Echinococcus granulosus Antigen B Structure: Subunit Composition and Oligomeric States

Abstract

BackgroundAntigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states.Methodology/Principal FindingsThe subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability.Conclusions/SignificanceFor the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.

An Easy and Efficient Method for Native and Immunoreactive Echinococcus granulosus Antigen 5 Enrichment from Hydatid Cyst Fluid

“…More suitable to routine analysis is the indirect ELISA, which combines reduced turnaround times, ease of execution and result evaluation, with an increase in sensitivity, enabling its application also for the wide screening of high-risk categories, in differential diagnosis during early phase of infection, and in patient follow-up for recurrence of infections. In addition, the native Ag5 complex is a large molecule; size exclusion chromatography and subsequent characterization of the fraction by mass spectrometry and western blot analysis revealed an Ag5 apparent molecular weight of at least 600 kDa, as recently suggested by Monteiro and coworkers [47], instead of the generally reported 400 kDa [19]–[20]. In fact, Ag5 has also been identified in very high molecular weight bands (bands a and b of Figure 5A), confirming the stability of the oligomer that does no dissociate even in the strongly denaturing conditions adopted for SDS-PAGE experiments.…”

Immunoblotting with Human Native Antigen Shows Stage-Related Sensitivity in the Serodiagnosis of Hepatic Cystic Echinococcosis

“…[17][18][19][20] There is also evidence that AgB genes are differentially expressed in the different stages of the parasite life cycle, and parasite tissues during development in the host. 9,16,21,22 Ag5 is an immunogenic glycoprotein expressed in all stages of the parasite life cycle, and is secreted from the surface of the worm. 9,23 -25 Ag5 is present in elevated concentration in HCF, and it may have a relevant function in the development of the metacestode.…”

Characterisation of Antigen B Protein Species Present in the Hydatid Cyst Fluid of Echinococcus canadensis G7 Genotype

“…multilocularis genomes confirmed that this scenario is highly conserved among Echinococcus species [21]. The mature protein products of these genes are small (around 8 kDa in mass), α helix-rich secreted polypeptides, with ability to self-assembly generating high-molecular-mass oligomers [22,23]; they are thus named AgB8/1 to AgB8/5 subunits. The native antigen, the recombinant AgB8/1 and AgB8/2 subunits, as well as various synthetic peptides derived from them, have shown to be valuable for CE diagnosis [24–26]; all of them have shown similar diagnostic performance in comparison with crude HF preparations, but in some clinical studies recombinant AgB8 subunits (rAgB8) seem to yield better specificity with little or no loss in sensitivity [27–30].…”

“…In the larva of E . granulosus s.s. all AgB genes were found to be expressed at mRNA level [31], even though only AgB8/1 to AgB8/4 protein products have been certainly detected in HF [23]. On the other hand, no evidence of AgB5 expression or of the generation of AgB8/2 and AgB8/5 was achieved in E .…”

“…Furthermore, in contrast with E . granulosus s.s. [23], AgB8/2 was not detected in E . canadensis G7 genotype, supporting the concept that AgB2 is a pseudogene in this species.…”