. brucei (TbIC).This reduced complexity of ISWI regulation and the presence ofnovel ISWI partners highlights the early divergence of trypanosomesin evolution.
Denninger V, Rudenko G, 2014, FACT plays a major role in histone dynamics affecting VSG expression site control in Trypanosoma brucei, MOLECULAR MICROBIOLOGY, Vol: 94, Pages: 945-962, ISSN: 0950-382X
Ooi C-P, Rudenko G, 2014, Do trypanosome turncoats wait before they commit?, eLife, Vol: 3, ISSN: 2050-084X
The strategy that sleeping sickness parasites use to evade the mammalian immune system may be linked to the metamorphosis that allows them to transfer from mammals into tsetse flies.
Denninger V, Fullbrook A, Bessat M, et al., 2014, The FACT subunit TbSpt16 is involved in cell cycle specific control of VSG expression sites in Trypanosoma brucei, MOLECULAR MICROBIOLOGY, Vol: 92, Pages: 901-901, ISSN: 0950-382X
Narayanan MS, Rudenko G, 2013, TDP1 is an HMG chromatin protein facilitating RNA polymerase I transcription in African trypanosomes, NUCLEIC ACIDS RESEARCH, Vol: 41, Pages: 2981-2992, ISSN: 0305-1048
Povelones ML, Gluenz E, Dembek M, et al., 2012, Histone H1 Plays a Role in Heterochromatin Formation and VSG Expression Site Silencing in Trypanosoma brucei, PLOS PATHOGENS, Vol: 8, ISSN: 1553-7374
Vink C, Rudenko G, Seifert HS, 2012, Microbial antigenic variation mediated by homologous DNA recombination, FEMS MICROBIOLOGY REVIEWS, Vol: 36, Pages: 917-948, ISSN: 0168-6445
Rudenko G, 2012, High-throughput whole genome analysis provides insight into how the major drugs against African sleeping sickness operate, PATHOGENS AND GLOBAL HEALTH, Vol: 106, Pages: 79-79, ISSN: 2047-7724
Stanne TM, Kushwaha M, Wand M, et al., 2011, TbISWI Regulates Multiple Polymerase I (Pol I)-Transcribed Loci and Is Present at Pol II Transcription Boundaries in Trypanosoma brucei, EUKARYOTIC CELL, Vol: 10, Pages: 964-976, ISSN: 1535-9778
Narayanan MS, Kushwaha M, Ersfeld K, et al., 2011, NLP is a novel transcription regulator involved in VSG expression site control in Trypanosoma brucei, NUCLEIC ACIDS RESEARCH, Vol: 39, Pages: 2018-2031, ISSN: 0305-1048
Rudenko G, 2011, African trypanosomes: the genome and adaptations for immune evasion, ESSAYS IN BIOCHEMISTRY: MOLECULAR PARASITOLOGY, Vol: 51, Pages: 47-62, ISSN: 0071-1365
Denninger V, Fullbrook A, Bessat M, et al., 2010, The FACT subunit TbSpt16 is involved in cell cycle specific control of VSG expression sites in Trypanosoma brucei, MOLECULAR MICROBIOLOGY, Vol: 78, Pages: 459-474, ISSN: 0950-382X
Stanne TM, Rudenko G, 2010, Active VSG Expression Sites in Trypanosoma brucei Are Depleted of Nucleosomes, EUKARYOTIC CELL, Vol: 9, Pages: 136-147, ISSN: 1535-9778
Rudenko G, 2010, Epigenetics and transcriptional control in African trypanosomes, ESSAYS IN BIOCHEMISTRY: EPIGENETICS, DISEASE AND BEHAVIOUR, Vol: 48, Pages: 201-219, ISSN: 0071-1365
Smith TK, Vasileva N, Gluenz E, et al., 2009, Blocking Variant Surface Glycoprotein Synthesis in Trypanosoma brucei Triggers a General Arrest in Translation Initiation, PLOS ONE, Vol: 4, ISSN: 1932-6203
Hertz-Fowler C, Figueiredo LM, Quail MA, et al., 2008, Telomeric expression sites are highly conserved in trypanosoma brucei, PLoS ONE, Vol: 3, ISSN: 1932-6203
Subtelomeric regions are often under-represented in genome sequences of eukaryotes. One of the best known examples of theuse of telomere proximity for adaptive purposes are the bloodstream expression sites (BESs) of the African trypanosomeTrypanosoma brucei. To enhance our understanding of BES structure and function in host adaptation and immune evasion, theBES repertoire from the Lister 427 strain ofT. bruceiwere independently tagged and sequenced. BESs are polymorphic in sizeand structure but reveal a surprisingly conserved architecture in the context of extensive recombination. Very small BESs doexist and many functioning BESs do not contain the full complement of expression site associated genes (ESAGs). Theconsequences of duplicated or missingESAGs, includingESAG9,anewlynamedESAG12, and additional variant surfaceglycoprotein genes (VSGs) were evaluated by functional assays after BESs were tagged with a drug-resistance gene.Phylogenetic analysis of constituentESAGfamilies suggests that BESs are sequence mosaics and that extensive recombinationhas shaped the evolution of the BES repertoire. This work opens important perspectives in understanding the molecularmechanisms of antigenic variation, a widely used strategy for immune evasion in pathogens, and telomere biology.
Young R, Taylor JE, Kurioka A, et al., 2008, Isolation and analysis of the genetic diversity of repertoires of VSG expression site containing telomeres from Trypanosoma brucei gambiense, T. b. brucei and T. equiperdum, BMC Genomics, Vol: 9, ISSN: 1471-2164
BackgroundAfrican trypanosomes (including Trypanosoma brucei) are unicellular parasites which multiply in the mammalian bloodstream. T. brucei has about twenty telomeric bloodstream form Variant Surface Glycoprotein (VSG) expression sites (BESs), of which one is expressed at a time in a mutually exclusive fashion. BESs are polycistronic transcription units, containing a variety of families of expression site associated genes (ESAG s) in addition to the telomeric VSG. These polymorphic ESAG families are thought to play a role in parasite-host adaptation, and it has been proposed that ESAG diversity might be related to host range. Analysis of the genetic diversity of these telomeric gene families has been confounded by the underrepresentation of telomeric sequences in standard libraries. We have previously developed a method to selectively isolate sets of trypanosome BES containing telomeres using Transformation associated recombination (TAR) cloning in yeast.ResultsHere we describe the isolation of repertoires of BES containing telomeres from three trypanosome subspecies: Trypanosoma brucei gambiense DAL 972 (causative agent of West-African trypanosomiasis), T. b. brucei EATRO 2340 (a nonhuman infective strain) and T. equiperdum STIB 818 (which causes a sexually transmitted disease in equines). We have sequenced and analysed the genetic diversity at four BES loci (BES promoter region, ESAG6, ESAG5 and ESAG2) from these three trypanosome BES repertoires.ConclusionWith the exception of ESAG2, the BES sequence repertoires derived from T. b. gambiense are both less diverse than and nearly reciprocally monophyletic relative to those from T. b. brucei and T. equiperdum. Furthermore, although we find evidence for adaptive evolution in all three ESAG repertoires in T. b. brucei and T. equiperdum, only ESAG2 appears to be under diversifying selection in T. b. gambiense. This low level of variation in the T. b. gambiense BES sequence repertoires is consistent both with the rel
Hughes K, Wand M, Foulston L, et al., 2007, A novel ISWI is involved in VSG expression site downregulation in African trypanosomes, EMBO JOURNAL, Vol: 26, Pages: 2400-2410, ISSN: 0261-4189
Taylor JE, Rudenko G, 2006, Switching trypanosome coats: what's in the wardrobe?, TRENDS IN GENETICS, Vol: 22, Pages: 614-620, ISSN: 0168-9525
Rudenko G, 2005, Maintaining the protective variant surface glycoprotein coat of African trypanosomes, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 33, Pages: 981-982, ISSN: 0300-5127
Aitcheson N, Talbot S, Shapiro J, et al., 2005, VSG switching in Trypanosoma brucei: antigenic variation analysed using RNAi in the absence of immune selection, MOLECULAR MICROBIOLOGY, Vol: 57, Pages: 1608-1622, ISSN: 0950-382X
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