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 1.4Å resolution crystal structure of variant VSG3 manifests heretofore unappreciated divergence in the tertiary fold and oligomeric state. The structure also reveals an O linked carbohydrate on the top surface of VSG3, a modification previously unknown in African trypanosomes. Mass spectrometric analysis indicates that this O -glycosylation site is heterogeneously occupied in VSG3 by 0 to 3 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 posttranslational modifications with immunomodulatory impact

Antibody isotype analysis of malaria-nematode co-infection: problems and solutions associated with cross-reactivity

<p>Abstract</p> <p>Background</p> <p>Antibody isotype responses can be useful as indicators of immune bias during infection. In studies of parasite co-infection however, interpretation of immune bias is complicated by the occurrence of cross-reactive antibodies. To confidently attribute shifts in immune bias to the presence of a co-infecting parasite, we suggest practical approaches to account for antibody cross-reactivity. The potential for cross-reactive antibodies to influence disease outcome is also discussed.</p> <p>Results</p> <p>Utilising two murine models of malaria-helminth co-infection we analysed antibody responses of mice singly- or co-infected with <it>Plasmodium chabaudi chabaudi </it>and <it>Nippostrongylus brasiliensis </it>or <it>Litomosoides sigmodontis</it>. We observed cross-reactive antibody responses that recognised antigens from both pathogens irrespective of whether crude parasite antigen preparations or purified recombinant proteins were used in ELISA. These responses were not apparent in control mice. The relative strength of cross-reactive versus antigen-specific responses was determined by calculating antibody titre. In addition, we analysed antibody binding to periodate-treated antigens, to distinguish responses targeted to protein versus carbohydrate moieties. Periodate treatment affected both antigen-specific and cross-reactive responses. For example, malaria-induced cross-reactive IgG1 responses were found to target the carbohydrate component of the helminth antigen, as they were not detected following periodate treatment. Interestingly, periodate treatment of recombinant malaria antigen Merozoite Surface Protein-1₁₉(MSP-1₁₉) resulted in increased detection of antigen-specific IgG2a responses in malaria-infected mice. This suggests that glycosylation may have been masking protein epitopes and that periodate-treated MSP-1₁₉may more closely reflect the natural non-glycosylated antigen seen during infection.</p> <p>Conclusions</p> <p>In order to utilize antibody isotypes as a measure of immune bias during co-infection studies, it is important to dissect antigen-specific from cross-reactive antibody responses. Calculating antibody titre, rather than using a single dilution of serum, as a measure of the relative strength of the response, largely accomplished this. Elimination of the carbohydrate moiety of an antigen that can often be the target of cross-reactive antibodies also proved useful.</p

Metabolic labeling of surface neo-sialylglyconjugates catalyzed by trypanosoma cruzi trans-sialidase

Trypanosoma cruzi, the protozoan agent of Chagas disease, has evolved an innovative metabolic pathway by which protective sialic acid (SA) residues are scavenged from host sialylglycoconjugates and transferred onto parasite surface mucin-like molecules (or surface glycoconjugates from host target cells) by means of a unique trans-sialidase (TS) enzyme. TS-induced changes in the glycoprotein sialylation profile of both parasite and host cells are crucial for the establishment of a persistent T. cruzi infection and for the development of Chagas disease-associated pathogenesis. In this chapter, we describe a novel metabolic labeling method developed in our labs that enables straightforward identification and molecular characterization of SA acceptors of the TS-catalyzed reaction.Fil: Carlevaro, Giannina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Lantos, Andrés Bernardo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Laboratorio Dr. Lantos; ArgentinaFil: Canepa, Gaspar Exequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Laboratory of Malaria and Vector Research National Institute of Allergy and Infectious Diseases, National Institutes of Health; Estados UnidosFil: Camara, Maria de Los Milagros. Universidad Argentina de la Empresa; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Somoza, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Buscaglia, Carlos Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Campetella, Oscar Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Mucci, Juan Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin