Specificity of serological screening tests and reference laboratory tests to diagnose gambiense human African trypanosomiasis: a prospective clinical performance study.

BACKGROUND: Serological screening tests play a crucial role to diagnose gambiense human African trypanosomiasis (gHAT). Presently, they preselect individuals for microscopic confirmation, but in future "screen and treat" strategies they will identify individuals for treatment. Variability in reported specificities, the development of new rapid diagnostic tests (RDT) and the hypothesis that malaria infection may decrease RDT specificity led us to evaluate the specificity of 5 gHAT screening tests. METHODS: During active screening, venous blood samples from 1095 individuals from Côte d'Ivoire and Guinea were tested consecutively with commercial (CATT, HAT Sero-K-SeT, Abbott Bioline HAT 2.0) and prototype (DCN HAT RDT, HAT Sero-K-SeT 2.0) gHAT screening tests and with a malaria RDT. Individuals with ≥ 1 positive gHAT screening test underwent microscopy and further immunological (trypanolysis with T.b. gambiense LiTat 1.3, 1.5 and 1.6; indirect ELISA/T.b. gambiense; T.b. gambiense inhibition ELISA with T.b. gambiense LiTat 1.3 and 1.5 VSG) and molecular reference laboratory tests (PCR TBRN3, 18S and TgsGP; SHERLOCK 18S Tids, 7SL Zoon, and TgsGP; Trypanozoon S2-RT-qPCR 18S2, 177T, GPI-PLC and TgsGP in multiplex; RT-qPCR DT8, DT9 and TgsGP in multiplex). Microscopic trypanosome detection confirmed gHAT, while other individuals were considered gHAT free. Differences in fractions between groups were assessed by Chi square and differences in specificity between 2 tests on the same individuals by McNemar. RESULTS: One gHAT case was diagnosed. Overall test specificities (n = 1094) were: CATT 98.9% (95% CI: 98.1-99.4%); HAT Sero-K-SeT 86.7% (95% CI: 84.5-88.5%); Bioline HAT 2.0 82.1% (95% CI: 79.7-84.2%); DCN HAT RDT 78.2% (95% CI: 75.7-80.6%); and HAT Sero-K-SeT 2.0 78.4% (95% CI: 75.9-80.8%). In malaria positives, gHAT screening tests appeared less specific, but the difference was significant only in Guinea for Abbott Bioline HAT 2.0 (P = 0.03) and HAT Sero-K-Set 2.0 (P = 0.0006). The specificities of immunological and molecular laboratory tests in gHAT seropositives were 98.7-100% (n = 399) and 93.0-100% (n = 302), respectively. Among 44 reference laboratory test positives, only the confirmed gHAT patient and one screening test seropositive combined immunological and molecular reference laboratory test positivity. CONCLUSIONS: Although a minor effect of malaria cannot be excluded, gHAT RDT specificities are far below the 95% minimal specificity stipulated by the WHO target product profile for a simple diagnostic tool to identify individuals eligible for treatment. Unless specificity is improved, an RDT-based "screen and treat" strategy would result in massive overtreatment. In view of their inconsistent results, additional comparative evaluations of the diagnostic performance of reference laboratory tests are indicated for better identifying, among screening test positives, those at increased suspicion for gHAT. TRIAL REGISTRATION: The trial was retrospectively registered under NCT05466630 in clinicaltrials.gov on July 15 2022.

Structural similarities between the metacyclic and bloodstream form variant surface glycoproteins of the African trypanosome.

During infection of mammalian hosts, African trypanosomes thwart immunity using antigenic variation of the dense Variant Surface Glycoprotein (VSG) coat, accessing a large repertoire of several thousand genes and pseudogenes, and switching to antigenically distinct copies. The parasite is transferred to mammalian hosts by the tsetse fly. In the salivary glands of the fly, the pathogen adopts the metacyclic form and expresses a limited repertoire of VSG genes specific to that developmental stage. It has remained unknown whether the metacyclic VSGs possess distinct properties associated with this particular and discrete phase of the parasite life cycle. We present here three novel metacyclic form VSG N-terminal domain crystal structures (mVSG397, mVSG531, and mVSG1954) and show that they mirror closely in architecture, oligomerization, and surface diversity the known classes of bloodstream form VSGs. These data suggest that the mVSGs are unlikely to be a specialized subclass of VSG proteins, and thus could be poor candidates as the major components of prophylactic vaccines against trypanosomiasis.

SHERLOCK4HAT: A CRISPR-based tool kit for diagnosis of Human African Trypanosomiasis.

BACKGROUND: To achieve elimination of Human African Trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense (gHAT), the development of highly sensitive diagnostics is needed. We have developed a CRISPR based diagnostic for HAT using SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) that is readily adaptable to a field-based setting. METHODS: We adapted SHERLOCK for the detection of T. brucei species. We targeted 7SLRNA, TgSGP and SRA genes and tested SHERLOCK against RNA from blood, buffy coat, dried blood spots (DBS), and clinical samples. FINDINGS: The pan-Trypanozoon 7SLRNA and T. b. gambiense-specific TgSGP SHERLOCK assays had a sensitivity of 0.1 parasite/µL and a limit of detection 100 molecules/µL. T. b. rhodesiense-specific SRA had a sensitivity of 0.1 parasite/µL and a limit of detection of 10 molecules/µL. TgSGP SHERLOCK and SRA SHERLOCK detected 100% of the field isolated strains. Using clinical specimens from the WHO HAT cryobank, the 7SLRNA SHERLOCK detected trypanosomes in gHAT samples with 56.1%, 95% CI [46.25-65.53] sensitivity and 98.4%, 95% CI [91.41-99.92] specificity, and rHAT samples with 100%, 95% CI [83.18-100] sensitivity and 94.1%, 95% CI [80.91-98.95] specificity. The species-specific TgSGP and SRA SHERLOCK discriminated between the gambiense/rhodesiense HAT infections with 100% accuracy. INTERPRETATION: The 7SLRNA, TgSGP and SRA SHERLOCK discriminate between gHAT and rHAT infections, and could be used for epidemiological surveillance and diagnosis of HAT in the field after further technical development. FUNDING: Institut Pasteur (PTR-175 SHERLOCK4HAT), French Government's Investissement d'Avenir program Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (LabEx IBEID), and Agence Nationale pour la Recherche (ANR-PRC 2021 SherPa).

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.

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

In 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. IMPORTANCE The single-cell parasite Trypanosoma brucei causes the fatal disease human African trypanosomiasis and is able to colonize the blood, fat, skin, and central nervous system. Trypanosomes survive in the mammalian host owing to a dense protective protein coat that consists of a single-variant surface glycoprotein species. Stochastic switching of one VSG for an immunologically distinct one enables the parasite to escape recognition by the host immune system. We have disrupted monoallelic antigen expression by expressing a second VSG and report that following DSB-triggered VSG switching, the DNA sequence of the ectopic VSG is lost in a transcription-dependent manner. We propose that there are strict requirements to ensure that only one variant antigen is expressed following a VSG switch, which has important implications for understanding how the parasite survives in the mammalian host.

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.

The MRN complex promotes DNA repair by homologous recombination and restrains antigenic variation in African trypanosomes.

Homologous recombination dominates as the major form of DNA repair in Trypanosoma brucei, and is especially important for recombination of the subtelomeric variant surface glycoprotein during antigenic variation. RAD50, a component of the MRN complex (MRE11, RAD50, NBS1), is central to homologous recombination through facilitating resection and governing the DNA damage response. The function of RAD50 in trypanosomes is untested. Here we report that RAD50 and MRE11 are required for RAD51-dependent homologous recombination and phosphorylation of histone H2A following a DNA double strand break (DSB), but neither MRE11 nor RAD50 substantially influence DSB resection at a chromosome-internal locus. In addition, we reveal intrinsic separation-of-function between T. brucei RAD50 and MRE11, with only RAD50 suppressing DSB repair using donors with short stretches of homology at a subtelomeric locus, and only MRE11 directing DSB resection at the same locus. Finally, we show that loss of either MRE11 or RAD50 causes a greater diversity of expressed VSG variants following DSB repair. We conclude that MRN promotes stringent homologous recombination at subtelomeric loci and restrains antigenic variation.

Spatial integration of transcription and splicing in a dedicated compartment sustains monogenic antigen expression in African trypanosomes.

Highly selective gene expression is a key requirement for antigenic variation in several pathogens, allowing evasion of host immune responses and maintenance of persistent infections1. African trypanosomes-parasites that cause lethal diseases in humans and livestock-employ an antigenic variation mechanism that involves monogenic antigen expression from a pool of >2,600 antigen-coding genes2. In other eukaryotes, the expression of individual genes can be enhanced by mechanisms involving the juxtaposition of otherwise distal chromosomal loci in the three-dimensional nuclear space3-5. However, trypanosomes lack classical enhancer sequences or regulated transcription initiation6,7. In this context, it has remained unclear how genome architecture contributes to monogenic transcription elongation and transcript processing. Here, we show that the single expressed antigen-coding gene displays a specific inter-chromosomal interaction with a major messenger RNA splicing locus. Chromosome conformation capture (Hi-C) revealed a dynamic reconfiguration of this inter-chromosomal interaction upon activation of another antigen. Super-resolution microscopy showed the interaction to be heritable and splicing dependent. We found a specific association of the two genomic loci with the antigen exclusion complex, whereby VSG exclusion 1 (VEX1) occupied the splicing locus and VEX2 occupied the antigen-coding locus. Following VEX2 depletion, loss of monogenic antigen expression was accompanied by increased interactions between previously silent antigen genes and the splicing locus. Our results reveal a mechanism to ensure monogenic expression, where antigen transcription and messenger RNA splicing occur in a specific nuclear compartment. These findings suggest a new means of post-transcriptional gene regulation.

Forward Genetics in African Trypanosomes.

Forward genetic screens in Trypanosoma brucei have enabled researchers to move from a candidate-gene based approach to one where we are able to studying all genes required for a single process simultaneously. In this protocol, we describe how to generate RNAi library strains in bloodstream form trypanosomes, run a screen by selecting for drug resistance or using a reporter gene and process the high-throughput sequencing data for a genome scale RNAi library screen.

mSphere of Influence: Expanding the CRISPR Sphere with Single-Locus Proteomics.

Lucy Glover's research focuses on the role of DNA repair and recombination in antigenic variation in the parasite Trypanosoma brucei, the causative agent of both human and animal African trypanosomiasis. In this mSphere of Influence article, she reflects on how "A CRISPR-based approach for proteomic analysis of a single genomic locus" by Z. J. Waldrip, S. D. Byrum, A. J. Storey, J. Gao, et al. (Epigenetics 9:1207-1211, 2014, https://doi.org/10.4161/epi.29919) made an impact on her research by taking the precision of CRISPR-Cas9 and repurposing it to look at single-locus proteomics. By using this technology in trypanosomes, Dr. Glover and her colleagues could study the dynamic accumulation of repair proteins after specific damage and gain insight into how the location of a double-strand break (DSB) dictates repair pathway choice and how this may influence immune evasion in these parasites.

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

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.

Persistent DNA Damage Foci and DNA Replication with a Broken Chromosome in the African Trypanosome.

Damaged DNA typically imposes stringent controls on eukaryotic cell cycle progression, ensuring faithful transmission of genetic material. Some DNA breaks, and the resulting rearrangements, are advantageous, however. For example, antigenic variation in the parasitic African trypanosome, Trypanosoma brucei, relies upon homologous recombination-based rearrangements of telomeric variant surface glycoprotein (VSG) genes, triggered by breaks. Surprisingly, trypanosomes with a severed telomere continued to grow while progressively losing subtelomeric DNA, suggesting a nominal telomeric DNA damage checkpoint response. Here, we monitor the single-stranded DNA-binding protein replication protein A (RPA) in response to induced, locus-specific DNA breaks in T. brucei RPA foci accumulated at nucleolar sites following a break within ribosomal DNA and at extranucleolar sites following a break elsewhere, including adjacent to transcribed or silent telomeric VSG genes. As in other eukaryotes, RPA foci were formed in S phase and γH2A and RAD51 damage foci were disassembled prior to mitosis. Unlike in other eukaryotes, however, and regardless of the damaged locus, RPA foci persisted through the cell cycle, and these cells continued to replicate their DNA. We conclude that a DNA break, regardless of the damaged locus, fails to trigger a stringent cell cycle checkpoint in T. brucei This DNA damage tolerance may facilitate the generation of virulence-enhancing genetic diversity, within subtelomeric domains in particular. Stringent checkpoints may be similarly lacking in some other eukaryotic cells.IMPORTANCE Chromosome damage must be repaired to prevent the proliferation of defective cells. Alternatively, cells with damage must be eliminated. This is true of human and several other cell types but may not be the case for single-celled parasites, such as trypanosomes. African trypanosomes, which cause lethal diseases in both humans and livestock, can actually exploit chromosomal damage to activate new surface coat proteins and to evade host immune responses, for example. We monitored responses to single chromosomal breaks in trypanosomes using a DNA-binding protein that, in response to DNA damage, forms nuclear foci visible using a microscope. Surprisingly, and unlike what is seen in mammalian cells, these foci persist while cells continue to divide. We also demonstrate chromosome replication even when one chromosome is broken. These results reveal a remarkable degree of damage tolerance in trypanosomes, which may suit the lifestyle of a single-celled parasite, potentially facilitating adaptation and enhancing virulence.

Monoallelic expression and epigenetic inheritance sustained by a Trypanosoma brucei variant surface glycoprotein exclusion complex.

The largest gene families in eukaryotes are subject to allelic exclusion, but mechanisms underpinning single allele selection and inheritance remain unclear. Here, we describe a protein complex sustaining variant surface glycoprotein (VSG) allelic exclusion and antigenic variation in Trypanosoma brucei parasites. The VSG-exclusion-1 (VEX1) protein binds both telomeric VSG-associated chromatin and VEX2, an ortholog of nonsense-mediated-decay helicase, UPF1. VEX1 and VEX2 assemble in an RNA polymerase-I transcription-dependent manner and sustain the active, subtelomeric VSG-associated transcription compartment. VSG transcripts and VSG coats become highly heterogeneous when VEX proteins are depleted. Further, the DNA replication-associated chromatin assembly factor, CAF-1, binds to and specifically maintains VEX1 compartmentalisation following DNA replication. Thus, the VEX-complex controls VSG-exclusion, while CAF-1 sustains VEX-complex inheritance in association with the active-VSG. Notably, the VEX2-orthologue and CAF-1 in mammals are also implicated in exclusion and inheritance functions. In trypanosomes, these factors sustain a highly effective and paradigmatic immune evasion strategy.

A post-transcriptional respiratome regulon in trypanosomes.

Post-transcriptional regulons coordinate the expression of groups of genes in eukaryotic cells, yet relatively few have been characterized. Parasitic trypanosomatids are particularly good models for studies on such mechanisms because they exhibit almost exclusive polycistronic, and unregulated, transcription. Here, we identify the Trypanosoma brucei ZC3H39/40 RNA-binding proteins as regulators of the respiratome; the mitochondrial electron transport chain (complexes I-IV) and the FoF1-ATP synthase (complex V). A high-throughput RNAi screen initially implicated both ZC3H proteins in variant surface glycoprotein (VSG) gene silencing. This link was confirmed and both proteins were shown to form a cytoplasmic ZC3H39/40 complex. Transcriptome and mRNA-interactome analyses indicated that the impact on VSG silencing was indirect, while the ZC3H39/40 complex specifically bound and stabilized transcripts encoding respiratome-complexes. Quantitative proteomic analyses revealed specific positive control of >20 components from complexes I, II and V. Our findings establish a link between the mitochondrial respiratome and VSG gene silencing in bloodstream form T. brucei. They also reveal a major respiratome regulon controlled by the conserved trypanosomatid ZC3H39/40 RNA-binding proteins.

CRISPR in Parasitology: Not Exactly Cut and Dried!

CRISPR/Cas9 technology has been developing rapidly in the field of parasitology, allowing for the dissection of molecular processes with unprecedented efficiency. Optimization and implementation of a new technology like CRISPR, especially in nonmodel organisms, requires communication and collaboration throughout the field. Recently, a 'CRISPR in Parasitology' symposium was held at the Institut Pasteur Paris, bringing together scientists studying Leishmania, Plasmodium, Trypanosoma, and Anopheles. Here we share technological advances and challenges in using CRISPR/Cas9 in the parasite and vector systems that were discussed. As CRISPR/Cas9 continues to be applied to diverse parasite systems, the community should now focus on improvement and standardization of the technique as well as expanding the CRISPR toolkit to include Cas9 alternatives/derivatives for more advanced applications like genome-wide functional screens.

Insights into antitrypanosomal drug mode-of-action from cytology-based profiling.

Chemotherapy continues to have a major impact on reducing the burden of disease caused by trypanosomatids. Unfortunately though, the mode-of-action (MoA) of antitrypanosomal drugs typically remains unclear or only partially characterised. This is the case for four of five current drugs used to treat Human African Trypanosomiasis (HAT); eflornithine is a specific inhibitor of ornithine decarboxylase. Here, we used a panel of T. brucei cellular assays to probe the MoA of the current HAT drugs. The assays included DNA-staining followed by microscopy and quantitative image analysis, or flow cytometry; terminal dUTP nick end labelling to monitor mitochondrial (kinetoplast) DNA replication; antibody-based detection of sites of nuclear DNA damage; and fluorescent dye-staining of mitochondria or lysosomes. We found that melarsoprol inhibited mitosis; nifurtimox reduced mitochondrial protein abundance; pentamidine triggered progressive loss of kinetoplast DNA and disruption of mitochondrial membrane potential; and suramin inhibited cytokinesis. Thus, current antitrypanosomal drugs perturb distinct and specific cellular compartments, structures or cell cycle phases. Further exploiting the findings, we show that putative mitogen-activated protein-kinases contribute to the melarsoprol-induced mitotic defect, reminiscent of the mitotic arrest associated signalling cascade triggered by arsenicals in mammalian cells, used to treat leukaemia. Thus, cytology-based profiling can rapidly yield novel insight into antitrypanosomal drug MoA.

Genome-wide RNAi selection identifies a regulator of transmission stage-enriched gene families and cell-type differentiation in Trypanosoma brucei.

Trypanosoma brucei, causing African sleeping-sickness, exploits quorum-sensing (QS) to generate the 'stumpy forms' necessary for the parasite's transmission to tsetse-flies. These quiescent cells are generated by differentiation in the bloodstream from proliferative slender forms. Using genome-wide RNAi selection we screened for repressors of transmission stage-enriched mRNAs in slender forms, using the stumpy-elevated ESAG9 transcript as a model. This identified REG9.1, whose RNAi-silencing alleviated ESAG9 repression in slender forms and tsetse-midgut procyclic forms. Interestingly, trypanosome surface protein Family 5 and Family 7 mRNAs were also elevated, which, like ESAG9, are T. brucei specific and stumpy-enriched. We suggest these contribute to the distinct transmission biology and vector tropism of T. brucei from other African trypanosome species. As well as surface family regulation, REG9.1-depletion generated QS-independent development to stumpy forms in vivo, whereas REG9.1 overexpression in bloodstream forms potentiated spontaneous differentiation to procyclic forms in the absence of an external signal. Combined, this identifies REG9.1 as a regulator of developmental cell fate, controlling the expression of Trypanosoma brucei-specific molecules elevated during transmission.

High-resolution analysis of multi-copy variant surface glycoprotein gene expression sites in African trypanosomes.

BACKGROUND: African trypanosomes cause lethal diseases in humans and animals and escape host immune attack by switching the expression of Variant Surface Glycoprotein (VSG) genes. The expressed VSGs are located at the ends of telomeric, polycistronic transcription units known as VSG expression sites (VSG-ESs). Each cell has many VSG-ESs but only one is transcribed in bloodstream-form parasites and all of them are inactive upon transmission to the insect vector mid-gut; a subset of monocistronic metacyclic VSG-ESs are then activated in the insect salivary gland. Deep-sequence analyses have been informative but assigning sequences to individual VSG-ESs has been challenging because they each contain closely related expression-site associated genes, or ESAGs, thought to contribute to virulence. RESULTS: We utilised ART, an in silico short read simulator to demonstrate the feasibility of accurately aligning reads to VSG-ESs. Then, using high-resolution transcriptomes from isogenic bloodstream and insect-stage Lister 427 Trypanosoma brucei, we uncover increased abundance in the insect mid-gut stage of mRNAs from metacyclic VSG-ESs and of mRNAs from the unusual ESAG, ESAG10. Further, we show that the silencing associated with allelic exclusion involves repression focussed at the ends of the VSG-ESs. We also use the approach to report relative fitness costs following ESAG RNAi from a genome-scale screen. CONCLUSIONS: By assigning sequences to individual VSG-ESs we provide new insights into VSG-ES transcription control, allelic exclusion and impacts on fitness. Thus, deeper insights into the expression and function of regulated multi-gene families are more accessible than previously anticipated.

The nuclear envelope and gene organization in parasitic protozoa: Specializations associated with disease.

The parasitic protozoa Trypanosoma brucei and Plasmodium falciparum are lethal human parasites that have developed elegant strategies of immune evasion by antigenic variation. Despite the vast evolutionary distance between the two taxa, both parasites employ strict monoallelic expression of their membrane proteins, variant surface glycoproteins in Trypanosomes and the var, rif and stevor genes in Plasmodium, in order to evade their host's immune system. Additionally, both telomeric location and epigenetic controls are prominent features of these membrane proteins. As such, telomeres, chromatin structure and nuclear organization all contribute to control of gene expression and immune evasion. Here, we discuss the importance of epigenetics and sub-nuclear context for the survival of these disease-causing parasites.

VEX1 controls the allelic exclusion required for antigenic variation in trypanosomes.

Allelic exclusion underpins antigenic variation and immune evasion in African trypanosomes. These bloodstream parasites use RNA polymerase-I (pol-I) to transcribe just one telomeric variant surface glycoprotein (VSG) gene at a time, producing superabundant and switchable VSG coats. We identified trypanosome VSG exclusion-1 (VEX1) using a genetic screen for defects in telomere-exclusive expression. VEX1 was sequestered by the active VSG and silencing of other VSGs failed when VEX1 was either ectopically expressed or depleted, indicating positive and negative regulation, respectively. Positive regulation affected VSGs and nontelomeric pol-I-transcribed genes, whereas negative regulation primarily affected VSGs. Negative regulation by VEX1 also affected telomeric pol-I-transcribed reporter constructs, but only when they contained blocks of sequence sharing homology with a pol-I-transcribed locus. We conclude that restricted positive regulation due to VEX1 sequestration, combined with VEX1-dependent, possibly homology-dependent silencing, drives a "winner-takes-all" mechanism of allelic exclusion.