Lipid-free Antigen B subunits from echinococcus granulosus: oligomerization, ligand binding, and membrane interaction properties

Background: The hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied.<p></p> Methodology/Principal Findings: Lipid-free EgAgB subunits obtained by reverse-phase HPLC were used to analyse their oligomerization, ligand binding and membrane interaction properties. Size exclusion chromatography and cross-linking experiments showed that EgAgB8/2 and EgAgB8/3 can self-associate, suggesting that lipids are not required for oligomerization. Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues. Analysis of fatty acid transfer to phospholipid vesicles demonstrated that EgAgB8/2 and EgAgB8/3 are potentially capable of transferring fatty acids to membranes, and that the efficiency of transfer is dependent on the surface charge of the vesicles.<p></p> Conclusions/Significance: We show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid acquisition and/or transport between parasite tissues. These results may therefore indicate vulnerabilities open to targeting by new types of drugs for hydatidosis therapy.<p></p&gt

Diversity in the structures and ligand binding sites of nematode fatty acid and retinol binding proteins revealed by Na-FAR-1 from Necator americanus

Fatty acid and retinol binding proteins (FARs) comprise a family of unusual α-helix rich lipid binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein (Ce-FAR-7) is from a subfamily of FARs that does not appear to be important at the host-parasite interface. We have therefore examined Na-FAR-1 from the blood-feeding intestinal parasite of humans, Necator americanus . The three dimensional structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, respectively, reveals an a-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand binding cavity and an additional C-terminal a-helix. Titration of apo -Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein:ligand complexes can be formed. Na-FAR-1, and possibly other FARs, may have a wider repertoire for hydrophobic ligand binding, as confirmed here by our finding that a range of neutral and polar lipids co-purify with the bacterial recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male

Structure and ligand binding of As-p18, an extracellular fatty acid binding protein from the eggs of a parasitic nematode

Intracellular lipid-binding proteins (iLBPs) of the fatty acid-binding protein (FABP) family of animals transport, mainly fatty acids or retinoids, are confined to the cytosol and have highly similar 3D structures. In contrast, nematodes possess fatty acid-binding proteins (nemFABPs) that are secreted into the perivitelline fluid surrounding their developing embryos. We report structures of As-p18, a nemFABP of the large intestinal roundworm Ascaris suum, with ligand bound, determined using X-ray crystallography and nuclear magnetic resonance spectroscopy. In common with other FABPs, As-p18 comprises a ten β-strand barrel capped by two short α-helices, with the carboxylate head group of oleate tethered in the interior of the protein. However, As-p18 exhibits two distinctive longer loops amongst β-strands not previously seen in a FABP. One of these is adjacent to the presumed ligand entry portal, so it may help to target the protein for efficient loading or unloading of ligand. The second, larger loop is at the opposite end of the molecule and has no equivalent in any iLBP structure yet determined. As-p18 preferentially binds a single 18-carbon fatty acid ligand in its central cavity but in an orientation that differs from iLBPs. The unusual structural features of nemFABPs may relate to resourcing of developing embryos of nematodes

Vigilancia epidemiológica de parasitosis zoonóticas en un área centinela desde la perspectiva de Una Salud

El abordaje de las enfermedades zoonóticas debe realizarse desde un enfoque multisectorial. Desde el concepto de Una Salud, las acciones realizadas por un solo sector no resultan eficaces para controlar este tipo de infecciones. En ese sentido, los distintos agentes de enfermedades transmisibles zoonóticas, afectan tanto a animales como a humanos y habitualmente el ecosistema actúa como reservorio o transmisor directo o indirecto, en la interfaz hombre-animal-ambiente. Los caninos pueden diseminar con sus heces enteroparásitos transmisibles a humanos y como animales centinela, pueden utilizarse para realizar vigilancia de la circulación de patógenos. Algunas helmintiasis, varias protozoosis y el alga parásita Blastocystis sp., son comunes en caninos y humanos. Nematodes del género Toxocara spp., enteroparásito de animales, ocasionan en personas toxocarosis, enfermedad de elevada seroprevalencia en la ciudad de La Plata y otras regiones. Sus formas neurológica y ocular tienen generalmente serias consecuencias. Su presencia en humanos se ve influenciada favorablemente por el lugar de residencia y su tejido suburbano. Giardia lamblia, enteroparásito zoonótico, ocasiona síndrome de malabsorción y modificación de moléculas de fármacos, también se correlaciona giardiasis con enfermedad de Whipple y otros disturbios gastroentéricos. El enfoque "Una salud" tiene en su estructura elementos esenciales, ellos son voluntad política (compromiso con las normas internacionales y los Objetivos de Desarrollo Sostenible), planes de financiación sostenibles; comunicación (entre sectores y disciplinas a nivel internacional, regional, nacional y subnacional). Los aprendizajes socialmente productivos y significativos “resignifica el papel social, cultural y económico social del conocimiento” y son aquellos “modifican a los sujetos enseñándoles a transformar su naturaleza y su cultura, enriqueciendo el capital cultural de la sociedad y de las comunidades” (Orozco B, 2009: 88). Los objetivos de este trabajo fueron: Realizar vigilancia y alertas tempranas en cuanto a parasitosis humanas animales y zoonóticas. Ejecutar acciones tendientes a su control. Implementar aprendizajes socialmente significativos.Facultad de Ciencias Veterinaria

The unusual lipid binding proteins of parasitic helminths and their potential roles in parasitism and as therapeutic targets

In this review paper we aim at presenting the current knowledge on structural aspects of soluble lipid binding proteins (LBPs) found in parasitic helminths and to discuss their potential role as novel drug targets. Helminth parasites produce and secrete a great variety of LBPs that may participate in the acquisition of nutrients from their host, such as fatty acids and cholesterol. It is also postulated that LBPs might interfere in the regulation of the host׳s immune response by sequestering lipidic intermediates or delivering bioactive lipids. A detailed comprehension of the structure of these proteins, as well as their interactions with ligands and membranes, is important to understand host–parasite relationships that they may mediate. This information could also contribute to determining the role that these proteins may play in the biology of parasitic helminths and how they modulate the immune systems of their hosts, and also towards the development of new therapeutics and prevention of the diseases caused by these highly pathogenic parasites

Effect of acceptor membrane concentration on 12-AS transfer from EgAgB8/2 and EgAgB8/3 to different SUVs.

<p>Transfer of 12-AS from EgAgB8/2 or EgAgB8/3 to SUVs was monitored by adding SUVs in a molar ratio of 10:1, 20:1 and 40:1 (SUVs/Protein) to the complex EgAgB8/2:12AS or EgAgB8/3:12AS (15:1 mol:mol). <b>(A)</b> Representative kinetic trace obtained when combining EgAgB8–12AS with NBD-PC-containing vesicles (molar ratio SUV/Protein of 10:1). Photobleaching control adding TBS instead of NBD-PC/SUVs is shown. <b>(B)</b> SUVs containing 100% EPC; <b>(C)</b> 75% EPC, 25% PS or <b>(D)</b> 75% EPC, 25% CL were used. For each experimental condition at least five replicates of the kinetic curves were done. All curves were well-described by a single exponential function to obtain each transfer rate value employing SigmaPlot software. Transfer rates (mean ± SD) of three independent experiments are reported. Statistical analysis of the data was carried out employing ANOVA followed by Tukey's Post Hoc Test from GraphPad Prism software.</p

Far UV CD-spectra of lipid-free EgAgB8 subunits.

<p>After purification and delipidation, rEgAgB8/2 and rEgAgB8/3 were analysed by CD for secondary structural content. Both subunits showed a predominant alpha-helical structure with double minima at 208 and 222 nm in agreement with their predicted structures [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003552#pntd.0003552.ref014" target="_blank">14</a>,<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003552#pntd.0003552.ref033" target="_blank">33</a>,<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003552#pntd.0003552.ref059" target="_blank">59</a>]. One representative experiment of two is shown for both EgAgB8 subunits.</p

Equilibrium partition of 12-AS between EgAgB8 subunits and SUVs.

<p>The K_p for 12-AS partitioning was determined by measuring 12-AS fluorescence after addition of SUVs into a solution containing 7.5 μM EgAgB8/2 or EgAgB8/3 and 0.5 μM 12-AS in buffer TBS at 25°C (15:1 mol:mol). <b>(A)</b>, <b>(B)</b> Emission spectra changes of 12AS bound to EgAgB8/2 or EgAgB8/3 respectively, upon incremental addition of EPC-SUVs containing NBD-PC. <b>(C)</b>, <b>(D)</b> Changes in relative 12-AS fluorescence were used to obtained K_p values, fitting the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003552#pntd.0003552.e002" target="_blank">Equation 2</a> described in Materials and Methods to the data, employing <i>Solver Add-In</i> from Excel (solid line). K_p values of 0.62 ± 0.09 and 0.88 ± 0.15 were obtained for rEgAgB8/2 and rEgAgB8/3, respectively. One representative experiment of two is shown for both EgAgB8 subunits.</p