Variant Surface Glycoprotein Research Articles

Pedal to the Metal: Nuclear Splicing Bodies Turbo-Charge VSG mRNA Production in African Trypanosomes.

The African trypanosome Trypanosoma brucei is a parasite of the mammalian bloodstream and tissues, where an antigenically variable Variant Surface Glycoprotein (VSG) coat protects it from immune attack. This dense layer comprised of ∼107 VSG proteins, makes VSG by far the most abundant mRNA (7–10% total) and protein (∼10% total) in the bloodstream form trypanosome. How can such prodigious amounts of VSG be produced from a single VSG gene? Extremely high levels of RNA polymerase I (Pol I) transcription of the active VSG provide part of the explanation. However, recent discoveries highlight the role of pre-mRNA processing, both in maintaining high levels of VSG transcription, as well as its monoallelic expression. Trypanosome mRNAs are matured through trans-splicing a spliced leader (SL) RNA to the 5’ end of precursor transcripts, meaning abundant SL RNA is required throughout the nucleus. However, requirement for SL RNA in the vicinity of the active VSG gene is so intense, that the cell reconfigures its chromatin architecture to facilitate interaction between the SL RNA genes and the active VSG. This presumably ensures that sufficient localised SL RNA is available, and not limiting for VSG mRNA expression. Recently, novel nuclear splicing bodies which appear to provide essential trans-splicing components, have been identified associating with the active VSG. These observations highlight the underappreciated role of pre-mRNA processing in modulating gene expression in trypanosomes. Dissecting the function of these nuclear RNA processing bodies should help us elucidate the mechanisms of both VSG expression and monoallelic exclusion in T. brucei.

Black-necked spitting cobra (Naja nigricollis) phospholipases A2 may cause Trypanosoma brucei death by blocking endocytosis through the flagellar pocket

African trypanosomes, such as Trypanosoma brucei, are flagellated protozoa which proliferate in mammals and cause a variety of diseases in people and animals. In a mammalian host, the external face of the African trypanosome plasma membrane is covered by a densely packed coat formed of variant surface glycoprotein (VSG), which counteracts the host's adaptive immune response by antigenic variation. The VSG is attached to the external face of the plasma membrane by covalent attachment of the C-terminus to glycosylphosphatidylinositol. As the trypanosome grows, newly synthesised VSG is added to the plasma membrane by vesicle fusion to the flagellar pocket, the sole location of exo- and endocytosis. Snake venoms contain dozens of components, including proteases and phospholipases A2. Here, we investigated the effect of Naja nigricollis venom on T. brucei with the aim of describing the response of the trypanosome to hydrolytic attack on the VSG. We found no evidence for VSG hydrolysis, however, N. nigricollis venom caused: (i) an enlargement of the flagellar pocket, (ii) the Rab11 positive endosomal compartments to adopt an abnormal dispersed localisation, and (iii) cell cycle arrest prior to cytokinesis. Our results indicate that a single protein family, the phospholipases A2 present in N. nigricollis venom, may be necessary and sufficient for the effects. This study provides new molecular insight into T. brucei biology and possibly describes mechanisms that could be exploited for T. brucei targeting.

VEX1 Influences mVSG Expression During the Transition to Mammalian Infectivity in Trypanosoma brucei.

The Trypanosoma (T) brucei life cycle alternates between the tsetse fly vector and the mammalian host. In the insect, T. brucei undergoes several developmental stages until it reaches the salivary gland and differentiates into the metacyclic form, which is capable of infecting the next mammalian host. Mammalian infectivity is dependent on expression of the metacyclic variant surface glycoprotein genes as the cells develop into mature metacyclics. The VEX complex is essential for monoallelic variant surface glycoprotein expression in T. brucei bloodstream form, however, initiation of expression of the surface proteins genes during metacyclic differentiation is poorly understood. To better understand the transition to mature metacyclics and the control of metacyclic variant surface glycoprotein expression we examined the role of VEX1 in this process. We show that modulating VEX1 expression leads to a dysregulation of variant surface glycoprotein expression during metacyclogenesis, and that following both in vivo and in vitro metacyclic differentiation VEX1 relocalises from multiple nuclear foci in procyclic cells to one to two distinct nuclear foci in metacyclic cells - a pattern like the one seen in mammalian infective bloodstream forms. Our data suggest a role for VEX1 in the metacyclic differentiation process and their capacity to become infectious to the mammalian host.

Detrimental Effect ofTrypanosoma brucei bruceiInfection on Memory B Cells and Host Ability to Recall Protective B-cell Responses

Trypanosoma brucei brucei evades host immune responses by multiple means, including the disruption of B-cell homeostasis. This hampers anti-trypanosome vaccine development. Because the cellular mechanism underlying this pathology has never been addressed, our study focuses on the fate of memory B cells (MBCs) in vaccinated mice upon trypanosome challenge. A trypanosome variant surface glycoprotein (VSG) and fluorescent phycoerythrin were used as immunization antigens. Functional and cellular characteristics of antigen-specific MBCs were studied after homologous and heterologous parasite challenge. Immunization with AnTat1.1 VSG triggers a specific antibody response and isotype-switched CD73+CD273+CD80+ MBCs, delivering 90% sterile protection against a homologous parasite challenge. As expected, AnTat1.1 VSG immunization does not protect against infection with heterologous VSG-switched parasites. After successful curative drug treatment, mice were shown to have completely lost their previously induced protective immunity against the homologous parasites, coinciding with the loss of vaccine-induced MBCs. A phycoerythrin immunization approach confirmed that trypanosome infections cause the general loss of antigen-specific splenic and bone marrow MBCs and a reduction in antigen-specific immunoglobulin G. Trypanosomosis induces general immunological memory loss. This benefits the parasites by reducing the stringency for antigenic variation requirements.

CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes

Proteins of interest are frequently expressed with a fusion-tag to facilitate experimental analysis. In trypanosomatids, which are typically diploid, a tag-encoding DNA fragment is typically fused to one native allele. However, since recombinant cells represent ≪0.1% of the population following transfection, these DNA fragments also incorporate a marker cassette for positive selection. Consequently, native mRNA untranslated regions (UTRs) are replaced, potentially perturbing gene expression; in trypanosomatids, UTRs often impact gene expression in the context of widespread and constitutive polycistronic transcription. We sought to develop a tagging strategy that preserves native UTRs in bloodstream-form African trypanosomes, and here we describe a CRISPR/Cas9-based knock-in approach to drive precise and marker-free tagging of essential genes. Using simple tag-encoding amplicons, we tagged four proteins: a histone acetyltransferase, HAT2; a histone deacetylase, HDAC3; a cleavage and polyadenylation specificity factor, CPSF3; and a variant surface glycoprotein exclusion factor, VEX2. The approach maintained the native UTRs and yielded clonal strains expressing functional recombinant proteins, typically with both alleles tagged. We demonstrate utility for both immunofluorescence-based localisation and for enriching protein complexes; GFPHAT2 or GFPHDAC3 complexes in this case. This precision tagging approach facilitates the assembly of strains expressing essential recombinant genes with their native UTRs preserved.

N6-methyladenosine in poly(A) tails stabilize VSG transcripts.

RNA modifications are important regulators of gene expression1. In Trypanosoma brucei, transcription is polycistronic and thus most regulation happens post-transcriptionally2. N6-methyladenosine (m6A) has been detected in this parasite, but its function remains unknown3. Here we found that m6A is enriched in 342 transcripts using RNA immunoprecipitation, with an enrichment in transcripts encoding variant surface glycoproteins (VSGs). Approximately 50% of the m6A is located in the poly(A) tail of the actively expressed VSG transcripts. m6A residues are removed from the VSG poly(A) tail before deadenylation and mRNA degradation. Computational analysis revealed an association between m6A in the poly(A) tail and a 16-mer motif in the 3' untranslated region of VSG genes. Using genetic tools, we show that the 16-mer motif acts as a cis-acting motif that is required for inclusion of m6A in the poly(A) tail. Removal of this motif from the 3' untranslated region of VSG genes results in poly(A) tails lacking m6A, rapid deadenylation and mRNA degradation. To our knowledge, this is the first identification of an RNA modification in the poly(A) tail of any eukaryote, uncovering a post-transcriptional mechanism of gene regulation.

Control of Variant Surface Glycoprotein Expression by CFB2 in Trypanosoma brucei and Quantitative Proteomic Connections to Translation and Cytokinesis.

ABSTRACTVariant surface glycoproteins (VSGs) coat parasitic African trypanosomes and underpin antigenic variation and immune evasion. These VSGs are superabundant virulence factors that are subject to posttranscriptional gene expression controls mediated via the VSG 3′ untranslated region (UTR). To identify positive VSG regulators in bloodstream-form Trypanosoma brucei, we used genome-scale screening data to prioritize mRNA binding protein (mRBP) knockdowns that phenocopy VSG mRNA knockdown, displaying loss of fitness and precytokinesis accumulation. The top three candidates were CFB2 (cyclin F-box protein 2) (Tb927.1.4650), MKT1 (Tb927.6.4770), and PBP1 (polyadenylate binding protein 1) (Tb927.8.4540). Notably, CFB2 was recently found to regulate VSG transcript stability, and all three proteins were found to associate. We used data-independent acquisition for accurate label-free quantification and deep proteome coverage to quantify the expression profiles following the depletion of each mRBP. Only CFB2 knockdown significantly reduced VSG expression and the expression of a reporter under the control of the VSG 3′ UTR. CFB2 knockdown also triggered the depletion of cytoplasmic ribosomal proteins, consistent with translation arrest observed when VSG synthesis is blocked. In contrast, PBP1 knockdown triggered the depletion of CFB2, MKT1, and other components of the PBP1 complex. Finally, all three knockdowns triggered the depletion of cytokinesis initiation factors, consistent with a cytokinesis defect, which was confirmed here for all three knockdowns. Thus, genome-scale knockdown data sets facilitate the triage and prioritization of candidate regulators. Quantitative proteomic analysis confirms the 3′-UTR-dependent positive control of VSG expression by CFB2 and interactions with additional mRBPs. Our results also reveal new insights into the connections between VSG expression control by CFB2, ribosomal protein expression, and cytokinesis.IMPORTANCE VSG expression represents a key parasite virulence mechanism and an example of extreme biology. Posttranscriptional gene expression controls in trypanosomatids also continue to be the subject of substantial research interest. We have identified three candidate VSG regulators and used knockdown and quantitative proteomics, in combination with other approaches, to assess their function. CFB2 is found to control VSG expression via the VSG 3′ untranslated region, while other data support the view that MKT1 and PBP1 also form part of a CFB2 mRNA binding complex. Remarkably, we also find the depletion of cytoplasmic ribosomal proteins upon CFB2 knockdown, consistent with translation arrest observed when VSG synthesis is blocked. Proteomic profiles following knockdown further yield insights into cytokinesis defects. Taken together, our findings confirm and elaborate the role of CFB2 in controlling VSG expression and reveal new insights into connectivity with translation and cytokinesis controls.

Single-cell transcriptomics reveals expression profiles of Trypanosoma brucei sexual stages.

Early diverging lineages such as trypanosomes can provide clues to the evolution of sexual reproduction in eukaryotes. In Trypanosoma brucei, the pathogen that causes Human African Trypanosomiasis, sexual reproduction occurs in the salivary glands of the insect host, but analysis of the molecular signatures that define these sexual forms is complicated because they mingle with more numerous, mitotically-dividing developmental stages. We used single-cell RNA-sequencing (scRNAseq) to profile 388 individual trypanosomes from midgut, proventriculus, and salivary glands of infected tsetse flies allowing us to identify tissue-specific cell types. Further investigation of salivary gland parasite transcriptomes revealed fine-scale changes in gene expression over a developmental progression from putative sexual forms through metacyclics expressing variant surface glycoprotein genes. The cluster of cells potentially containing sexual forms was characterized by high level transcription of the gamete fusion protein HAP2, together with an array of surface proteins and several genes of unknown function. We linked these expression patterns to distinct morphological forms using immunofluorescence assays and reporter gene expression to demonstrate that the kinetoplastid-conserved gene Tb927.10.12080 is exclusively expressed at high levels by meiotic intermediates and gametes. Further experiments are required to establish whether this protein, currently of unknown function, plays a role in gamete formation and/or fusion.

Transcription Dependent Loss of an Ectopically Expressed Variant Surface Glycoprotein during Antigenic Variation in Trypanosoma brucei.

ABSTRACTIn the mammalian host, Trypanosoma brucei is coated in a single-variant surface glycoprotein (VSG) species. Stochastic switching of the expressed VSG allows the parasite to escape detection by the host immune system. DNA double-strand breaks (DSB) trigger VSG switching, and repair via gene conversion results in an antigenically distinct VSG being expressed from the single active bloodstream-form expression site (BES). The single active BES is marked by VSG exclusion 2 (VEX2) protein. Here, we have disrupted monoallelic VSG expression by stably expressing a second telomeric VSG from a ribosomal locus. We found that cells expressing two VSGs contained one VEX2 focus that was significantly larger in size than the wild-type cells; this therefore suggests the ectopic VSG is expressed from the same nuclear position as the active BES. Unexpectedly, we report that in the double VSG-expressing cells, the DNA sequence of the ectopic copy is lost following a DSB in the active BES, despite it being spatially separated in the genome. The loss of the ectopic VSG is dependent on active transcription and does not disrupt the number or variety of templates used to repair a BES DSB and elicit a VSG switch. We propose that there are stringent mechanisms within the cell to reinforce monoallelic expression during antigenic variation.

Fragment-Based Drug Discovery for Trypanosoma brucei Glycosylphosphatidylinositol-Specific Phospholipase C through Biochemical and WaterLOGSY-NMR Methods.

Glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) of Trypanosoma brucei, the causative protozoan parasite of African trypanosomiasis, is a membrane-bound enzyme essential for antigenic variation, because it catalyses the release of the membrane-bound form of variable surface glycoproteins. Here, we performed a fragment-based drug discovery of TbGPI-PLC inhibitors using a combination of enzymatic inhibition assay and water ligand observed via gradient spectroscopy (WaterLOGSY) NMR experiment. The TbGPI-PLC was cloned and overexpressed using an Escherichia coli expression system followed by purification using three-phase partitioning and gel filtration. Subsequently, the inhibitory activity of 873 fragment compounds against the recombinant TbGPI-PLC led to the identification of 66 primary hits. These primary hits were subjected to the WaterLOGSY NMR experiment where 10 fragment hits were confirmed to directly bind to the TbGPI-PLC. These included benzothiazole, chlorobenzene, imidazole, indole, pyrazol and quinolinone derivatives. Molecular docking simulation indicated that six of them share a common binding site, which corresponds to the catalytic pocket. The present study identified chemically diverse fragment hits that could directly bind and inhibit the TbGPI-PLC activity, which constructed a framework for fragment optimization or linking towards the design of novel drugs for African trypanosomiasis.

Silent Trypanosoma evansi infection in humans from India revealed by serological and molecular surveys, and characterized by variable surface glycoprotein gene sequences

BackgroundThe importance of emerging atypical human trypanosomosis is gaining momentum due to increasing detection and its possible impact on human health. A cross sectional study of atypical human trypanosomosis due to Trypanosoma evansi was carried out in Kolkata and Canning area of West Bengal state of India where previously a death was reported. MethodsIn this study blood and serum samples from 173 individuals were collected during August to December 2014. To check the presence of antibodies against T. evansi, card agglutination test and for the presence of T. evansi specific DNA, PCR were conducted. ResultsT. evansi infection was identified in 5.2% (9/173) human blood samples by CATT serological test (Card agglutination test for trypanosomosis). PCR targeting VSG gene sequences suggested active T. evansi infection in 2.89% (5/173). VSG gene sequences herein determined for five isolates from human cases shared high similarity (89.4–100%). Phylogenetic inference clustered the human isolates with other isolates from different host species from India and other countries, forming a clade exclusive of Indian isolates (84.0 to 100% sequence similarity). ConclusionFirst report of symptomless human T. evansi infection detected by combined serological and PCR assays. First phylogenetic analysis of VSG gene sequences including human isolates of T. evansi in which Indian isolates of T. evansi from human and other hosts clustered in a single clade.

Signatures of hybridization in Trypanosoma brucei.

Genetic exchange among disease-causing micro-organisms can generate progeny that combine different pathogenic traits. Though sexual reproduction has been described in trypanosomes, its impact on the epidemiology of Human African Trypanosomiasis (HAT) remains controversial. However, human infective and non-human infective strains of Trypanosoma brucei circulate in the same transmission cycles in HAT endemic areas in subsaharan Africa, providing the opportunity for mating during the developmental cycle in the tsetse fly vector. Here we investigated inheritance among progeny from a laboratory cross of T. brucei and then applied these insights to genomic analysis of field-collected isolates to identify signatures of past genetic exchange. Genomes of two parental and four hybrid progeny clones with a range of DNA contents were assembled and analysed by k-mer and single nucleotide polymorphism (SNP) frequencies to determine heterozygosity and chromosomal inheritance. Variant surface glycoprotein (VSG) genes and kinetoplast (mitochondrial) DNA maxi- and minicircles were extracted from each genome to examine how each of these components was inherited in the hybrid progeny. The same bioinformatic approaches were applied to an additional 37 genomes representing the diversity of T. brucei in subsaharan Africa and T. evansi. SNP analysis provided evidence of crossover events affecting all 11 pairs of megabase chromosomes and demonstrated that polyploid hybrids were formed post-meiotically and not by fusion of the parental diploid cells. VSGs and kinetoplast DNA minicircles were inherited biparentally, with approximately equal numbers from each parent, whereas maxicircles were inherited uniparentally. Extrapolation of these findings to field isolates allowed us to distinguish clonal descent from hybridization by comparing maxicircle genotype to VSG and minicircle repertoires. Discordance between maxicircle genotype and VSG and minicircle repertoires indicated inter-lineage hybridization. Significantly, some of the hybridization events we identified involved human infective and non-human infective trypanosomes circulating in the same geographic areas.

Visualisation of experimentally determined and predicted protein N-glycosylation and predicted glycosylphosphatidylinositol anchor addition in Trypanosoma brucei.

Background: Trypanosoma brucei is a protozoan parasite and the etiological agent of human and animal African trypanosomiasis. The organism cycles between its mammalian host and tsetse vector. The host-dwelling bloodstream form of the parasite is covered with a monolayer of variant surface glycoprotein (VSG) that enables it to escape both the innate and adaptive immune systems. Within this coat reside lower-abundance surface glycoproteins that function as receptors and/or nutrient transporters. The glycosylation of the Trypanosoma brucei surface proteome is essential to evade the immune response and is mediated by three oligosaccharyltransferase genes; two of which, TbSTT3A and TbSTT3B, are expressed in the bloodstream form of the parasite. Methods: We processed a recent dataset of our laboratory to visualise putative glycosylation sites of the Trypanosoma brucei proteome. We provided a visualisation for the predictions of glycosylation carried by TbSTT3A and TbSTT3B, and we augmented the visualisation with predictions for Glycosylphosphatidylinositol anchoring sites, domains and topology of the Trypanosoma brucei proteome. Conclusions: We created a web service to explore the glycosylation sites of the Trypanosoma brucei oligosaccharyltransferases substrates, using data described in a recent publication of our laboratory. We also made a machine learning algorithm available as a web service, described in our recent publication, to distinguish between TbSTT3A and TbSTT3B substrates.

Visualisation of experimentally determined and predicted protein N-glycosylation and predicted glycosylphosphatidylinositol anchor addition in Trypanosoma brucei.

Background: Trypanosoma brucei is a protozoan parasite and the etiological agent of human and animal African trypanosomiasis. The organism cycles between its mammalian host and tsetse vector. The host-dwelling bloodstream form of the parasite is covered with a monolayer of variant surface glycoprotein (VSG) that enables it to escape both the innate and adaptive immune systems. Within this coat reside lower-abundance surface glycoproteins that function as receptors and/or nutrient transporters. The glycosylation of the Trypanosoma brucei surface proteome is essential to evade the immune response and is mediated by three oligosaccharyltransferase genes; two of which, TbSTT3A and TbSTT3B, are expressed in the bloodstream form of the parasite. Methods: We processed a recent dataset of our laboratory to visualise putative glycosylation sites of the Trypanosoma brucei proteome. We provided a visualisation for the predictions of glycosylation carried by TbSTT3A and TbSTT3B, and we augmented the visualisation with predictions for Glycosylphosphatidylinositol anchoring sites, domains and topology of the Trypanosoma brucei proteome. Conclusions: We created a web service to explore the glycosylation sites of the Trypanosoma brucei oligosaccharyltransferases substrates, using data described in a recent publication of our laboratory. We also made a machine learning algorithm available as a web service, described in our recent publication, to distinguish between TbSTT3A and TbSTT3B substrates.

Molecular characterization of Trypanosoma evansi, T. vivax and T. congolense in camels (Camelus dromedarius) of KSA

BackgroundTrypanosoma evansi is the leading infectious Trypanosoma spp. in camels (Camelus dromedarius) present in the Kingdom of Saudi Arabia (KSA) that could lead to extensive economic losses. The present study was aimed to assess the prevalence rate of T. evansi in Taif governorate, Makkah province, KSA using parasitological and molecular evaluations, and analyze their genetic relationship targeting internal transcribed spacer 1 (ITS1) and variable surface glycoprotein (VSG) genes. For evaluation, we have used 102 blood samples of camels obtained from three different regions in Taif.ResultsResults show a considerable prevalence rate of trypanosomosis 2/102 (2.0%) according to Giemsa-stained buffy coat smear, and 16/102 (15.7%) according to touchdown PCR. T. evansi (n = 10/102, 9.8%) was the main infectious species found in camels then T. vivax (n = 3/102, 2.9%). Mixed infections were detected in three camels with T. evansi, T. vivax, and T. congolense (n = 3/102, 2.9%). Regarding gender, the results indicate that female camels (11/66, 16.7%) show higher prevalence of Trypanosoma than males (5/36, 13.9%). Sequencing and phylogenetic analyses of ITS1 and VSG showed their relationships with T. evansi in other hosts from different countries.ConclusionsIn our peer knowledge, it is the first time to report a research-based prevalence of trypanosomosis in the camels of Taif governorate, Makkah province, KSA.

An assembly of nuclear bodies associates with the active VSG expression site in African trypanosomes

A Variant Surface Glycoprotein (VSG) coat protects bloodstream form Trypanosoma brucei. Prodigious amounts of VSG mRNA (~7-10% total) are generated from a single RNA polymerase I (Pol I) transcribed VSG expression site (ES), necessitating extremely high levels of localised splicing. We show that splicing is required for processive ES transcription, and describe novel ES-associated T. brucei nuclear bodies. In bloodstream form trypanosomes, the expression site body (ESB), spliced leader array body (SLAB), NUFIP body and Cajal bodies all frequently associate with the active ES. This assembly of nuclear bodies appears to facilitate the extraordinarily high levels of transcription and splicing at the active ES. In procyclic form trypanosomes, the NUFIP body and SLAB do not appear to interact with the Pol I transcribed procyclin locus. The congregation of a restricted number of nuclear bodies at a single active ES, provides an attractive mechanism for how monoallelic ES transcription is mediated.

First molecular survey of animal trypanosomes in Paraguayan horses.

Despite the epidemic situation of animal trypanosomosis caused by Trypanosoma evansi, Trypanosoma equiperdum and Trypanosoma vivax in South American countries, there are no reports for the prevalence of animal trypanosomes in Paraguay. In this study, 408 blood samples were obtained from apparently healthy horses from sixteen departments of Paraguay, for routine medical check-up from August to September 2019, and a polymerase chain reaction (PCR)-based cross-sectional study was carried out to identify trypanosome prevalence. The prevalence of Trypanozoon (T. evansi and T. equiperdum) and T. vivax was 7.11% (29/408) and 26.23% (107/408), respectively. Mixed infections were detected in 4.90% (20/408) of the samples. Some of the selected trypanosome positive samples were confirmed as T. vivax and T. evansi Type A by sequence analysis of the internal transcribe spacer region and RoTat1.2 variant surface glycoprotein gene, respectively. In conclusion, we found higher prevalence of T. vivax than Trypanozoon in Paraguayan horses. However, the genotypic variation should be verified in further studies.

Homology modelling of Trypanosoma brucei major surface proteases and molecular docking of variant surface glycoproteins and inhibitor ligands for drug design

Trypanosomes, which cause animal African trypanosomiasis, escape host immune responses by renewing their variable surface glycoprotein (VSG) coat. Chemotherapy is currently the only form of external intervention available. However, the efficacy of current trypanocides is poor due to overuse leading to an increase in drug resistance. Major surface proteases (MSPs) of trypanosomes, which are zinc-dependent metalloproteases, are possible drug targets. A Trypanosoma brucei MSP-B (TbMSP-B) mediates parasite antigenic variation via cleavage of 60% of VSG molecules. Whilst TbMSP-A has no apparent role in VSG cleavage; it is not known if TbMSP-C is involved in VSG cleavage. In this study, three-dimensional structures of TbMSP-A, TbMSP-B and TbMSP-C were modelled. By comparing the docking poses of the C-terminal domains of VSG substrates into the models, TbMSP-C showed an affinity for similar VSG substrate sites as TbMSP-B, but these sites differed from those recognised by TbMSP-A. This observation suggests that TbMSP-C may be involved in VSG cleavage during antigenic variation. Furthermore, by docking small inhibitor ligands into the TbMSP-B and TbMSP-C homology models, followed by molecular dynamics simulations, ligands with potential anti-trypanosomal activity were identified. Docking studies also revealed the depth of the S1’ pockets of TbMSP-B and TbMSP-C, which is influential in ligand and substrate binding, thereby identifying the protease subsite pocket that should be targeted in drug design.

DNA double strand break position leads to distinct gene expression changes and regulates VSG switching pathway choice.

Antigenic variation is an immune evasion strategy used by Trypanosoma brucei that results in the periodic exchange of the surface protein coat. This process is facilitated by the movement of variant surface glycoprotein genes in or out of a specialized locus known as bloodstream form expression site by homologous recombination, facilitated by blocks of repetitive sequence known as the 70-bp repeats, that provide homology for gene conversion events. DNA double strand breaks are potent drivers of antigenic variation, however where these breaks must fall to elicit a switch is not well understood. To understand how the position of a break influences antigenic variation we established a series of cell lines to study the effect of an I-SceI meganuclease break in the active expression site. We found that a DNA break within repetitive regions is not productive for VSG switching, and show that the break position leads to a distinct gene expression profile and DNA repair response which dictates how antigenic variation proceeds in African trypanosomes.

A DOT1B/Ribonuclease H2 Protein Complex Is Involved in R-Loop Processing, Genomic Integrity, and Antigenic Variation in Trypanosoma brucei.

ABSTRACTThe parasite Trypanosoma brucei periodically changes the expression of protective variant surface glycoproteins (VSGs) to evade its host’s immune system in a process known as antigenic variation. One route to change VSG expression is the transcriptional activation of a previously silent VSG expression site (ES), a subtelomeric region containing the VSG genes. Homologous recombination of a different VSG from a large reservoir into the active ES represents another route. The conserved histone methyltransferase DOT1B is involved in transcriptional silencing of inactive ES and influences ES switching kinetics. The molecular machinery that enables DOT1B to execute these regulatory functions remains elusive, however. To better understand DOT1B-mediated regulatory processes, we purified DOT1B-associated proteins using complementary biochemical approaches. We identified several novel DOT1B interactors. One of these was the RNase H2 complex, previously shown to resolve RNA-DNA hybrids, maintain genome integrity, and play a role in antigenic variation. Our study revealed that DOT1B depletion results in an increase in RNA-DNA hybrids, accumulation of DNA damage, and ES switching events. Surprisingly, a similar pattern of VSG deregulation was observed in RNase H2 mutants. We propose that both proteins act together in resolving R-loops to ensure genome integrity and contribute to the tightly regulated process of antigenic variation.