Echinococcus granulosus Antigen B Structure: Subunit Composition and Oligomeric States

Antigen B (AgB) is the major secretory protein of the Echinococcus granulosus hydatid cyst, the causative agent of cystic hydatid disease. Structurally, AgB is a multisubunit protein formed by 8-kDa subunits, but it is not known which subunits are secreted by a single parasite (cyst) and how they interact in the formation of distinct AgB oligomeric states. Here, we investigated AgB subunit composition and oligomeric states in individual samples from bovine and human cysts. We identified AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits in AgB oligomers of all samples analyzed. Quantitative and qualitative differences in the expression of AgB subunits were observed within and between samples. Using recombinant subunits as models, we showed that AgB subunits form distinct oligomeric states, with a rAgB8/3>rAgB8/2>rAgB8/1 maximum size relation. We also demonstrated by different experimental approaches that rAgB8/3 oligomers are more similar, both in size and morphology, to those observed for E. granulosus AgB. Overall, we provided experimental evidences that AgB is composed of different subunits within a single cyst, and that subunits have different abundances and oligomerization properties. These issues are important for the understanding of AgB expression and structure variations, and their impact for the host-parasite cross-talk

Serological diagnosis of echinococcosis: the diagnostic potential of native antigens

PURPOSE: Human alveolar (AE) and cystic echinococcosis (CE) caused by the metacestode stages of Echinococcus multilocularis and E. granulosus, respectively, lack pathognomonic clinical signs. Diagnosis therefore relies on the results of imaging and serological studies. The primary goal of this study was to evaluate the efficacy of several easy-to-produce crude or partially purified E. granulosus and E. multilocularis metacestode-derived antigens as tools for the serological diagnosis and differential diagnosis of patients suspicious for AE or CE. METHODS: The sera of 51 treatment-naïve AE and 32 CE patients, 98 Swiss blood donors and 38 patients who were initially suspicious for echinococcosis but suffering from various other liver diseases (e.g., liver neoplasia, etc.) were analysed. RESULTS: According to the results of enzyme-linked immunosorbent assays (ELISA), metacestode-derived antigens of E. granulosus had sensitivities varying from 81 to 97% and >99.9% for the diagnosis of CE and AE, respectively. Antigens derived from E. multilocularis metacestodes had sensitivities ranging from 84 to 91% and >99.9% for the diagnosis of CE and AE, respectively. Specificities ranged from 92 to >99.9%. Post-test probabilities for the differential diagnosis of AE from liver neoplasias, CE from cystic liver lesions, and screening for AE in Switzerland were around 95, 86 and 2.2%, respectively. Cross-reactions with antibodies in sera of patients with other parasitic affections (fasciolosis, schistosomosis, amebosis, cysticercosis, and filarioses) did occur at variable frequencies, but could be eliminated through the use of confirmatory testing. CONCLUSIONS: Different metacestode-derived antigens of E. granulosus and E. multilocularis are valuable, widely accessible, and cost-efficient tools for the serological diagnosis of echinococcosis. However, confirmatory testing is necessary, due to the lack of species specificity and the occurrence of cross-reactions to other helminthic diseases

Serological Diagnosis of Lung Cystic Hydatid Disease Using the Synthetic p176 Peptide

Cystic hydatid disease (CHD) is a worldwide zoonosis caused by the larval stage of the dog tapeworm Echinococcus granulosus. Diagnosis is based on imagenological tools (abdominal ultrasound, chest X-rays, or computed tomography [CT] scan). Serological antibody-detecting assays, using diverse native antigens, have been used as a supportive diagnostic tool, but their sensitivities and specificities differ greatly. The use of synthetic peptides as antigens should provide more reliability and allow better assessment and comparison of test formats and case series. The synthetic peptide p176, corresponding to the N-terminal extreme of the subunit of antigen B (AgB8/1), has shown promising performances for diagnosis of CHD. We evaluated the performance of the synthetic peptide p176 for the diagnosis of pulmonary hydatid disease in an enzyme-linked immunosorbent assay (ELISA) format. Sixty-one serum samples from patients with a diagnosis of pulmonary hydatidosis confirmed by surgery and 128 from healthy volunteers were tested. The overall sensitivity and specificity of the p176 ELISA for lung CHD were 78.69% and 96.88%, respectively. On bivariate analysis, positive serum antibody reactions were associated with the presence of complications and with the number of cysts (single/multiple). Only the presence of persistent complications significantly associated with seropositivity on multivariate logistic regression analysis (odds ratio [OR], 9.58; 95% confidence interval [CI], 2.15 to 42.6; P = 0.003). The p176 ELISA performs well for the diagnosis of lung CHD and adds an easily reproducible diagnostic assay to the existing diagnostic tools

Characterisation of the Native Lipid Moiety of Echinococcus granulosus Antigen B

Antigen B (EgAgB) is the most abundant and immunogenic antigen produced by the larval stage (metacestode) of Echinococcus granulosus. It is a lipoprotein, the structure and function of which have not been completely elucidated. EgAgB apolipoprotein components have been well characterised; they share homology with a group of hydrophobic ligand binding proteins (HLBPs) present exclusively in cestode organisms, and consist of different isoforms of 8-kDa proteins encoded by a polymorphic multigene family comprising five subfamilies (EgAgB1 to EgAgB5). In vitro studies have shown that EgAgB apolipoproteins are capable of binding fatty acids. However, the identity of the native lipid components of EgAgB remains unknown. The present work was aimed at characterising the lipid ligands bound to EgAgB in vivo. EgAgB was purified to homogeneity from hydatid cyst fluid and its lipid fraction was extracted using chloroform∶methanol mixtures. This fraction constituted approximately 40–50% of EgAgB total mass. High-performance thin layer chromatography revealed that the native lipid moiety of EgAgB consists of a variety of neutral (mainly triacylglycerides, sterols and sterol esters) and polar (mainly phosphatidylcholine) lipids. Gas-liquid chromatography analysis showed that 16∶0, 18∶0 and 18∶1(n-9) are the most abundant fatty acids in EgAgB. Furthermore, size exclusion chromatography coupled to light scattering demonstrated that EgAgB comprises a population of particles heterogeneous in size, with an average molecular mass of 229 kDa. Our results provide the first direct evidence of the nature of the hydrophobic ligands bound to EgAgB in vivo and indicate that the structure and composition of EgAgB lipoprotein particles are more complex than previously thought, resembling high density plasma lipoproteins. Results are discussed considering what is known on lipid metabolism in cestodes, and taken into account the Echinococcus spp. genomic information regarding both lipid metabolism and the EgAgB gene family