Trypanosoma brucei Interaction with Host: Mechanism of VSG Release as Target for Drug Discovery for African Trypanosomiasis

The protozoan Trypanosoma brucei, responsible for animal and human trypanosomiasis, has a family of major surface proteases (MSPs) and phospholipase-C (PLC), both involved in some mechanisms of virulence during mammalian infections. During parasitism in the mammalian host, this protozoan is exclusively extracellular and presents a robust mechanism of antigenic variation that allows the persistence of infection. There has been incredible progress in our understanding of how variable surface glycoproteins (VSGs) are organised and expressed, and how expression is switched, particularly through recombination. The objective of this manuscript is to create a reflection about the mechanisms of antigenic variation in T. brucei, more specifically, in the process of variable surface glycoprotein (VSG) release. We firstly explore the mechanism of VSG release as a potential pathway and target for the development of anti-T. brucei drugs.

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Variable Surface Glycoprotein from Trypanosoma brucei Undergoes Cleavage by Matrix Metalloproteinases: An in silico Approach

Pathogens ◽

10.3390/pathogens8040178 ◽

2019 ◽

Vol 8 (4) ◽

pp. 178

Author(s):

Cláudia Jassica Gonçalves Moreno ◽

Taffarel Torres ◽

Marcelo Sousa Silva

Keyword(s):

Matrix Metalloproteinases ◽

Trypanosoma Brucei ◽

Cleavage Site ◽

Surface Protein ◽

Matrix Metalloproteases ◽

Surface Glycoprotein ◽

Mammalian Host ◽

Variable Surface Glycoprotein ◽

Biological Studies ◽

Variable Surface

In order to survive as extracellular parasites in the mammalian host environment, Trypanosoma brucei has developed efficient mechanisms of immune system evasion, which include the abundant expression of a variable surface glycoprotein (VSG) coat. VSGs are anchored in the parasite membrane by covalent C-terminal binding to glycosylphosphatidylinositol and may be periodically removed by a phospholipase C (PLC) and a major surface protein (TbMSP). VSG molecules show extraordinary antigenic diversity and a comparative analysis of protein sequences suggests that conserved elements may be a suitable target against African trypanosomiasis. However, the cleavage mechanisms of these molecules remain unclear. Moreover, in protozoan infections, including those caused by Trypanosoma brucei, it is possible to observe an increased expression of the matrix metalloproteinases (MMPs). To address the cleavage mechanism of VSGs, the PROSPER server was used for the identification of VSG sequence cleavage sites. After data compilation, it was observed that 64 VSG consensus sequences showed a high conservation of hydrophobic residues, such as valine (V), methionine (M), leucine (L) and isoleucine (I) in the fifth position—the exact location of the cleavage site. In addition, the PROSPER server identified conserved cleavage site portions of VSG proteins recognized by three matrix metalloproteases (gelatinases: MMP-2, MMP-3 and MMP-9). However, further biological studies are needed in order to analyze and confirm this prediction.

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Subcellular localization of a variable surface glycoprotein phosphatidylinositol-specific phospholipase-C in African trypanosomes.

The Journal of Cell Biology ◽

10.1083/jcb.105.2.737 ◽

1987 ◽

Vol 105 (2) ◽

pp. 737-746 ◽

Cited By ~ 33

Author(s):

D J Grab ◽

P Webster ◽

S Ito ◽

W R Fish ◽

Y Verjee ◽

...

Keyword(s):

Phospholipase C ◽

Trypanosoma Brucei ◽

Specific Activity ◽

Surface Glycoprotein ◽

Trypanosoma Vivax ◽

Trypanosome Species ◽

Phospholipase Activity ◽

African Trypanosomes ◽

Variable Surface Glycoprotein ◽

Variable Surface

African trypanosomes contain a membrane-bound enzyme capable of removing dimyristylglycerol from the membrane-attached form of the variable surface glycoprotein (mfVSG; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968). Although mfVSG phospholipase-C has been implicated in the removal of the VSG from the trypanosome surface (Cardoso de Almeida, M. L., and M. J. Turner, 1983, Nature (Lond.)., 302:349-352; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968), its precise function and subcellular location have not been determined. We have developed a procedure for the separation of the cell fractions and organelles of Trypanosoma brucei brucei (and other trypanosome species) by differential sucrose and isopycnic PercollR centrifugation. These fractions were tested for mfVSG phospholipase activity using Trypanosoma brucei mfVSG labeled with 3H-myristic acid as substrate. The highest enzyme-specific activity was associated with the flagella and evidence is presented to suggest that it is localized in the flagellar pocket. Some activity was also associated with the Golgi complex. These results suggest that the mfVSG phospholipase is localized primarily in the membrane of the flagella pocket and possibly other membrane organelles derived from and associated with this structure, and may be part of the VSG-membrane recycling system in African trypanosomes. The activity of mfVSG phospholipase amongst various trypanosome species was determined. We show that, in contrast to the bloodstream forms of Trypanosoma brucei, cultured procyclic Trypanosoma brucei and bloodstream Trypanosoma vivax had little or no mfVSG phospholipase activity. The activity found in bloodstream forms of Trypanosoma congolense was intermediate between Trypanosoma vivax and Trypanosoma brucei.

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Immunodominant surface epitopes power immune evasion in the African trypanosome

10.1101/2021.07.20.453071 ◽

2021 ◽

Author(s):

Anastasia Gkeka ◽

Francisco Aresta-Branco ◽

Gianna Triller ◽

Evi P Vlachou ◽

Mirjana Lilic ◽

...

Keyword(s):

Immune Evasion ◽

Antigenic Variation ◽

Cross Reactivity ◽

Surface Glycoprotein ◽

Mammalian Host ◽

African Trypanosome ◽

Repetitive Nature ◽

Variable Surface ◽

Major Surface ◽

Immunodominant Epitopes

The African trypanosome survives the immune response of its mammalian host by antigenic variation of its major surface antigen (the Variable Surface Glycoprotein, or VSG). Here we describe the antibody repertoires elicited by different VSGs. We show that the repertoires are highly restricted, directed predominantly to epitopes on the surface of the VSGs. They are also highly discriminatory: minor alterations within these exposed epitopes confer antigenically-distinct properties to these VSGs and elicit different repertoires. We propose that the patterned and repetitive nature of the VSG coat focuses host immunity to a restricted set of immunodominant epitopes per VSG, eliciting a highly stereotyped response, minimizing cross reactivity between different VSGs and facilitating prolonged immune evasion through epitope variation.

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The GPI-Phospholipase C of Trypanosoma brucei Is Nonessential But Influences Parasitemia in Mice

The Journal of Cell Biology ◽

10.1083/jcb.139.1.103 ◽

1997 ◽

Vol 139 (1) ◽

pp. 103-114 ◽

Cited By ~ 66

Author(s):

Helena Webb ◽

Nicola Carnall ◽

Luc Vanhamme ◽

Sylvie Rolin ◽

Jakke Van Den Abbeele ◽

...

Keyword(s):

Phospholipase C ◽

Trypanosoma Brucei ◽

Antigenic Variation ◽

Surface Glycoprotein ◽

Covalent Attachment ◽

Mammalian Host ◽

Surface Coat ◽

Null Mutant

In the mammalian host, the cell surface of Trypanosoma brucei is protected by a variant surface glycoprotein that is anchored in the plasma membrane through covalent attachment of the COOH terminus to a glycosylphosphatidylinositol. The trypanosome also contains a phospholipase C (GPI-PLC) that cleaves this anchor and could thus potentially enable the trypanosome to shed the surface coat of VSG. Indeed, release of the surface VSG can be observed within a few minutes on lysis of trypanosomes in vitro. To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC null mutant trypanosome has been generated by targeted gene deletion. The mutant trypanosomes are fully viable; they can go through an entire life cycle and maintain a persistent infection in mice. Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation. However, mice infected with the mutant trypanosomes have a reduced parasitemia and survive longer than those infected with control trypanosomes. This phenotype is partially alleviated when the null mutant is modified to express low levels of GPI-PLC.

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Trypanosoma brucei: posttranscriptional control of the variable surface glycoprotein gene expression site

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.4018-4021.1989 ◽

1989 ◽

Vol 9 (9) ◽

pp. 4018-4021

Author(s):

E Pays ◽

H Coquelet ◽

A Pays ◽

P Tebabi ◽

M Steinert

Keyword(s):

Gene Expression ◽

Trypanosoma Brucei ◽

Transcription Initiation ◽

Tsetse Fly ◽

Surface Glycoprotein ◽

Insect Vector ◽

Variable Surface Glycoprotein ◽

A Genome ◽

Variable Surface ◽

Expression Site

The arrest of variable surface glycoprotein (VSG) synthesis is one of the first events accompanying the differentiation of Trypanosoma brucei bloodstream forms into procyclic forms, which are characteristic of the insect vector. This is because of a very fast inhibition of VSG gene transcription which occurs as soon as the temperature is lowered. We report that this effect is probably not controlled at the level of transcription initiation, since the beginning of the VSG gene expression site, about 45 kilobases upstream from the antigen gene, remains transcribed in procyclic forms. The permanent activity of the promoter readily accounts for the systematic reappearance, upon return to the bloodstream form after cyclical transmission, of the antigen type present before passage to the tsetse fly. The abortive transcription of the VSG gene expression site appears linked to RNA processing abnormalities. Such posttranscriptional controls may allow the modulation of gene expression in a genome organized in large multigenic transcription units.

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Trypanosoma brucei: enrichment by UV of intergenic transcripts from the variable surface glycoprotein gene expression site

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.4022-4025.1989 ◽

1989 ◽

Vol 9 (9) ◽

pp. 4022-4025

Author(s):

H Coquelet ◽

P Tebabi ◽

A Pays ◽

M Steinert ◽

E Pays

Keyword(s):

Gene Expression ◽

Trypanosoma Brucei ◽

Uv Irradiation ◽

Surface Glycoprotein ◽

Putative Promoter Region ◽

Variable Surface Glycoprotein ◽

Polycistronic Transcription ◽

Variable Surface ◽

Intergenic Regions ◽

Expression Site

The expression site for the variable surface glycoprotein (VSG) gene AnTat 1.3A of Trypanosoma brucei is 45 kilobases long and encompasses seven expression site-associated genes (ESAGs) (E. Pays, P. Tebabi, A. Pays, H. Coquelet, P. Revelard, D. Salmon, and M. Steinert, Cell 57:835-845, 1989). After UV irradiation, several large transcripts from the putative promoter region were strongly enriched. We report that one such major transcript starts near the poly(A) addition site of the first gene (ESAG 7), spans the intergenic region, and extends to the poly(A) addition site of the second gene (ESAG 6), thus bypassing the normal 3' splice site of the ESAG 6 mRNA. Since this transcript is spliced, we conclude that UV irradiation does not inhibit splicing but stabilizes unstable processing products. This demonstrates that at least some intergenic regions of the VSG gene expression site are continuously transcribed in accordance with a polycistronic transcription model.

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

Pathogens ◽

10.3390/pathogens10080967 ◽

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.

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The intracellular pathway and assembly of newly formed variable surface glycoprotein of Trypanosoma brucei.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.81.24.7703 ◽

1984 ◽

Vol 81 (24) ◽

pp. 7703-7707 ◽

Cited By ~ 8

Author(s):

D. J. Grab ◽

P. Webster ◽

Y. Verjee

Keyword(s):

Trypanosoma Brucei ◽

Surface Glycoprotein ◽

Variable Surface Glycoprotein ◽

Variable Surface ◽

Intracellular Pathway

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Trypanosomal variant surface glycoprotein expression in human African trypanosomiasis patients

10.1101/2021.09.09.459620 ◽

2021 ◽

Author(s):

Jaime So ◽

Sarah Sudlow ◽

Abeer Sayeed ◽

Tanner Grudda ◽

Stijn Deborggraeve ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Human African Trypanosomiasis ◽

Antigenic Variation ◽

Surface Glycoprotein ◽

Variant Surface Glycoprotein ◽

Tsetse Flies ◽

African Trypanosomiasis ◽

Trypanosoma Brucei Gambiense ◽

Terminal Domain ◽

Human Infections

AbstractTrypanosoma brucei gambiense, an extracellular protozoan parasite, is the primary causative agent of human African Trypanosomiasis. T. b. gambiense is endemic to West and Central Africa where it is transmitted by the bite of infected tsetse flies. In the bloodstream of an infected host, the parasite evades antibody recognition by altering the Variant Surface Glycoprotein (VSG) that forms a dense coat on its cell surface through a process known as antigenic variation. Each VSG has a variable N-terminal domain that is exposed to the host and a less variable C-terminal domain that is at least partially hidden from host antibodies. Our lab developed VSG-seq, a targeted RNA-seq method, to study VSG expression in T. brucei. Studies using VSG-seq to characterize antigenic variation in a mouse model have revealed marked diversity in VSG expression within parasite populations, but this finding has not yet been validated in a natural human infection. Here, we used VSG-seq to analyze VSGs expressed in the blood of twelve patients infected with T. b. gambiense. The number of VSGs identified per patient ranged from one to fourteen and, notably, two VSGs were shared by more than one patient. Analysis of expressed VSG N-terminal domain types revealed that 82% of expressed VSGs encoded a type B N-terminus, a bias not seen in datasets from other T. brucei subspecies. C-terminal types in T. b. gambiense infection were also restricted. These results demonstrate a bias either in the underlying VSG repertoire of T. b. gambiense or in the selection of VSGs from the repertoire during infection. This work demonstrates the feasibility of using VSG-seq to study antigenic variation in human infections and highlights the importance of understanding VSG repertoires in the field.Author SummaryHuman African Trypanosomiasis is a neglected tropical disease largely caused by the extracellular parasite known as Trypanosoma brucei gambiense. To avoid elimination by the host, these parasites repeatedly replace their dense surface coat of Variant Surface Glycoprotein (VSG). Despite the important role of VSGs in prolonging infection, VSG expression during natural human infections is poorly understood. A better understanding of natural VSG expression dynamics can clarify the mechanisms which T. brucei uses to alter its VSG coat and improve how trypanosomiasis is diagnosed in humans. We analyzed the expressed VSGs detected in the blood of patients with trypanosomiasis. Our findings indicate that a diverse range of VSGs are expressed in both natural and experimental infections.

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