Functional insights from a surface antigen mRNA-bound proteome.

Trypanosoma brucei is the causative agent of human sleeping sickness. The parasites' variant surface glycoprotein (VSG) enables them to evade adaptive immunity via antigenic variation. VSG comprises 10% of total cell protein and the high stability of VSG mRNA is essential for trypanosome survival. To determine how VSG mRNA stability is maintained, we used mRNA affinity purification to identify all its associated proteins. CFB2 (cyclin F-box protein 2), an unconventional RNA-binding protein with an F-box domain, was specifically enriched with VSG mRNA. We demonstrate that CFB2 is essential for VSG mRNA stability, describe cis acting elements within the VSG 3'-untranslated region that regulate the interaction, identify trans-acting factors that are present in the VSG messenger ribonucleoprotein particle, and mechanistically explain how CFB2 stabilizes the mRNA of this key pathogenicity factor. Beyond T. brucei, the mRNP purification approach has the potential to supply detailed biological insight into metabolism of relatively abundant mRNAs in any eukaryote.

Structure of trypanosome coat protein VSGsur and function in suramin resistance.

Suramin has been a primary early-stage treatment for African trypanosomiasis for nearly 100 yr. Recent studies revealed that trypanosome strains that express the variant surface glycoprotein (VSG) VSGsur possess heightened resistance to suramin. Here, we show that VSGsur binds tightly to suramin but other VSGs do not. By solving high-resolution crystal structures of VSGsur and VSG13, we also demonstrate that these VSGs define a structurally divergent subgroup of the coat proteins. The co-crystal structure of VSGsur with suramin reveals that the chemically symmetric drug binds within a large cavity in the VSG homodimer asymmetrically, primarily through contacts of its central benzene rings. Structure-based, loss-of-contact mutations in VSGsur significantly decrease the affinity to suramin and lead to a loss of the resistance phenotype. Altogether, these data show that the resistance phenotype is dependent on the binding of suramin to VSGsur, establishing that the VSG proteins can possess functionality beyond their role in antigenic variation.

Variant antigen repertoires in Trypanosoma congolense populations and experimental infections can be profiled from deep sequence data using universal protein motifs.

African trypanosomes are vector-borne hemoparasites of humans and animals. In the mammal, parasites evade the immune response through antigenic variation. Periodic switching of the variant surface glycoprotein (VSG) coat covering their cell surface allows sequential expansion of serologically distinct parasite clones. Trypanosome genomes contain many hundreds of VSG genes, subject to rapid changes in nucleotide sequence, copy number, and chromosomal position. Thus, analyzing, or even quantifying, VSG diversity over space and time presents an enormous challenge to conventional techniques. Indeed, previous population genomic studies have overlooked this vital aspect of pathogen biology for lack of analytical tools. Here we present a method for analyzing population-scale VSG diversity in Trypanosoma congolense from deep sequencing data. Previously, we suggested that T. congolense VSGs segregate into defined "phylotypes" that do not recombine. In our data set comprising 41 T. congolense genome sequences from across Africa, these phylotypes are universal and exhaustive. Screening sequence contigs with diagnostic protein motifs accurately quantifies relative phylotype frequencies, providing a metric of VSG diversity, called the "variant antigen profile." We applied our metric to VSG expression in the tsetse fly, showing that certain, rare VSG phylotypes may be preferentially expressed in infective, metacyclic-stage parasites. Hence, variant antigen profiling accurately and rapidly determines the T. congolense VSG gene and transcript repertoire from sequence data, without need for manual curation or highly contiguous sequences. It offers a tractable approach to measuring VSG diversity across strains and during infections, which is imperative to understanding the host-parasite interaction at population and individual scales.

African trypanosomes evade immune clearance by O-glycosylation of the VSG surface coat.

The African trypanosome Trypanosoma brucei spp. is a paradigm for antigenic variation, the orchestrated alteration of cell surface molecules to evade host immunity. The parasite elicits robust antibody-mediated immune responses to its variant surface glycoprotein (VSG) coat, but evades immune clearance by repeatedly accessing a large genetic VSG repertoire and 'switching' to antigenically distinct VSGs. This persistent immune evasion has been ascribed exclusively to amino-acid variance on the VSG surface presented by a conserved underlying protein architecture. We establish here that this model does not account for the scope of VSG structural and biochemical diversity. The 1.4-Å-resolution crystal structure of the variant VSG3 manifests divergence in the tertiary fold and oligomeric state. The structure also reveals an O-linked carbohydrate on the top surface of VSG3. Mass spectrometric analysis indicates that this O-glycosylation site is heterogeneously occupied in VSG3 by zero to three hexose residues and is also present in other VSGs. We demonstrate that this O-glycosylation increases parasite virulence by impairing the generation of protective immunity. These data alter the paradigm of antigenic variation by the African trypanosome, expanding VSG variability beyond amino-acid sequence to include surface post-translational modifications with immunomodulatory impact.

Synthesis of Galactosylated Glycosylphosphatidylinositol Derivatives from Trypanosoma brucei.

Trypanosoma brucei uses variant surface glycoproteins (VSGs) to evade the host immune system and ensure parasitic longevity in animals and humans. VSGs are attached to the cell membrane by complex glycosylphosphatidylinositol anchors (GPI). Distinguishing structural feature of VSG GPIs are multiple α- and ß-galactosides attached to the conserved GPI core structure. T. brucei GPIs have been associated with macrophage activation and alleviation of parasitemia during infection, acting as disease onset delaying antigens. Literature reports that link structural modifications in the GPIs to changes in biological activity are contradictory. We have established a synthetic route to prepare structurally overlapping GPI derivatives bearing different T. brucei characteristic structural modifications. The GPI collection will be used to assess the effect of galactosylation and phosphorylation on T. brucei GPI immunomodulatory activity, and to perform an epitope mapping of this complex glycolipid as potential diagnostic marker for Trypanosomiasis. A strategy for the synthesis of a complete α-tetragalactoside using the 2-naphthylmethyl protecting group and for subsequent attachment of GPI fragments to peptides is presented.

Structural basis for the shielding function of the dynamic trypanosome variant surface glycoprotein coat.

The most prominent defence of the unicellular parasite Trypanosoma brucei against the host immune system is a dense coat that comprises a variant surface glycoprotein (VSG). Despite the importance of the VSG family, no complete structure of a VSG has been reported. Making use of high-resolution structures of individual VSG domains, we employed small-angle X-ray scattering to elucidate the first two complete VSG structures. The resulting models imply that the linker regions confer great flexibility between domains, which suggests that VSGs can adopt two main conformations to respond to obstacles and changes of protein density, while maintaining a protective barrier at all times. Single-molecule diffusion measurements of VSG in supported lipid bilayers substantiate this possibility, as two freely diffusing populations could be detected. This translates into a highly flexible overall topology of the surface VSG coat, which displays both lateral movement in the plane of the membrane and variation in the overall thickness of the coat.

Identification of the ISWI Chromatin Remodeling Complex of the Early Branching Eukaryote Trypanosoma brucei.

ISWI chromatin remodelers are highly conserved in eukaryotes and are important for the assembly and spacing of nucleosomes, thereby controlling transcription initiation and elongation. ISWI is typically associated with different subunits, forming specialized complexes with discrete functions. In the unicellular parasite Trypanosoma brucei, which causes African sleeping sickness, TbISWI down-regulates RNA polymerase I (Pol I)-transcribed variant surface glycoprotein (VSG) gene expression sites (ESs), which are monoallelically expressed. Here, we use tandem affinity purification to determine the interacting partners of TbISWI. We identify three proteins that do not show significant homology with known ISWI-associated partners. Surprisingly, one of these is nucleoplasmin-like protein (NLP), which we had previously shown to play a role in ES control. In addition, we identify two novel ISWI partners, regulator of chromosome condensation 1-like protein (RCCP) and phenylalanine/tyrosine-rich protein (FYRP), both containing protein motifs typically found on chromatin proteins. Knockdown of RCCP or FYRP in bloodstream form T. brucei results in derepression of silent variant surface glycoprotein ESs, as had previously been shown for TbISWI and NLP. All four proteins are expressed and interact with each other in both major life cycle stages and show similar distributions at Pol I-transcribed loci. They are also found at Pol II strand switch regions as determined with ChIP. ISWI, NLP, RCCP, and FYRP therefore appear to form a single major ISWI complex in T. brucei (TbIC). This reduced complexity of ISWI regulation and the presence of novel ISWI partners highlights the early divergence of trypanosomes in evolution.

Mapping antigenic motifs in the trypomastigote small surface antigen from Trypanosoma cruzi.

The trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease.

How Does the VSG Coat of Bloodstream Form African Trypanosomes Interact with External Proteins?

Variations on the statement "the variant surface glycoprotein (VSG) coat that covers the external face of the mammalian bloodstream form of Trypanosoma brucei acts a physical barrier" appear regularly in research articles and reviews. The concept of the impenetrable VSG coat is an attractive one, as it provides a clear model for understanding how a trypanosome population persists; each successive VSG protects the plasma membrane and is immunologically distinct from previous VSGs. What is the evidence that the VSG coat is an impenetrable barrier, and how do antibodies and other extracellular proteins interact with it? In this review, the nature of the extracellular surface of the bloodstream form trypanosome is described, and past experiments that investigated binding of antibodies and lectins to trypanosomes are analysed using knowledge of VSG sequence and structure that was unavailable when the experiments were performed. Epitopes for some VSG monoclonal antibodies are mapped as far as possible from previous experimental data, onto models of VSG structures. The binding of lectins to some, but not to other, VSGs is revisited with more recent knowledge of the location and nature of N-linked oligosaccharides. The conclusions are: (i) Much of the variation observed in earlier experiments can be explained by the identity of the individual VSGs. (ii) Much of an individual VSG is accessible to antibodies, and the barrier that prevents access to the cell surface is probably at the base of the VSG N-terminal domain, approximately 5 nm from the plasma membrane. This second conclusion highlights a gap in our understanding of how the VSG coat works, as several plasma membrane proteins with large extracellular domains are very unlikely to be hidden from host antibodies by VSG.

[Oral transmission of Trypanosoma cruzi : a new epidemiological scenario for Chagas' disease in Colombia and other South American countries]. / Transmisión oral de Trypanosoma cruzi: una nueva situación epidemiológica de la enfermedad de Chagas en Colombia y otros países suramericanos.

Many cases of infection caused by the oral transmission of Trypanosoma cruzi have been reported during the last decade. These have been due to the contamination of food by faeces from sylvatic triatomines or by leakage from reservoirs in areas where domiciliated vectors have been controlled or where there has been no prior background of domiciliation. The United Nations Food and Agriculture Organization (FAO) and the World Health Organization (WHO) have used epidemiological, clinical and socioeconomic criteria for ranking parasites transmitted by the contamination of food in different areas of the world; T. cruzi was placed tenth in importance amongst a group of 24 parasites in such ranking. Environmental changes such as deforestation and global warming have affected ecotopes and the behaviour of T. cruzi vectors and reservoirs so that these have become displaced to new areas, thereby leading to such new transmission scenario caused by the contamination of food, which requires evaluation in Colombia. The current review deals with the oral transmission of Chagas' disease, emphasising studies aimed at identifying the pertinent risk factors, the triatomine species involved, the physiopathology of oral infection, the parasite's genotypes implicated in this type of transmission in Colombia and other Latin American regions, as well as the need for ongoing epidemiological surveillance and control policies.

Transmisión oral de Trypanosoma cruzi: una nueva situación epidemiológica de la enfermedad de Chagas en Colombia y otros países suramericanos / Oral transmission of Trypanosoma cruzi : a new epidemiological scenario for Chagas´ disease in Colombia and other South American countries

Durante la última década se han reportado numerosos casos de infección por Trypanosoma cruzi por vía oral, debidos a la contaminación de alimentos con heces de triatominos silvestres o con secreciones de reservorios en áreas donde los vectores domiciliados han sido controlados o no hay antecedentes de domiciliación. Con base en criterios epidemiológicos, clínicos y socioeconómicos, la Organización de las Naciones Unidas para la Agricultura y la Alimentación (FAO) y la Organización Mundial de la Salud (OMS) establecieron una clasificación de los parásitos transmitidos por contaminación de alimentos en diferentes regiones del mundo, en la cual T. cruzi ocupó el décimo lugar de importancia en un grupo de 24 parásitos. Los cambios ambientales, como la deforestación y el calentamiento global, han afectado los ecotopos y el comportamiento de los vectores y de los reservorios de T. cruzi , de manera que estos se han desplazado a nuevas zonas, generando una nueva forma de transmisión por contaminación de alimentos que requiere su evaluación en el país. La presente revisión aborda la transmisión oral de la enfermedad de Chagas con énfasis en los estudios orientados a identificar los factores de riesgo, las especies de triatominos involucrados, la fisiopatología de la infección oral y los genotipos del parásito que están implicados en esta forma de transmisión en Colombia y en otras regiones de América Latina, así como la necesidad de adoptar políticas para su control y vigilancia epidemiológica.

Many cases of infection caused by the oral transmission of Trypanosoma cruzi have been reported during the last decade. These have been due to the contamination of food by faeces from sylvatic triatomines or by leakage from reservoirs in areas where domiciliated vectors have been controlled or where there has been no prior background of domiciliation. The United Nations Food and Agriculture Organization (FAO) and the World Health Organization (WHO) have used epidemiological, clinical and socioeconomic criteria for ranking parasites transmitted by the contamination of food in different areas of the world; T. cruzi was placed tenth in importance amongst a group of 24 parasites in such ranking. Environmental changes such as deforestation and global warming have affected ecotopes and the behaviour of T. cruzi vectors and reservoirs so that these have become displaced to new areas, thereby leading to such new transmission scenario caused by the contamination of food, which requires evaluation in Colombia. The current review deals with the oral transmission of Chagas´ disease, emphasising studies aimed at identifying the pertinent risk factors, the triatomine species involved, the physiopathology of oral infection, the parasite´s genotypes implicated in this type of transmission in Colombia and other Latin American regions, as well as the need for ongoing epidemiological surveillance and control policies.

Capturing the variant surface glycoprotein repertoire (the VSGnome) of Trypanosoma brucei Lister 427.

Trypanosoma brucei evades the adaptive immune response through the expression of antigenically distinct Variant Surface Glycoprotein (VSG) coats. To understand the progression and mechanisms of VSG switching, and to identify the VSGs expressed in populations of trypanosomes, it is desirable to predetermine the available repertoire of VSG genes (the 'VSGnome'). To date, the catalog of VSG genes present in any strain is far from complete and the majority of current information regarding VSGs is derived from the TREU927 strain that is not commonly used as an experimental model. We have assembled, annotated and analyzed 2563 distinct and previously unsequenced genes encoding complete and partial VSGs of the widely used Lister 427 strain of T. brucei. Around 80% of the VSGnome consists of incomplete genes or pseudogenes. Read-depth analysis demonstrated that most VSGs exist as single copies, but 360 exist as two or more indistinguishable copies. The assembled regions include five functional metacyclic VSG expression sites. One third of minichromosome sub-telomeres contain a VSG (64-67 VSGs on ∼96 minichromosomes), of which 85% appear to be functionally competent. The minichromosomal repertoire is very dynamic, differing among clones of the same strain. Few VSGs are unique along their entire length: frequent recombination events are likely to have shaped (and to continue to shape) the repertoire. In spite of their low sequence conservation and short window of expression, VSGs show evidence of purifying selection, with ∼40% of non-synonymous mutations being removed from the population. VSGs show a strong codon-usage bias that is distinct from that of any other group of trypanosome genes. VSG sequences are generally very divergent between Lister 427 and TREU927 strains of T. brucei, but those that are highly similar are not found in 'protected' genomic environments, but may reflect genetic exchange among populations.

Development of peptide-based lineage-specific serology for chronic Chagas disease: geographical and clinical distribution of epitope recognition.

BACKGROUND: Chagas disease, caused by infection with the protozoan Trypanosoma cruzi, remains a serious public health issue in Latin America. Genetically diverse, the species is sub-divided into six lineages, known as TcI-TcVI, which have disparate geographical and ecological distributions. TcII, TcV, and TcVI are associated with severe human disease in the Southern Cone countries, whereas TcI is associated with cardiomyopathy north of the Amazon. T. cruzi persists as a chronic infection, with cardiac and/or gastrointestinal symptoms developing years or decades after initial infection. Identifying an individual's history of T. cruzi lineage infection directly by genotyping of the parasite is complicated by the low parasitaemia and sequestration in the host tissues. METHODOLOGY/PRINCIPAL FINDINGS: We have applied here serology against lineage-specific epitopes of the T. cruzi surface antigen TSSA, as an indirect approach to allow identification of infecting lineage. Chagasic sera from chronic patients from a range of endemic countries were tested by ELISA against synthetic peptides representing lineage-specific TSSA epitopes bound to avidin-coated ELISA plates via a biotin labelled polyethylene glycol-glycine spacer to increase rotation and ensure each amino acid side chain could freely interact with their antibodies. 79/113 (70%) of samples from Brazil, Bolivia, and Argentina recognised the TSSA epitope common to lineages TcII/TcV/TcVI. Comparison with clinical information showed that a higher proportion of Brazilian TSSApep-II/V/VI responders had ECG abnormalities than non-responders (38% vs 17%; p<0.0001). Among northern chagasic sera 4/20 (20%) from Ecuador reacted with this peptide; 1/12 Venezuelan and 1/34 Colombian samples reacted with TSSApep-IV. In addition, a proposed TcI-specific epitope, described elsewhere, was demonstrated here to be highly conserved across lineages and therefore not applicable to lineage-specific serology. CONCLUSIONS/SIGNIFICANCE: These results demonstrate the considerable potential for synthetic peptide serology to investigate the infection history of individuals, geographical and clinical associations of T. cruzi lineages.

The gp82 surface molecule of Trypanosoma cruzi metacyclic forms.

Gp82 is a surface glycoprotein expressed in Trypanosoma cruzi metacyclic trypomastigotes, the parasite forms from the insect vector that initiate infection in the mammalian host. Studies with metacyclic forms generated in vitro, as counterparts of insect-borne parasites, have shown that gp82 plays an essential role in host cell invasion and in the establishment of infection by the oral route. Among the gp82 properties relevant for infection are the gastric mucin-binding capacity and the ability to induce the target cell signaling cascades that result in actin cytoskeleton disruption and lysosome exocytosis, events that facilitate parasite internalization. The gp82 sequences from genetically divergent T. cruzi strains are highly conserved, displaying >90 % identity. Both the host cell-binding sites, as well as the gastric mucin-binding sequence of gp82, are localized in the C-terminal domain of the molecule. In the gp82 structure model, the main cell-binding site consists of an α-helix, which connects the N-terminal ß-propeller domain to the C-terminal ß-sandwich domain, where the second cell binding site is nested. The two cell binding sites are fully exposed on gp82 surface. Downstream and close to the α-helix is the gp82 gastric mucin-binding site, which is partially exposed. All available data support the notion that gp82 is structurally suited for metacyclic trypomastigote invasion of host cells and for initiating infection by the oral route.

Trypanosoma vivax GM6 antigen: a candidate antigen for diagnosis of African animal trypanosomosis in cattle.

BACKGROUND: Diagnosis of African animal trypanosomosis is vital to controlling this severe disease which hampers development across 10 million km(2) of Africa endemic to tsetse flies. Diagnosis at the point of treatment is currently dependent on parasite detection which is unreliable, and on clinical signs, which are common to several other prevalent bovine diseases. METHODOLOGY/PRINCIPLE FINDINGS: the repeat sequence of the GM6 antigen of Trypanosoma vivax (TvGM6), a flagellar-associated protein, was analysed from several isolates of T. vivax and found to be almost identical despite the fact that T. vivax is known to have high genetic variation. The TvGM6 repeat was recombinantly expressed in E. coli and purified. An indirect ELISA for bovine sera based on this antigen was developed. The TvGM6 indirect ELISA had a sensitivity of 91.4% (95% CI: 91.3 to 91.6) in the period following 10 days post experimental infection with T. vivax, which decreased ten-fold to 9.1% (95% CI: 7.3 to 10.9) one month post treatment. With field sera from cattle infected with T. vivax from two locations in East and West Africa, 91.5% (95% CI: 83.2 to 99.5) sensitivity and 91.3% (95% CI: 78.9 to 93.1) specificity was obtained for the TvGM6 ELISA using the whole trypanosome lysate ELISA as a reference. For heterologous T. congolense field infections, the TvGM6 ELISA had a sensitivity of 85.1% (95% CI: 76.8 to 94.4). CONCLUSION/SIGNIFICANCE: this study is the first to analyse the GM6 antigen of T. vivax and the first to test the GM6 antigen on a large collection of sera from experimentally and naturally infected cattle. This study demonstrates that the TvGM6 is an excellent candidate antigen for the development of a point-of-treatment test for diagnosis of T. vivax, and to a lesser extent T. congolense, African animal trypanosomosis in cattle.

Mechanism of Trypanosoma brucei gambiense resistance to human serum.

The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease in cattle. Human immunity to some African trypanosomes is due to two serum complexes designated trypanolytic factors (TLF-1 and -2), which both contain haptoglobin-related protein (HPR) and apolipoprotein LI (APOL1). Whereas HPR association with haemoglobin (Hb) allows TLF-1 binding and uptake via the trypanosome receptor TbHpHbR (ref. 5), TLF-2 enters trypanosomes independently of TbHpHbR (refs 4, 5). APOL1 kills trypanosomes after insertion into endosomal/lysosomal membranes. Here we report that T. b. gambiense resists TLFs via a hydrophobic ß-sheet of the T. b. gambiense-specific glycoprotein (TgsGP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According to such a multifactorial defence mechanism, transgenic expression of T. b. brucei TbHpHbR in T. b. gambiense did not cause parasite lysis in normal human serum. However, these transgenic parasites were killed in hypohaptoglobinaemic serum, after high TLF-1 uptake in the absence of haptoglobin (Hp) that competes for Hb and receptor binding. TbHpHbR inactivation preventing high APOL1 loading in hypohaptoglobinaemic serum may have evolved because of the overlapping endemic area of T. b. gambiense infection and malaria, the main cause of haemolysis-induced hypohaptoglobinaemia in western and central Africa.

Carbohydrate-binding agents act as potent trypanocidals that elicit modifications in VSG glycosylation and reduced virulence in Trypanosoma brucei.

The surface of Trypanosoma brucei is covered by a dense coat of glycosylphosphatidylinositol-anchored glycoproteins. The major component is the variant surface glycoprotein (VSG) which is glycosylated by both paucimannose and oligomannose N-glycans. Surface glycans are poorly accessible and killing mediated by peptide lectin-VSG complexes is hindered by active endocytosis. However, contrary to previous observations, here we show that high-affinity carbohydrate binding agents bind to surface glycoproteins and abrogate growth of T. brucei bloodstream forms. Specifically, binding of the mannose-specific Hippeastrum hybrid agglutinin (HHA) resulted in profound perturbations in endocytosis and parasite lysis. Prolonged exposure to HHA led to the loss of triantennary oligomannose structures in surface glycoproteins as a result of genetic rearrangements that abolished expression of the oligosaccharyltransferase TbSTT3B gene and yielded novel chimeric enzymes. Mutant parasites exhibited markedly reduced infectivity thus demonstrating the importance of specific glycosylation patterns in parasite virulence.

Structure of the trypanosome haptoglobin-hemoglobin receptor and implications for nutrient uptake and innate immunity.

African trypanosomes are protected by a densely packed surface monolayer of variant surface glycoprotein (VSG). A haptoglobin-hemoglobin receptor (HpHbR) within this VSG coat mediates heme acquisition. HpHbR is also exploited by the human host to mediate endocytosis of trypanolytic factor (TLF)1 from serum, contributing to innate immunity. Here, the crystal structure of HpHbR from Trypanosoma congolense has been solved, revealing an elongated three α-helical bundle with a small membrane distal head. To understand the receptor in the context of the VSG layer, the dimensions of Trypanosoma brucei HpHbR and VSG have been determined by small-angle X-ray scattering, revealing the receptor to be more elongated than VSG. It is, therefore, likely that the receptor protrudes above the VSG layer and unlikely that the VSG coat can prevent immunoglobulin binding to the receptor. The HpHb-binding site has been mapped by single-residue mutagenesis and surface plasmon resonance. This site is located where it is readily accessible above the VSG layer. A single HbHpR polymorphism unique to human infective T. brucei gambiense has been shown to be sufficient to reduce binding of both HpHb and TLF1, modulating ligand affinity in a delicate balancing act that allows nutrient acquisition but avoids TLF1 uptake.

Structural basis of the interaction of a Trypanosoma cruzi surface molecule implicated in oral infection with host cells and gastric mucin.

Host cell invasion and dissemination within the host are hallmarks of virulence for many pathogenic microorganisms. As concerns Trypanosoma cruzi, which causes Chagas disease, the insect vector-derived metacyclic trypomastigotes (MT) initiate infection by invading host cells, and later blood trypomastigotes disseminate to diverse organs and tissues. Studies with MT generated in vitro and tissue culture-derived trypomastigotes (TCT), as counterparts of insect-borne and bloodstream parasites, have implicated members of the gp85/trans-sialidase superfamily, MT gp82 and TCT Tc85-11, in cell invasion and interaction with host factors. Here we analyzed the gp82 structure/function characteristics and compared them with those previously reported for Tc85-11. One of the gp82 sequences identified as a cell binding site consisted of an α-helix, which connects the N-terminal ß-propeller domain to the C-terminal ß-sandwich domain where the second binding site is nested. In the gp82 structure model, both sites were exposed at the surface. Unlike gp82, the Tc85-11 cell adhesion sites are located in the N-terminal ß-propeller region. The gp82 sequence corresponding to the epitope for a monoclonal antibody that inhibits MT entry into target cells was exposed on the surface, upstream and contiguous to the α-helix. Located downstream and close to the α-helix was the gp82 gastric mucin binding site, which plays a central role in oral T. cruzi infection. The sequences equivalent to Tc85-11 laminin-binding sites, which have been associated with the parasite ability to overcome extracellular matrices and basal laminae, was poorly conserved in gp82, compatible with its reduced capacity to bind laminin. Our study indicates that gp82 is structurally suited for MT to initiate infection by the oral route, whereas Tc85-11, with its affinity for laminin, would facilitate the parasite dissemination through diverse organs and tissues.

Characterization of Trypanosoma brucei gambiense variant surface glycoprotein LiTat 1.5.

At present, all available diagnostic antibody detection tests for Trypanosoma brucei gambiense human African trypanosomiasis are based on predominant variant surface glycoproteins (VSGs), such as VSG LiTat 1.5. During investigations aiming at replacement of the native VSGs by recombinant proteins or synthetic peptides, the sequence of VSG LiTat 1.5 was derived from cDNA and direct N-terminal amino acid sequencing. Characterization of the VSG based on cysteine distribution in the amino acid sequence revealed an unusual cysteine pattern identical to that of VSG Kinu 1 of T. b. brucei. Even though both VSGs lack the third of four conserved cysteines typical for type A N-terminal domains, they can be classified as type A.