Structural analysis of an Echinococcus granulosus actin-fragmenting protein by small-angle x-ray scattering studies and molecular modeling

The Echinococcus granulosus actin filament-fragmenting protein (EgAFFP) is a three domain member of the gelsolin family of proteins, which is antigenic to human hosts. These proteins, formed by three or six conserved domains, are involved in the dynamic rearrangements of the cytoskeleton, being responsible for severing and capping actin filaments and promoting nucleation of actin monomers. Various structures of six domain gelsolin-related proteins have been investigated, but little information on the structure of three domain members is available. In this work, the solution structure of the three domain EgAFFP has been investigated through small-angle x-ray scattering (SAXS) studies. EgAFFP exhibits an elongated molecular shape. The radius of gyration and the maximum dimension obtained by SAXS were, respectively, 2.52 +/- 0.01 nm and 8.00 +/- 1.00 nm, both in the absence and presence of Ca2+. Two different molecular homology models were built for EgAFFP, but only one was validated through SAXS studies. The predicted structure for EgAFFP consists of three repeats of a central beta-sheet sandwiched between one short and one long alpha-helix. Possible implications of the structure of EgAFFP upon actin binding are discussed.     

Echinococcus granulosus antigen B structure: subunit composition and oligomeric states

Background

Antigen 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 Findings

The 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/Significance

For 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.     

Hydrogen peroxide production and myo‐inositol metabolism as important traits for virulence of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia. In our previous work, we reconstructed the metabolic models of this species along with two other mycoplasmas from the respiratory tract of swine: Mycoplasma hyorhinis, considered less pathogenic but which nonetheless causes disease and Mycoplasma flocculare, a commensal bacterium. We identified metabolic differences that partially explained their different levels of pathogenicity. One important trait was the production of hydrogen peroxide from the glycerol metabolism only in the pathogenic species. Another important feature was a pathway for the metabolism of myo‐inositol in M. hyopneumoniae. Here, we tested these traits to understand their relation to the different levels of pathogenicity, comparing not only the species but also pathogenic and attenuated strains of M. hyopneumoniae. Regarding the myo‐inositol metabolism, we show that only M. hyopneumoniae assimilated this carbohydrate and remained viable when myo‐inositol was the primary energy source. Strikingly, only the two pathogenic strains of M. hyopneumoniae produced hydrogen peroxide in complex medium. We also show that this production was dependent on the presence of glycerol. Although further functional tests are needed, we present in this work two interesting metabolic traits of M. hyopneumoniae that might be directly related to its enhanced virulence.     

Drosophila mediopunctata P elements: a new example of horizontal transfer.

Sequences homologous to the P element of Drosophila melanogaster were previously identified in Drosophila mediopunctata, a member of the tripunctata group, subgenus Drosophila. We report here that the P element is present in about three to five copies in the D. mediopunctata genome. While one of the insertion sites appears to be fixed, others may be polymorphic, indicating relatively recent P element activity. Phylogenetic analysis revealed that the D. mediopunctata element belongs to the canonical subfamily of P elements and that divergence of the D. mediopunctata element from other members of this subfamily ranges from 2% to 5% at the nucleotide level. This is the first report of a canonical P element outside the subgenus Sophophora. Based primarily on the striking incongruence between P element and host species phylogenies, the presence of a canonical P element in D. mediopunctata is most likely explained by horizontal transfer between species.     

Self-assembly and structural characterization of Echinococcus granulosus antigen B recombinant subunit oligomers

Echinococcus granulosus antigen B is an oligomeric protein of 120-160 kDa composed by 8-kDa (AgB8) subunits. Here, we demonstrated that the AgB8 recombinant subunits AgB8/1, AgB8/2 and AgB8/3 are able to self-associate into high order homo-oligomers, showing similar properties to that of parasite-produced AgB, making them valuable tools to study AgB structure. Dynamic light scattering, size exclusion chromatography and cross-linking assays revealed approximately 120- to 160-kDa recombinant oligomers, with a tendency to form populations with different aggregation states. Recombinant oligomers showed helical circular dichroism spectra and thermostability similar to those of purified AgB. Cross-linking and limited proteolysis experiments indicated different degrees of stability and compactness between the recombinant oligomers, with the AgB8/3 one showing a more stable and compact structure. We have also built AgB8 subunit structural models in order to predict the surfaces possibly involved in electrostatic and hydrophobic interactions during oligomerization.     

The Treatment with Interleukin 17 Inhibitors and Immune-Mediated Inflammatory Diseases

IL-17 inhibitors (IL-17i) are medicines used to treat dermatological and rheumatic diseases They belong to a class of medicines called biological disease-modifying anti-rheumatic drugs (bDMARDs). This class of drugs has had a major impact on the therapy of autoimmune diseases, being much safer and more effective than treatment with small molecules. At the same time, they have highly beneficial effects on skin and joint changes, and their efficacy has been extensively monitored and demonstrated in numerous clinical trials. More and more such drugs are still being discovered today to ensure the best possible treatment of these patients, but more frequently and relatively constantly three agents are used. Two of them (Secukinumab and Ixekizumab) inhibit IL-17A directly, and the third, Brodamulab, inhibits the IL-17A receptor. Although they are extremely effective in the treatment of these diseases, sometimes their administration has been associated with paradoxical effects, i.e., there is an exacerbation of the inflammatory process. Tough, clinical trials of IL-17i have described cases of exacerbation or even onset of inflammatory bowel disease (IBD), such as Crohn’s disease and ulcerative colitis, after administration of these drugs in patients previously diagnosed with psoriasis (PS), psoriatic arthritis (PsA), or ankylosing spondylitis (AS). The pathophysiological mechanism of action is not well understood at present. One explanation would be that this hyperreactive inflammatory process would be triggered by Interferon 1 derived from dendritic plasma cells. Even though there are many reports in the recent literature about the role of IL17i in the onset of IBD, conclusions of studies do not converge. Some of them show an increased incidence of IBD in patients treated with IL17i, while some others affirm their safety of them. In the near future we will surely have more data emerging from ongoing meta-analyses regarding safety of use IL17i in patients who are at risk of developing IBD. Clinical and paraclinical evaluation (inflammatory intestinal markers) are carefully advised before recommending treatment with IL-17i and after initiation of treatment, and prospective surveillance by clinical and biomarkers of patients treated with IL-17i is absolutely essential to capture the onset of IBD.     

The Echinococcus granulosus antigen B shows a high degree of genetic variability

Echinococcus granulosus larvae secret a polymeric lipoprotein known as antigen B (AgB) into the metacestode hydatid fluid. Three similar AgB subunits have been previously identified (AgB1, AgB2, and AgB3), and their respective genes isolated, but the actual number of genes encoding AgB subunits remains uncertain. In this study, we characterize the variability of genes encoding the AgB2 subunit, using PCR and RT-PCR followed by cloning and sequencing. We have analyzed 32 cDNA and 34 genomic sequences from a single metacestode, showing a high degree of sequence polymorphism. In addition, we have identified a possibly new AgB subunit, which we call AgB4. Additionally, we describe an AgB2 genomic clone lacking (i) a segment corresponding to the intron and (ii) a short, 45 bp sequence within exon II. The 45 bp segment encompasses the conserved splicing signals and corresponds to a highly conserved insect promoter motif.