Variant Surface Antigens Research Articles

Designing a VAR2CSA-based vaccine to prevent placental malaria

Placental malaria (PM) due to Plasmodium falciparum is a major cause of maternal, fetal and infant mortality, but the mechanisms of pathogenesis and protective immunity are relatively well-understood for this condition, providing a path for vaccine development. P. falciparum parasites bind to chondroitin sulfate A (CSA) to sequester in the placenta, and women become resistant over 1–2 pregnancies as they acquire antibodies that block adhesion to CSA. The protein VAR2CSA, a member of the PfEMP1 variant surface antigen family, mediates parasite adhesion to CSA, and is the leading target for a vaccine to prevent PM. Obstacles to PM vaccine development include the large size (∼350kD), high cysteine content, and sequence variation of VAR2CSA. A number of approaches have been taken to identify the combination of VAR2CSA domains and alleles that can induce broadly active antibodies that block adhesion of heterologous parasite isolates to CSA. This review summarizes these approaches, which have examined VAR2CSA fragments for binding activity, antigenicity with naturally acquired antibodies, and immunogenicity in animals for inducing anti-adhesion or surface-reactive antibodies. Two products are expected to enter human clinical studies in the near future based on N-terminal VAR2CSA fragments that have high binding affinity for CSA, and additional proteins preferentially expressed by placental parasites are also being examined for their potential contribution to a PM vaccine.

Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates.

BackgroundThe diarrhea-causing protozoan Giardia intestinalis makes up a species complex of eight different assemblages (A-H), where assemblage A and B infect humans. Comparative whole-genome analyses of three of these assemblages have shown that there is significant divergence at the inter-assemblage level, however little is currently known regarding variation at the intra-assemblage level. We have performed whole genome sequencing of two sub-assemblage AII isolates, recently axenized from symptomatic human patients, to study the biological and genetic diversity within assemblage A isolates.ResultsSeveral biological differences between the new and earlier characterized assemblage A isolates were identified, including a difference in growth medium preference. The two AII isolates were of different sub-assemblage types (AII-1 [AS175] and AII-2 [AS98]) and showed size differences in the smallest chromosomes. The amount of genetic diversity was characterized in relation to the genome of the Giardia reference isolate WB, an assemblage AI isolate. Our analyses indicate that the divergence between AI and AII is approximately 1 %, represented by ~100,000 single nucleotide polymorphisms (SNP) distributed over the chromosomes with enrichment in variable genomic regions containing surface antigens. The level of allelic sequence heterozygosity (ASH) in the two AII isolates was found to be 0.25–0.35 %, which is 25–30 fold higher than in the WB isolate and 10 fold higher than the assemblage AII isolate DH (0.037 %). 35 protein-encoding genes, not found in the WB genome, were identified in the two AII genomes. The large gene families of variant-specific surface proteins (VSPs) and high cysteine membrane proteins (HCMPs) showed isolate-specific divergences of the gene repertoires. Certain genes, often in small gene families with 2 to 8 members, localize to the variable regions of the genomes and show high sequence diversity between the assemblage A isolates. One of the families, Bactericidal/Permeability Increasing-like protein (BPIL), with eight members was characterized further and the proteins were shown to localize to the ER in trophozoites.ConclusionsGiardia genomes are modular with highly conserved core regions mixed up by variable regions containing high levels of ASH, SNPs and variable surface antigens. There are significant genomic variations in assemblage A isolates, in terms of chromosome size, gene content, surface protein repertoire and gene polymorphisms and these differences mainly localize to the variable regions of the genomes. The large genetic differences within one assemblage of G. intestinalis strengthen the argument that the assemblages represent different Giardia species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1893-6) contains supplementary material, which is available to authorized users.

Mosquitoes Reset Malaria Parasites.

Serial blood passage of Plasmodium universally increases parasite virulence, which can be reversed by mosquito transmission. How mosquitoes reset Plasmodium virulence has been unknown. We have shown that mosquito transmission modifies expression of Plasmodium subtelomeric multigene families, including those that code for variant surface antigens (VSA), and transforms the systemic immune response to blood-stage infection. In this way, the mosquito regulates malaria disease severity. Here, we present a model in which expression of multigene families is reset by epigenetic reprogramming of Plasmodium within the mosquito. This prepares the malaria parasite for entry into a new unknown host and transforms the early parasite–host interactions that shape disease severity. Studying the molecular mechanisms that operate outside the human host to regulate Plasmodium virulence is therefore a priority.

BH3 domain-independent apolipoprotein L1 toxicity rescued by BCL2 prosurvival proteins

The potent trypanolytic properties of human apolipoprotein L1 (APOL1) can be neutralized by the trypanosome variant surface antigen gene product known as serum resistance-associated protein. However, two common APOL1 haplotypes present uniquely in individuals of West African ancestry each encode APOL1 variants resistant to serum resistance-associated protein, and each confers substantial resistance to human African sleeping sickness. In contrast to the dominantly inherited anti-trypanosomal activity of APOL1, recessive inheritance of these two trypanoprotective APOL1 alleles predisposes to kidney disease. Proposed mechanisms of APOL1 toxicity have included BH3 domain-dependent autophagy and/or ion channel activity. We probed these potential mechanisms by expressing APOL1 in Xenopus laevis oocytes. APOL1 expression in oocytes increased ion permeability and caused profound morphological deterioration (toxicity). Coexpression of BCL2 family members rescued APOL1-associated oocyte toxicity in the order MCL1 ∼ BCLW > BCLXL ∼ BCL2A1 ≫ BCL2. Deletion of nine nominal core BH3 domain residues abolished APOL1-associated toxicity, but missense substitution of the same residues abolished neither oocyte toxicity nor its rescue by coexpressed MCL1. The APOL1 BH3 domain was similarly dispensable for the ability of APOL1 to rescue intact mice from lethal trypanosome challenge. Replacement of most extracellular Na(+) by K(+) also reduced APOL1-associated oocyte toxicity, allowing demonstration of APOL1-associated increases in Ca(2+) and Cl(-) fluxes and oocyte ion currents, which were similarly reduced by MCL1 coexpression. Thus APOL1 toxicity in Xenopus oocytes is BH3-independent, but can nonetheless be rescued by some BCL2 family proteins.

Induction of endoplasmic reticulum-derived oxidative stress by an occult infection related S surface antigen variant.

To investigate the mechanism of endoplasmic reticulum (ER) stress induction by an occult infection related hepatitis B virus S surface antigen (HBsAg) variant. We used an HBsAg variant with lower secretion capacity, which was a KD variant from a Korean subject who was occultly infected with the genotype C. We compared the expression profiles of ER stress-related proteins between HuH-7 cells transfected with HBsAg plasmids of a wild-type and a KD variant using Western blot. Confocal microscopy indicated that the KD variant had higher levels of co-localization with ER than the wild-type HBsAg. The KD variant up-regulated ER stress-related proteins and induced reactive oxygen species (ROS) compared to the wild-type via an increase in calcium. The KD variant also down-regulated anti-oxidant proteins (HO-1, catalase and SOD) compared to the wild-type, which indicates positive amplification loops of the ER-ROS axis. The KD variant also induced apoptotic cell death via the up-regulation of caspase proteins (caspase 6, 9 and 12). Furthermore, the KD variant induced a higher level of nitric oxide than wild-type HBsAg via the up-regulation of the iNOS protein. Our data indicate that occult infection related HBsAg variants can lead to ER-derived oxidative stress and liver cell death in HuH-7 cells.

PTU-062 Endoscopy workload following the introduction of faecal calprotectin (FCP) – does fcp testing reduce lower gi endoscopies? – part of a nice nhs adoption site study in wandsworth:

As transmission intensity has declined in Senegal, so has the genetic complexity of circulating <i>Plasmodium falciparum</i> parasites, resulting in specific genotypes emerging and persisting over years. We address whether changes in parasite genetic signatures can alter the immune repertoire to variant surface antigens, and whether such responses can influence the expansion or contraction of specific parasite genotypes in the population. We characterize parasites within genotypic clusters, defined as identical by a 24-SNP molecular barcode and a haplotype identifier for other highly polymorphic loci; we measure expression of variant surface antigens (VSA) such as PfEMP-1 by transcript expression typing and expressed <i>var</i> DBL1<i>α</i> sequencing in ex vivo and short-term adapted RNA samples; and we measure IgG responses against VSAs from short-term adapted parasites. We find that parasites within genotypic clusters are genetically identical at other highly polymorphic loci. These parasites express similar Ups <i>var</i> classes and largely the same dominant <i>var</i> DBL1<i>α</i> sequences ex vivo. These parasites are recognized similarly by anti-VSA antibodies after short-term adaptation to culture; however, antibody responses do not correlate with genotype frequencies over time. Both genotype-specific and multiple genotype-reactive surface IgG responses are observed in this population. Parasites with identical genomes are extremely similar in their expression and host antibody recognition of VSAs. Monitoring changes in population-level parasite genomics and transmission dynamics is critical, as fluctuations will influence the breadth of resulting host immune responses to circulating parasite genotypes. These findings suggest shared immune recognition of genetically similar parasites, which has implications for both our understanding of immunity and vaccine development strategies in malaria elimination settings.

Plasmodium falciparum complicated malaria: Modulation and connectivity between exportome and variant surface antigen gene families

In temperate and sub-tropical regions of Asia and Latin America, complicated malaria manifested as hepatic dysfunction or renal dysfunction is seen in all age groups. There has been a concerted focus on understanding the patho-physiological and molecular basis of complicated malaria in children, much less is known about it in adults. We report here, the analysis of data from a custom, cross strain microarray (Agilent Platform) using material from adult patient samples, showing hepatic dysfunction or renal failure. These are the most common manifestations seen in adults along with cerebral malaria. The data has been analyzed with reference to variant surface antigens, encoded by the var, rifin and stevor gene families. The differential regulation profiles of key genes (comparison between Plasmodium falciparum complicated and uncomplicated isolates) have been observed. The exportome has been analyzed using similar parameters. Gene ontology term based functional enrichment of differentially regulated genes identified, up-regulated genes statistically enriched (P<0.05) to critical biological processes like generation of precursor metabolite and energy, chromosome organization and electron transport chain. Systems network based functional enrichment of overall differentially regulated genes yielded a similar result. We are reporting here, up-regulation of var group B and C genes whose proteins are predicted to interact with CD36 receptor in the host, the up-regulation of domain cassette 13 (DC13) containing var group A, as also the up-regulation of group A rifins and many of the stevors. This is contrary to most other reports from pediatric patients, with cerebral malaria where the up-regulation of mostly var A group genes have been seen. A protein–protein interaction based network has been created and analysis performed. This co-expression and text mining based network has shown overall connectivity between the variant surface antigens (VSA) and the exportome. The up-regulation of var group B and C genes encoding PfEMP1 with different domain architecture would be important for deciding strategies for disease prevention.

Measuring Cellular Immunity to Influenza: Methods of Detection, Applications and Challenges.

Influenza A virus is a respiratory pathogen which causes both seasonal epidemics and occasional pandemics; infection continues to be a significant cause of mortality worldwide. Current influenza vaccines principally stimulate humoral immune responses that are largely directed towards the variant surface antigens of influenza. Vaccination can result in an effective, albeit strain-specific antibody response and there is a need for vaccines that can provide superior, long-lasting immunity to influenza. Vaccination approaches targeting conserved viral antigens have the potential to provide broadly cross-reactive, heterosubtypic immunity to diverse influenza viruses. However, the field lacks consensus on the correlates of protection for cellular immunity in reducing severe influenza infection, transmission or disease outcome. Furthermore, unlike serological methods such as the standardized haemagglutination inhibition assay, there remains a large degree of variation in both the types of assays and method of reporting cellular outputs. T-cell directed immunity has long been known to play a role in ameliorating the severity and/or duration of influenza infection, but the precise phenotype, magnitude and longevity of the requisite protective response is unclear. In order to progress the development of universal influenza vaccines, it is critical to standardize assays across sites to facilitate direct comparisons between clinical trials.

Discovery of a novel and conserved Plasmodium falciparum exported protein that is important for adhesion of PfEMP1 at the surface of infected erythrocytes.

Plasmodium falciparum virulence is linked to its ability to sequester in post-capillary venules in the human host. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the main variant surface antigen implicated in this process. Complete loss of parasite adhesion is linked to a large subtelomeric deletion on chromosome 9 in a number of laboratory strains such as D10 and T9-96. Similar to the cytoadherent reference line FCR3, D10 strain expresses PfEMP1 on the surface of parasitized erythrocytes, however without any detectable cytoadhesion. To investigate which of the deleted subtelomeric genes may be implicated in parasite adhesion, we selected 12 genes for D10 complementation studies that are predicted to code for proteins exported to the red blood cell. We identified a novel single copy gene (PF3D7_0936500) restricted to P. falciparum that restores adhesion to CD36, termed here virulence-associated protein 1 (Pfvap1). Protein knockdown and gene knockout experiments confirmed a role of PfVAP1 in the adhesion process in FCR3 parasites. PfVAP1 is co-exported with PfEMP1 into the host cell via vesicle-like structures called Maurer's clefts. This study identifies a novel highly conserved parasite molecule that contributes to parasite virulence possibly by assisting PfEMP1 to establish functional adhesion at the host cell surface.

Neisseria gonorrhoeae elicits extracellular traps in primary neutrophil culture while suppressing the oxidative burst.

ABSTRACT Neisseria gonorrhoeae (the gonococcus) causes gonorrhea and is uniquely adapted to survive within the human reproductive tract. Gonococci evade host immune surveillance in part by varying their pili and opacity-associated proteins. These variable surface antigens influence interactions with host epithelial and immune cells. A potent polymorphonuclear leukocyte (PMN) response is a hallmark of symptomatic gonococcal infection, with vast numbers of PMNs recruited to the site of infection. A large body of literature describes gonococcus-PMN interactions, but the factors driving the outcome of infection are not fully understood. Gonococci have been described to both induce and suppress the PMN oxidative burst, but we determined that gonococci differentially affect induction of the PMN oxidative burst depending on the multiplicity of infection (MOI). Infecting PMN at an MOI of <20 gonococci elicits an oxidative burst, while an MOI of >20 suppresses the burst. Oxidative burst in response to gonococci is enhanced by, but does not require, expression of pili or opacity proteins. Neutrophil extracellular traps (NETs) were observed in gonococcus-infected PMNs, a process which requires an oxidative burst, yet gonococci induced NETs under suppressing conditions. The NETs were unable to kill gonococci despite killing the common vaginal bacterium Lactobacillus crispatus. Thus, gonococci influence PMN biology to promote their own survival by suppressing the oxidative burst of PMNs and stimulating the formation of NETs, which do not effectively kill gonococci, illustrating how N. gonorrhoeae has evolved to modulate PMN responses to promote infection.

Occult infection related hepatitis B surface antigen variants showing lowered secretion capacity.

To elucidate the molecular mechanisms underlying hepatitis B virus (HBV) occult infection of genotype C. A total of 10 types of hepatitis B surface antigen (HBsAg) variants from a Korean occult cohort were used. After a complete HBV genome plasmid mutated such that it does not express HBsAg and plasmid encoding, each HBsAg variant was transiently co-transfected into HuH-7 cells. The secretion capacity and intracellular expression of the HBV virions and HBsAgs in their respective variants were analyzed using real-time quantitative polymerase chain reaction assays and commercial HBsAg enzyme-linked immunosorbent assays, respectively. All variants exhibited lower levels of HBsAg secretion into the medium compared with the wild type. In particular, in eight of the ten variants, very low levels of HBsAg secretion that were similar to the negative control were detected. In contrast, most variants (9/10) exhibited normal virion secretion capacities comparable with, or even higher than, the wild type. This provided new insight into the intrinsic nature of occult HBV infection, which leads to HBsAg sero-negativeness but has horizontal infectivity. Furthermore, most variants generated higher reactive oxidative species production than the wild type. This finding provides potential links between occult HBV infection and liver disease progression. The presently obtained data indicate that deficiency in the secretion capacity of HBsAg variants may have a pivotal function in the occult infections of HBV genotype C.

Evaluating IgG Antibody to Variant Surface Antigens Expressed on Plasmodium falciparum Infected Erythrocytes Using Flow Cytometry.

Constant exposure to Plasmodium falciparum leads to acquisition of malarial antibodies that can protect against the clinical consequences of infection. One important target of such antibodies is against the parasite-infected erythrocyte (IEs). Current established assays to test the efficacy of antibodies in preventing parasite growth include direct parasite growth inhibition, the agglutination of IEs, and the inhibition of adhesion of IEs (to quantify antibody that inhibits adhesion to purified receptors or cells).However, many of these assays are labor-intensive and low-output which limits study sizes to small cohorts. Here we present an alternative assay that measures the levels of protective antibodies to variant surface antigens (VSA) of IEs. This assay can be performed using a microtitre plate and requires only a small volume of test serum sample. Serum samples are incubated with IEs and are then analyzed using a semi-automated autosampler attached to a flow cytometer. The assay is accurate, quick, and reproducible. Ultimately this assay could be used on large population-based studies, which could increase the statistical power of clinical studies.

Next generation digital PCR measurement of hepatitis B virus copy number in formalin-fixed paraffin-embedded hepatocellular carcinoma tissue.

Hepatocellular carcinoma (HCC) is strongly associated with hepatitis B virus (HBV) infection. False-negative results are common in routine serological tests and quantitative real-time PCR because of HBV surface antigen (HBsAg) variation and low HBV copy number. Droplet digital PCR (ddPCR), a next generation digital PCR, is a novel, sensitive, and specific platform that can be used to improve HBV detection. A total of 131 HCC cases with different tumor stages and clinical features were initially classified with a serological test as HBsAg positive (n = 107) or negative (n = 24) for HBV infection. Next, DNA templates were prepared from the corresponding formalin-fixed paraffin-embedded (FFPE) tissues to determine HBV copy number by ddPCR. HBV copy numbers, successfully determined for all clinical FFPE tissues (n = 131), ranged from 1.1 to 175.5 copies/μL according to ddPCR. The copy numbers of HBV were positively correlated with tumor-nodes-metastasis (P = 0.008) and Barcelona-Clinic Liver Cancer (P = 0.045) classification. Moreover, serum cholinesterase correlated with hepatitis B viral load (P = 0.006). HBV infection is a key factor that influences tumorigenesis in HCC by regulating tumor occurrence and development. ddPCR improves the analytical sensitivity and specificity of measurements in nucleic acids at a single-molecule level and is suitable for HBV detection.

What helminth genomes have taught us about parasite evolution.

The genomes of more than 20 helminths have now been sequenced. Here we perform a meta-analysis of all sequenced genomes of nematodes and Platyhelminthes, and attempt to address the question of what are the defining characteristics of helminth genomes. We find that parasitic worms lack systems for surface antigenic variation, instead maintaining infections using their surfaces as the first line of defence against the host immune system, with several expanded gene families of genes associated with the surface and tegument. Parasite excretory/secretory products evolve rapidly, and proteases even more so, with each parasite exhibiting unique modifications of its protease repertoire. Endoparasitic flatworms show striking losses of metabolic capabilities, not matched by nematodes. All helminths do however exhibit an overall reduction in auxiliary metabolism (biogenesis of co-factors and vitamins). Overall, the prevailing pattern is that there are few commonalities between the genomes of independently evolved parasitic worms, with each parasite having undergone specific adaptations for their particular niche.

Genomic characterization of variable surface antigens reveals a telomere position effect as a prerequisite for RNA interference-mediated silencing in Paramecium tetraurelia.

ABSTRACTAntigenic or phenotypic variation is a widespread phenomenon of expression of variable surface protein coats on eukaryotic microbes. To clarify the mechanism behind mutually exclusive gene expression, we characterized the genetic properties of the surface antigen multigene family in the ciliate Paramecium tetraurelia and the epigenetic factors controlling expression and silencing. Genome analysis indicated that the multigene family consists of intrachromosomal and subtelomeric genes; both classes apparently derive from different gene duplication events: whole-genome and intrachromosomal duplication. Expression analysis provides evidence for telomere position effects, because only subtelomeric genes follow mutually exclusive transcription. Microarray analysis of cultures deficient in Rdr3, an RNA-dependent RNA polymerase, in comparison to serotype-pure wild-type cultures, shows cotranscription of a subset of subtelomeric genes, indicating that the telomere position effect is due to a selective occurrence of Rdr3-mediated silencing in subtelomeric regions. We present a model of surface antigen evolution by intrachromosomal gene duplication involving the maintenance of positive selection of structurally relevant regions. Further analysis of chromosome heterogeneity shows that alternative telomere addition regions clearly affect transcription of closely related genes. Consequently, chromosome fragmentation appears to be of crucial importance for surface antigen expression and evolution. Our data suggest that RNAi-mediated control of this genetic network by trans-acting RNAs allows rapid epigenetic adaptation by phenotypic variation in combination with long-term genetic adaptation by Darwinian evolution of antigen genes.

Immune characterization of Plasmodium falciparum parasites with a shared genetic signature in a region of decreasing transmission.

As the intensity of malaria transmission has declined, Plasmodium falciparum parasite populations have displayed decreased clonal diversity resulting from the emergence of many parasites with common genetic signatures (CGS). We have monitored such CGS parasite clusters from 2006 to 2013 in Thiès, Senegal, using the molecular barcode. The first, and one of the largest observed clusters of CGS parasites, was present in 24% of clinical isolates in 2008, declined to 3.4% of clinical isolates in 2009, and then disappeared. To begin to explore the relationship between the immune responses of the population and the emergence and decline of specific parasite genotypes, we have determined whether antibodies to CGS parasites correlate with their prevalence. We measured (i) antibodies capable of inhibiting parasite growth in culture and (ii) antibodies recognizing the surfaces of infected erythrocytes (RBCs). IgG obtained from volunteers in 2009 showed increased reactivity to the surfaces of CGS-parasitized erythrocytes over IgG from 2008. Since P. falciparum EMP-1 (PfEMP-1) is a major variant surface antigen, we used var Ups quantitative reverse transcription-PCR (qRT-PCR) and sequencing with degenerate DBL1α domain primers to characterize the var genes expressed by CGS parasites after short-term in vitro culture. CGS parasites show upregulation of UpsA var genes and 2-cysteine-containing PfEMP-1 molecules and express the same dominant var transcript. Our work indicates that the CGS parasites in this cluster express similar var genes, more than would be expected by chance in the population, and that there is year-to-year variation in immune recognition of surface antigens on CGS parasite-infected erythrocytes. This study lays the groundwork for detailed investigations of the mechanisms driving the expansion or contraction of specific parasite clones in the population.

Analysis of Plasmodium falciparum erythrocyte membrane protein 1- DBLα domain variants with respect to antigenic variations and docking interaction analysis with glycosaminoglycans

Background The variant surface antigen PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) encoded by the polymorphic multi-copy var gene family plays an important role in parasite biology and the host-parasite interactions. Sequestration and antigenic variation is an essential component in the survival and pathogenesis of Plasmodium falciparum and contributes to chronic infection . The DBLa domain of PfEMP1 is a potential target for immuno-epidemiological studies and has been visualized as a vaccine candidate against severe malaria. Extensive var gene diversity (DBLa domain of PfEMP1) is an important aspect of pathogenesis in parasite lines. Specific host receptors like heparin, heparan sulphate, blood group A and complement receptor 1 have been reported to bind DBLa domain. Although heparin has been experimentally shown to disrupt the parasite-host interaction and effectively disrupt rosetting, the binding sites for the DBLa domain and mechanism behind heparin-mediated rosette inhibition have not been elucidated.

Pathogenesis of cerebral malaria--inflammation and cytoadherence.

Despite decades of research on cerebral malaria (CM) there is still a paucity of knowledge about what actual causes CM and why certain people develop it. Although sequestration of P. falciparum infected red blood cells has been linked to pathology, it is still not clear if this is directly or solely responsible for this clinical syndrome. Recent data have suggested that a combination of parasite variant types, mainly defined by the variant surface antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1), its receptors, coagulation and host endothelial cell activation (or inflammation) are equally important. This makes CM a multi-factorial disease and a challenge to unravel its causes to decrease its detrimental impact.

STEVOR Is a Plasmodium falciparum Erythrocyte Binding Protein that Mediates Merozoite Invasion and Rosetting

Variant surface antigens play an important role in Plasmodium falciparum malaria pathogenesis and in immune evasion by the parasite. Although most work to date has focused on P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1), two other multigene families encoding STEVOR and RIFIN are expressed in invasive merozoites and on the infected erythrocyte surface. However, their role during parasite infection remains to be clarified. Here we report that STEVOR functions as an erythrocyte-binding protein that recognizes Glycophorin C (GPC) on the red blood cell (RBC) surface and that its binding correlates with the level of GPC on the RBC surface. STEVOR expression on the RBC leads to PfEMP1-independent binding of infected RBCs to uninfected RBCs (rosette formation), while antibodies targeting STEVOR in the merozoite can effectively inhibit invasion. Our results suggest a PfEMP1-independent role for STEVOR in enabling infected erythrocytes at the schizont stage to form rosettes and in promoting merozoite invasion.

Trypanosoma brucei TIF2 suppresses VSG switching by maintaining subtelomere integrity.

Subtelomeres consist of sequences adjacent to telomeres and contain genes involved in important cellular functions, as subtelomere instability is associated with several human diseases. Balancing between subtelomere stability and plasticity is particularly important for Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. T. brucei regularly switches its major variant surface antigen, variant surface glycoprotein (VSG), to evade the host immune response, and VSGs are expressed exclusively from subtelomeres in a strictly monoallelic fashion. Telomere proteins are important for protecting chromosome ends from illegitimate DNA processes. However, whether they contribute to subtelomere integrity and stability has not been well studied. We have identified a novel T. brucei telomere protein, T. brucei TRF-Interacting Factor 2 (TbTIF2), as a functional homolog of mammalian TIN2. A transient depletion of TbTIF2 led to an elevated VSG switching frequency and an increased amount of DNA double-strand breaks (DSBs) in both active and silent subtelomeric bloodstream form expression sites (BESs). Therefore, TbTIF2 plays an important role in VSG switching regulation and is important for subtelomere integrity and stability. TbTIF2 depletion increased the association of TbRAD51 with the telomeric and subtelomeric chromatin, and TbRAD51 deletion further increased subtelomeric DSBs in TbTIF2-depleted cells, suggesting that TbRAD51-mediated DSB repair is the underlying mechanism of subsequent VSG switching. Surprisingly, significantly more TbRAD51 associated with the active BES than with the silent BESs upon TbTIF2 depletion, and TbRAD51 deletion induced much more DSBs in the active BES than in the silent BESs in TbTIF2-depleted cells, suggesting that TbRAD51 preferentially repairs DSBs in the active BES.