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 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 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 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.

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 Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes

2015 ◽  
Vol 112 (21) ◽  
pp. E2803-E2812 ◽  
Author(s):  
Igor Cestari ◽  
Ken Stuart
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Inositol Phosphate ◽  
Rna Polymerase I ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Activator Protein 1 ◽  
African Trypanosomes ◽  
Polymerase I

African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing.

scholarly journals Experimental production of anti-membrane bound variable surface glycoglycoprotein (anti-VSGm) antibodies for in vitro and in situ immunostaining of pathogenic African trypanosomes

2020 ◽  
Author(s):  
Reymick Okwong-Oketch ◽  
Julius Nsubuga ◽  
Peter Ayebare ◽  
Zachary Nsadha ◽  
George William Lubega ◽  
...  
Keyword(s):  
Culture Media ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Homologous Region ◽  
Experimental Production ◽  
Artificial Culture ◽  
African Trypanosomes ◽  
Variable Surface ◽  
Membrane Bound

AbstractBackgroundThe variant surface glycoprotein (VSG) of the African trypanosomes is the major membrane protein of the plasma membrane of the bloodstream stage of the parasite. African trypanosomiasis (sleeping sickness in humans and nagana in animals) is caused by the systemic infection of the host by several sub-species of the extracellular haemoflagellate protozoa under genus Trypanosoma. As a defense barrier against the host immune response, the entire surface of the bloodstream form of trypanosome is covered with densely packed molecules of VSG that determines the antigenic phenotype of the parasite. Variant surface glycoprotein has a C-terminal domain that is highly conserved in various species of trypanosomes.MethodsThe membrane bound VSG (VSGm) protein was prepared without denaturing the homologous region and by including numerous variable antigen types from Trypanosoma brucei brucei parasites. The purified VSGm native trypanosome protein was used to produce anti-VSGm immune sera in rabbits. The indirect immunofluorescence assay (IFA) was used to detect trypanosomes from mice blood, artificial culture media and cattle histological sections.ResultsThe resultant immune sera were able to detect different strains and species of African trypanosomes from in vivo and in situ sources after immunostaining. Anti-VSGm antibodies also demonstrated a unique property to locate trypanosomes within the histological tissues even after the trypanosome’s morphology had been distorted.ConclusionThe produced immune sera can be utilized for immunohistochemistry to detect Trypanosoma species in various fluids and tissues.Author summary

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.

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