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 Targeting the Variable Surface of African Trypanosomes with Variant Surface Glycoprotein-Specific, Serum-Stable RNA Aptamers

2003 ◽  
Vol 2 (1) ◽  
pp. 84-94 ◽  
Author(s):  
Mihaela Lorger ◽  
Markus Engstler ◽  
Matthias Homann ◽  
H. Ulrich Göringer
Keyword(s):  
Antigenic Variation ◽  
Sleeping Sickness ◽  
Rna Aptamers ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Parasite Protein ◽  
African Trypanosomes ◽  
Variable Surface ◽  
New Strategy

ABSTRACT African trypanosomes cause sleeping sickness in humans and Nagana in cattle. The parasites multiply in the blood and escape the immune response of the infected host by antigenic variation. Antigenic variation is characterized by a periodic change of the parasite protein surface, which consists of a variant glycoprotein known as variant surface glycoprotein (VSG). Using a SELEX (systematic evolution of ligands by exponential enrichment) approach, we report the selection of small, serum-stable RNAs, so-called aptamers, that bind to VSGs with subnanomolar affinity. The RNAs are able to recognize different VSG variants and bind to the surface of live trypanosomes. Aptamers tethered to an antigenic side group are capable of directing antibodies to the surface of the parasite in vitro. In this manner, the RNAs might provide a new strategy for a therapeutic intervention to fight sleeping sickness.

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.

scholarly journals Gene conversions mediating antigenic variation in Trypanosoma brucei can occur in variant surface glycoprotein expression sites lacking 70-base-pair repeat sequences.

1997 ◽  
Vol 17 (2) ◽  
pp. 833-843 ◽  
Author(s):  
R McCulloch ◽  
G Rudenko ◽  
P Borst
Keyword(s):  
Gene Conversion ◽  
Antigenic Variation ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Mammalian Host ◽  
Recognition Sequence ◽  
African Trypanosomes ◽  
Repeat Sequences ◽  
Active Expression ◽  
Expression Site

African trypanosomes undergo antigenic variation of their variant surface glycoprotein (VSG) coat to avoid immune system-mediated killing by their mammalian host. An important mechanism for switching the expressed VSG gene is the duplicative transposition of a silent VSG gene into one of the telomeric VSG expression sites of the trypanosome, resulting in the replacement of the previously expressed VSG gene. This process appears to be a gene conversion reaction, and it has been postulated that sequences within the expression site may act to initiate and direct the reaction. All bloodstream form expression sites contain huge arrays (many kilobase pairs) of 70-bp repeat sequences that act as the 5' boundary of gene conversion reactions involving most silent VSG genes. For this reason, the 70-bp repeats seemed a likely candidate to be involved in the initiation of switching. Here, we show that deletion of the 70-bp repeats from the active expression site does not affect duplicative transposition of VSG genes from silent expression sites. We conclude that the 70-bp repeats do not appear to function as indispensable initiation sites for duplicative transposition and are unlikely to be the recognition sequence for a sequence-specific enzyme which initiates recombination-based VSG switching.

scholarly journals In Vitro Generation of Human High-Density-Lipoprotein-Resistant Trypanosoma brucei brucei

2006 ◽  
Vol 5 (8) ◽  
pp. 1276-1286 ◽  
Author(s):  
Sara D. Faulkner ◽  
Monika W. Oli ◽  
Rudo Kieft ◽  
Laura Cotlin ◽  
Justin Widener ◽  
...  
Keyword(s):  
High Density Lipoprotein ◽  
Trypanosoma Brucei ◽  
Density Lipoprotein ◽  
High Density ◽  
Surface Glycoprotein ◽  
Trypanosoma Brucei Brucei ◽  
African Trypanosomes ◽  
Expression Site ◽  
Human High Density Lipoprotein

ABSTRACT The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei.

scholarly journals Nuclear Phosphatidylinositol 5-Phosphatase Is Essential for Allelic Exclusion of Variant Surface Glycoprotein Genes in Trypanosomes

2018 ◽  
Vol 39 (3) ◽  
Author(s):  
Igor Cestari ◽  
Hilary McLeland-Wieser ◽  
Kenneth Stuart
Keyword(s):  
Antibody Response ◽  
Antigenic Variation ◽  
Chromatin Organization ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Allelic Exclusion ◽  
Activator Protein 1 ◽  
Multiprotein Complex ◽  
African Trypanosomes ◽  
Activator Protein

ABSTRACT Allelic exclusion of variant surface glycoprotein (VSG) genes is essential for African trypanosomes to evade the host antibody response by antigenic variation. The mechanisms by which this parasite expresses only one of its ∼2,000 VSG genes at a time are unknown. We show that nuclear phosphatidylinositol 5-phosphatase (PIP5Pase) interacts with repressor activator protein 1 (RAP1) in a multiprotein complex and functions in the control of VSG allelic exclusion. RAP1 binds PIP5Pase substrate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], and catalytic mutation of PIP5Pase that inhibits PI(3,4,5)P3 dephosphorylation results in simultaneous transcription of VSGs from all telomeric expression sites (ESs) and from silent subtelomeric VSG arrays. PIP5Pase and RAP1 bind to telomeric ESs, especially at 70-bp repeats and telomeres, and their binding is altered by PIP5Pase inactivation or knockdown, implying changes in ES chromatin organization. Our data suggest a model whereby PIP5Pase controls PI(3,4,5)P3 binding by RAP1 and, thus, RAP1 silencing of telomeric and subtelomeric VSG genes. Hence, allelic exclusion of VSG genes may entail control of nuclear phosphoinositides.

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.

What the genome sequence is revealing about trypanosome antigenic variation

2005 ◽  
Vol 33 (5) ◽  
pp. 986-989 ◽  
Author(s):  
J.D. Barry ◽  
L. Marcello ◽  
L.J. Morrison ◽  
A.F. Read ◽  
K. Lythgoe ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Humoral Immunity ◽  
Antigenic Variation ◽  
Genome Project ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Functional Genes ◽  
Gene Encoding ◽  
African Trypanosomes ◽  
Active Locus

African trypanosomes evade humoral immunity through antigenic variation, whereby they switch expression of the gene encoding their VSG (variant surface glycoprotein) coat. Switching proceeds by duplication of silent VSG genes into a transcriptionally active locus. The genome project has revealed that most of the silent archive consists of hundreds of subtelomeric VSG tandem arrays, and that most of these are not functional genes. Precedent suggests that they can contribute combinatorially to the formation of expressed, functional genes through segmental gene conversion. These findings from the genome project have major implications for evolution of the VSG archive and for transmission of the parasite in the field.

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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