scholarly journals Monoallelic antigen expression in trypanosomes requires a stage-specific transcription activator

2021 ◽  
Author(s):  
Lara Lopez Escobar ◽  
Benjamin Hanisch ◽  
Clare Halliday ◽  
Samuel Dean ◽  
Jack Sunter ◽  
...  
Keyword(s):  
Antigenic Variation ◽  
Single Gene ◽  
Antigen Expression ◽  
Specific Protein ◽  
Surface Glycoprotein ◽  
Monoallelic Expression ◽  
Transcription Activator ◽  
Transcript Processing ◽  
Pol I ◽  
Expression Site

Monoallelic expression of a single gene family member underpins a molecular “arms race” between many pathogens and their host, through host monoallelic immunoglobulin and pathogen monoallelic antigen expression. In Trypanosoma brucei, a single, abundant, variant surface glycoprotein (VSG) covers the entire surface of the bloodstream parasite and monoallelic VSG transcription underpins their archetypal example of antigenic variation. It is vital for pathogenicity, only occurring in mammalian infectious forms. Transcription of one VSG gene is achieved by RNA polymerase I (Pol I) in a singular nuclear structure: the expression site body (ESB). How monoallelic expression of the single VSG is achieved is incompletely understood and no specific ESB components are known. Here, using a protein localisation screen in bloodstream parasites, we discovered the first ESB-specific protein: ESB1. It is specific to VSG-expressing life cycle stages where it is necessary for VSG expression, and its overexpression activates inactive VSG promoters. This showed monoallelic VSG transcription requires a stage-specific activator. Furthermore, ESB1 is necessary for Pol I recruitment to the ESB, however transcript processing and inactive VSG gene exclusion ESB sub-domains do not require ESB1. This shows that the cellular solution for monoallelic transcription is a complex factory of functionally distinct and separably assembled sub-domains.

scholarly journals Dynamic colocalization of 2 simultaneously active VSG expression sites within a single expression-site body in Trypanosoma brucei

2019 ◽  
Vol 116 (33) ◽  
pp. 16561-16570 ◽  
Author(s):  
James Budzak ◽  
Louise E. Kerry ◽  
Aris Aristodemou ◽  
Belinda S. Hall ◽  
Kathrin Witmer ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Chromatin Immunoprecipitation ◽  
Surface Glycoprotein ◽  
Monoallelic Expression ◽  
Drug Selection ◽  
Wild Type ◽  
Pol I ◽  
Dna Base ◽  
Expression Site ◽  
Polymerase I

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.

scholarly journals Quantification of RNA Polymerase I transcriptional attenuation at the active VSG expression site in Trypanosoma brucei

2021 ◽  
Author(s):  
Nadine Weisert ◽  
Klara Thein ◽  
Helena Reis ◽  
Christian J Janzen
Keyword(s):  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Rna Polymerase I ◽  
Surface Glycoprotein ◽  
Transcriptional Elongation ◽  
Pol I ◽  
Expression Site ◽  
Polymerase I ◽  
Large Repertoire

The cell surface of the extracellular pathogen Trypanosoma brucei consists of a dense coat of variant surface glycoprotein (VSG), which enables the parasite to evade the immune system of the vertebrate host. Only one VSG gene from a large repertoire is expressed from a so-called bloodstream form expression site (BES) at a given timepoint. There are several BES in every parasite but only one is transcriptionally active. Other BES are silenced by transcriptional attenuation. Periodic activation of a previously-silenced BES results in differential VSG transcription and escape from the immune response. A process called antigenic variation. In contrast to gene transcription in other eukaryotes, the BES is transcribed by RNA polymerase I (Pol I). It was proposed that this highly-processive polymerase is needed to provide a sufficiently high transcription rate at the VSG gene. Surprisingly, we discovered a position-dependent Pol I activity and attenuation of transcriptional elongation also at the active BES. Transcription rates at the VSG gene appear to be comparable to Pol II-mediated transcription of house-keeping genes. Although these findings are in contradiction to the long-standing concept of continuously high transcription rates at the active BES in Trypanosoma brucei, they are complementary to recent groundbreaking findings about transcriptional regulation of VSG genes.

scholarly journals Escaping the immune system by DNA repair and recombination in African trypanosomes

Open Biology ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 190182 ◽  
Author(s):  
Núria Sima ◽  
Emilia Jane McLaughlin ◽  
Sebastian Hutchinson ◽  
Lucy Glover
Keyword(s):  
Immune Response ◽  
Dna Repair ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Surface Coat ◽  
African Trypanosomes ◽  
Active Expression ◽  
Expression Site

African trypanosomes escape the mammalian immune response by antigenic variation—the periodic exchange of one surface coat protein, in Trypanosoma brucei the variant surface glycoprotein (VSG), for an immunologically distinct one. VSG transcription is monoallelic, with only one VSG being expressed at a time from a specialized locus, known as an expression site. VSG switching is a predominantly recombination-driven process that allows VSG sequences to be recombined into the active expression site either replacing the currently active VSG or generating a ‘new’ VSG by segmental gene conversion. In this review, we describe what is known about the factors that influence this process, focusing specifically on DNA repair and recombination.

Identification of potential protein partners that bind to the variant surface glycoprotein in Trypanosoma equiperdum

Parasitology ◽  
2017 ◽  
Vol 144 (7) ◽  
pp. 923-936 ◽  
Author(s):  
LIOMARY M. CARRASQUEL ◽  
JOSÉ L. ESCALONA ◽  
ALVARO ACOSTA-SERRANO ◽  
YURONG GUO ◽  
JOSÉ BUBIS
Keyword(s):  
Antigenic Variation ◽  
Orthologous Gene ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Western Blots ◽  
Binding Partners ◽  
Protein Partners ◽  
Expression Site ◽  
Tryparedoxin Peroxidase ◽  
Trypanosoma Equiperdum

SUMMARYTrypanosoma equiperdum possesses a dense coat of a variant surface glycoprotein (VSG) that is used to evade the host immune response by a process known as antigenic variation. Soluble and membrane forms of the predominant VSG from the Venezuelan T. equiperdum TeAp-N/D1 strain (sVSG and mVSG, respectively) were purified to homogeneity; and antibodies against sVSG and mVSG were raised, isolated, and employed to produce anti-idiotypic antibodies that structurally mimic the VSG surface. Prospective VSG-binding partners were initially detected by far-Western blots, and then by immunoblots using the generated anti-idiotypic antibodies. Polypeptides of ~80 and 55 kDa were isolated when anti-idiotypic antibodies–Sepharose affinity matrixes were used as baits. Mass spectrometry sequencing yielded hits with various proteins from Trypanosoma brucei such as heat-shock protein 70, tryparedoxin peroxidase, VSG variants, expression site associated gene product 6, and two hypothetical proteins. In addition, a possible interaction with a protein homologous to the glutamic acid/alanine-rich protein from Trypanosoma congolense was also found. These results indicate that the corresponding orthologous gene products are candidates for VSG-interacting proteins in T. equiperdum.

scholarly journals Expression Pattern of the Pneumocystis jirovecii Major Surface Glycoprotein Superfamily in Patients with Pneumonia

2020 ◽  
Author(s):  
Emanuel Schmid-Siegert ◽  
Sophie Richard ◽  
Amanda Luraschi ◽  
Konrad Mühlethaler ◽  
Marco Pagni ◽  
...  
Keyword(s):  
Antigenic Variation ◽  
Single Gene ◽  
Host Cells ◽  
Surface Glycoprotein ◽  
Pneumocystis Jirovecii ◽  
Clinical Samples ◽  
Single Nucleotide Variants ◽  
Rna Sequences ◽  
Major Surface ◽  
Cell Surface Structure

Abstract Background The human pathogen Pneumocystis jirovecii harbors 6 families of major surface glycoproteins (MSGs) encoded by a single gene superfamily. MSGs are presumably responsible for antigenic variation and adhesion to host cells. The genomic organization suggests that a single member of family I is expressed at a given time per cell, whereas members of the other families are simultaneously expressed. Methods We analyzed RNA sequences expressed in several clinical samples, using specific weighted profiles for sorting of reads and calling of single-nucleotide variants to estimate the diversity of the expressed genes. Results A number of different isoforms of at least 4 MSG families were expressed simultaneously, including isoforms of family I, for which confirmation was obtained in the wet laboratory. Conclusion These observations suggest that every single P. jirovecii population is made of individual cells with distinct surface properties. Our results enhance our understanding of the unique antigenic variation system and cell surface structure of P. jirovecii.

scholarly journals Frequent Loss of the Active Site during Variant Surface Glycoprotein Expression Site Switching In Vitro inTrypanosoma brucei

1998 ◽  
Vol 18 (1) ◽  
pp. 198-205 ◽  
Author(s):  
Mike Cross ◽  
Martin C. Taylor ◽  
Piet Borst
Keyword(s):  
Active Site ◽  
Antigenic Variation ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Selection System ◽  
African Trypanosomes ◽  
Expression Site ◽  
Gene Switching

ABSTRACT African trypanosomes undergo antigenic variation of their variant surface glycoprotein (VSG) coat to avoid being killed by their mammalian hosts. The active VSG gene is located in one of many telomeric expression sites. Replacement of the VSG gene in the active site or switching between expression sites can give rise to a new VSG coat. To study Trypanosoma brucei VSG expression site inactivation rather than VSG gene switching, it is useful to have an in vitro negative-selection system independent of the VSG. We have achieved this aim by using a viral thymidine kinase (TK) gene. Following integration of the TK gene downstream of the 221a VSG expression site promoter, transformant cell lines became sensitive to the nucleoside analog 1-(2-deoxy-2-fluoro-8-d-arabinofuranosyl)-5-iodouracil. These TK trypanosomes were able to revert to resistance at a rate approaching 10−5 per cell per generation. The majority of revertants expressed a new VSG gene even though there had been no selection against the VSG itself. Analysis of these switched variants showed that some had shut down TK expression via an in situ expression site switch. However, most variants had the complete 221 expression site deleted and another VSG expression site activated. We speculate that a new VSG expression site cannot switch on without inactivation of the old site.

scholarly journals Cohesin regulates VSG monoallelic expression in trypanosomes

2009 ◽  
Vol 186 (2) ◽  
pp. 243-254 ◽  
Author(s):  
David Landeira ◽  
Jean-Mathieu Bart ◽  
Daria Van Tyne ◽  
Miguel Navarro
Keyword(s):  
Antigenic Variation ◽  
Epigenetic Inheritance ◽  
Surface Glycoprotein ◽  
Monoallelic Expression ◽  
Cohesin Complex ◽  
Sister Chromatids ◽  
Nuclear Site ◽  
Chromatid Cohesion ◽  
Polymerase I ◽  
Transcriptional State

Antigenic variation allows Trypanosoma brucei to evade the host immune response by switching the expression of 1 out of ∼15 telomeric variant surface glycoprotein (VSG) expression sites (ESs). VSG ES transcription is mediated by RNA polymerase I in a discrete nuclear site named the ES body (ESB). However, nothing is known about how the monoallelic VSG ES transcriptional state is maintained over generations. In this study, we show that during S and G2 phases and early mitosis, the active VSG ES locus remains associated with the single ESB and exhibits a delay in the separation of sister chromatids relative to control loci. This delay is dependent on the cohesin complex, as partial knockdown of cohesin subunits resulted in premature separation of sister chromatids of the active VSG ES. Cohesin depletion also prompted transcriptional switching from the active to previously inactive VSG ESs. Thus, in addition to maintaining sister chromatid cohesion during mitosis, the cohesin complex plays an essential role in the correct epigenetic inheritance of the active transcriptional VSG ES state.

scholarly journals Genetics of Surface Antigen Expression inPneumocystis carinii

2001 ◽  
Vol 69 (2) ◽  
pp. 627-639 ◽  
Author(s):  
James R. Stringer ◽  
Scott P. Keely
Keyword(s):  
Antigenic Variation ◽  
Molecular Genetic ◽  
Pneumocystis Carinii ◽  
Antigen Expression ◽  
Surface Glycoprotein ◽  
Major Surface Glycoprotein ◽  
Molecular Genetic Data ◽  
Surface Variation ◽  
Surface Antigen Expression ◽  
Major Surface

ABSTRACT This article reviews the molecular genetic data pertaining to the major surface glycoprotein (MSG) gene family of Pneumocystis carinii and its role in surface variation and compares this fungal system to antigenic variation systems in the protozoanTrypanosoma brucei and the bacteriaBorrelia spp.

scholarly journals Regulation of Antigenic Variation by Trypanosoma brucei Telomere Proteins Depends on Their Unique DNA Binding Activities

Pathogens ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 967
Author(s):  
Bibo Li ◽  
Yanxiang Zhao
Keyword(s):  
Nucleic Acid ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Dna Recombination ◽  
Surface Glycoprotein ◽  
Monoallelic Expression ◽  
Nucleic Acid Binding ◽  
Major Surface ◽  
Telomere Structure

Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, Variant Surface Glycoprotein (VSG), to evade the host immune response. Such antigenic variation is a key pathogenesis mechanism that enables T. brucei to establish long-term infections. VSG is expressed exclusively from subtelomere loci in a strictly monoallelic manner, and DNA recombination is an important VSG switching pathway. The integrity of telomere and subtelomere structure, maintained by multiple telomere proteins, is essential for T. brucei viability and for regulating the monoallelic VSG expression and VSG switching. Here we will focus on T. brucei TRF and RAP1, two telomere proteins with unique nucleic acid binding activities, and summarize their functions in telomere integrity and stability, VSG switching, and monoallelic VSG expression. Targeting the unique features of TbTRF and TbRAP1′s nucleic acid binding activities to perturb the integrity of telomere structure and disrupt VSG monoallelic expression may serve as potential therapeutic strategy against T. brucei.

scholarly journals Expression site attenuation mechanistically links antigenic variation and development in Trypanosoma brucei

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Christopher Batram ◽  
Nicola G Jones ◽  
Christian J Janzen ◽  
Sebastian M Markert ◽  
Markus Engstler
Keyword(s):  
Trypanosoma Brucei ◽  
Histone Methylation ◽  
Antigenic Variation ◽  
Tsetse Fly ◽  
Developmental Competence ◽  
Surface Glycoprotein ◽  
G1 Phase ◽  
African Trypanosomes ◽  
Developmental Progression ◽  
Expression Site

We have discovered a new mechanism of monoallelic gene expression that links antigenic variation, cell cycle, and development in the model parasite Trypanosoma brucei. African trypanosomes possess hundreds of variant surface glycoprotein (VSG) genes, but only one is expressed from a telomeric expression site (ES) at any given time. We found that the expression of a second VSG alone is sufficient to silence the active VSG gene and directionally attenuate the ES by disruptor of telomeric silencing-1B (DOT1B)-mediated histone methylation. Three conserved expression-site-associated genes (ESAGs) appear to serve as signal for ES attenuation. Their depletion causes G1-phase dormancy and reversible initiation of the slender-to-stumpy differentiation pathway. ES-attenuated slender bloodstream trypanosomes gain full developmental competence for transformation to the tsetse fly stage. This surprising connection between antigenic variation and developmental progression provides an unexpected point of attack against the deadly sleeping sickness.

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