Nanoparticle formulation for the development of a dog nanovaccine against Cystic Echinococcosis.

Cystic Echinococcosis is a cosmopolitan zoonosis closely linked to poverty and ignorance. It affects both cattle and humans, causing significant losses to both human and animal health. To date, there is no effective way to combat this. Our proposal focused on the formulation of poly (lactic-co-glycolic acid (PLGA) and Eudragit-RSPO polymeric nanoparticles, which are suitable to encapsulate an antigen for oral administration in dogs. This antigen, named EgFABP1, belonging to the family of fatty acid-binding proteins, was isolated from the larval form of the parasite Echinococcus granulosus. Several reports point to proteins from this family from parasitic flatworms as candidates for a successful vaccine, considering the restricted lipid metabolism of these organisms. The encapsulation of the antigen yielded an efficiency higher than 50 %, and the nanoparticles showed the expected size range. In addition, antigen integrity was conserved and the formulation was resistant to artificial gastric and intestinal fluid effects.

Fatty acid binding proteins have the potential to channel dietary fatty acids into enterocyte nuclei.

Intracellular lipid binding proteins, including fatty acid binding proteins (FABPs) 1 and 2, are highly expressed in tissues involved in the active lipid metabolism. A zebrafish model was used to demonstrate differential expression levels of fabp1b.1, fabp1b.2, and fabp2 transcripts in liver, anterior intestine, and brain. Transcription levels of fabp1b.1 and fabp2 in the anterior intestine were upregulated after feeding and modulated according to diet formulation. Immunofluorescence and electron microscopy immunodetection with gold particles localized these FABPs in the microvilli, cytosol, and nuclei of most enterocytes in the anterior intestinal mucosa. Nuclear localization was mostly in the interchromatin space outside the condensed chromatin clusters. Native PAGE binding assay of BODIPY-FL-labeled FAs demonstrated binding of BODIPY-FLC(12) but not BODIPY-FLC(5) to recombinant Fabp1b.1 and Fabp2. The binding of BODIPY-FLC(12) to Fabp1b.1 was fully displaced by oleic acid. In vivo experiments demonstrated, for the first time, that intestinal absorption of dietary BODIPY-FLC(12) was followed by colocalization of the labeled FA with Fabp1b and Fabp2 in the nuclei. These data suggest that dietary FAs complexed with FABPs are able to reach the enterocyte nucleus with the potential to modulate nuclear activity.

Echinococcus granulosus fatty acid binding proteins subcellular localization.

Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes.

Hard as stone: an exuberant form of calcinosis cutis.

Dermatomyositis (DM) is an immune-mediated myopathy characterized by proximal skeletal muscle weakness, muscle inflammation, and distinct skin manifestations. Calcinosis cutis, involving the deposition of insoluble calcium salts in the soft tissues, affects approximately 8% of DM patients1 and poses significant treatment challenges. It can complicate with inflammation, ulceration, pain, and local and systemic infections, resulting in considerable morbidity. We present the case of a 69-year-old-woman with dermatomyositis who developed an exuberant form of calcinosis cutis.

Modeling, molecular dynamics and docking studies of a full-length Echinococcus granulosus 2DBD nuclear receptor.

Nuclear receptors are ligand-activated transcription factors capable of regulating the expression of complex gene networks. The family includes seven subfamilies of protein with a wide phylogenetic distribution. A novel subfamily with two DNA-binding domains (2DBDs) has been first reported in Schistosoma mansoni (Platyhelminth, Trematoda). Employing an ab initio protocol and homology modeling methods, the full-length 3D structure of the Eg2DBDα.1 nuclear receptor from Echinococcus granulosus (Platyhelminth, Cestoda) was generated. The model analysis reveals the presence of the conserved three-layered alpha-helical sandwich structure in the ligand binding domain, and a particularly long and flexible hinge region. Molecular dynamics simulations were performed previous to dock a conformational library of fatty acids and retinoic acids. Our results indicate that oleic and linoleic acids are suitable ligands to this receptor. The ligand-protein complex is stabilized mainly by hydrogen bonds and hydrophobic interactions. The fact that 2DBD nuclear receptors have not been identified in vertebrates confers particular interest to these nuclear receptors, not only concerning their structure and function but as targets of new anthelmintic drugs.Communicated by Ramaswamy H. Sarma.

A general overview of the multifactorial adaptation to cold: biochemical mechanisms and strategies.

Cold environments are more frequent than people think. They include deep oceans, cold lakes, snow, permafrost, sea ice, glaciers, cold soils, cold deserts, caves, areas at elevations greater than 3000 m, and also artificial refrigeration systems. These environments are inhabited by a diversity of eukaryotic and prokaryotic organisms that must adapt to the hard conditions imposed by cold. This adaptation is multifactorial and includes (i) sensing the cold, mainly through the modification of the liquid-crystalline membrane state, leading to the activation of a two-component system that transduce the signal; (ii) adapting the composition of membranes for proper functions mainly due to the production of double bonds in lipids, changes in hopanoid composition, and the inclusion of pigments; (iii) producing cold-adapted proteins, some of which show modifications in the composition of amino acids involved in stabilizing interactions and structural adaptations, e.g., enzymes with high catalytic efficiency; and (iv) producing ice-binding proteins and anti-freeze proteins, extracellular polysaccharides and compatible solutes that protect cells from intracellular and extracellular ice. However, organisms also respond by reprogramming their metabolism and specifically inducing cold-shock and cold-adaptation genes through strategies such as DNA supercoiling, distinctive signatures in promoter regions and/or the action of CSPs on mRNAs, among others. In this review, we describe the main findings about how organisms adapt to cold, with a focus in prokaryotes and linking the information with findings in eukaryotes.

Development of an oral nanovaccine for dogs against Echinococcus granulosus.

Dogs are the main source of animal and human cystic echinococcosis caused by the Cestode parasite Echinococcus granulosus. Dog vaccination seems to be a good strategy to control this parasitic disease. Here we present the development of a polymeric nanoparticle-based oral vaccine for dogs against Echinococcus granulosus delivered in enteric-coated capsules. To achieve our target, we encapsulated two recombinant antigens into biodegradable polymeric nanoparticles in the presence of Monophosphoryl lipid A as an adjuvant to ensure efficient delivery and activation of a protective mucosal immune response. The formulated delivery system showed a nanoparticle size less than 200 nm with more than 80 % antigen encapsulation efficiency and conserved integrity and immunogenicity. The nanoparticle surface was coated with chitosan to enhance adhesion to the gut mucosa and a subsequent antigen delivery. Chitosan-coated nanoparticles showed a higher cell internalization in murine macrophages and dendritic cells as well as a higher penetration into Caco-2 cells in vitro. Antigen-loaded nanoparticles were freeze-dried and enteric-coated capsules were filled with the obtained powder. The obtained results show a promising nanoparticles delivery system for oral vaccination.

Structural and functional studies of ReP1-NCXSQ, a protein regulating the squid nerve Na+/Ca2+ exchanger.

The protein ReP1-NCXSQ was isolated from the cytosol of squid nerves and has been shown to be required for MgATP stimulation of the squid nerve Na(+)/Ca(2+) exchanger NCXSQ1. In order to determine its mode of action and the corresponding biologically active ligand, sequence analysis, crystal structures and mass-spectrometric studies of this protein and its Tyr128Phe mutant are reported. Sequence analysis suggests that it belongs to the CRABP family in the FABP superfamily. The X-ray structure at 1.28 Å resolution shows the FABP ß-barrel fold, with a fatty acid inside the barrel that makes a relatively short hydrogen bond to Tyr128 and shows a double bond between C9 and C10 but that is disordered beyond C12. Mass-spectrometric studies identified this fatty acid as palmitoleic acid, confirming the double bond between C9 and C10 and establishing a length of 16 C atoms in the aliphatic chain. This acid was caught inside during the culture in Escherichia coli and therefore is not necessarily linked to the biological activity. The Tyr128Phe mutant was unable to activate the Na(+)/Ca(2+) exchanger and the corresponding crystal structure showed that without the hydrogen bond to Tyr128 the palmitoleic acid inside the barrel becomes disordered. Native mass-spectrometric analysis confirmed a lower occupancy of the fatty acid in the Tyr128Phe mutant. The correlation between (i) the lack of activity of the Tyr128Phe mutant, (ii) the lower occupancy/disorder of the bound palmitoleic acid and (iii) the mass-spectrometric studies of ReP1-NCXSQ suggests that the transport of a fatty acid is involved in regulation of the NCXSQ1 exchanger, providing a novel insight into the mechanism of its regulation. In order to identify the biologically active ligand, additional high-resolution mass-spectrometric studies of the ligands bound to ReP1-NCXSQ were performed after incubation with squid nerve vesicles both with and without MgATP. These studies clearly identified palmitic acid as the fatty acid involved in regulation of the Na(+)/Ca(2+) exchanger from squid nerve.

Identification of a non-classical three-dimensional nuclear localization signal in the intestinal fatty acid binding protein.

The intestinal fatty acid binding protein (FABP) is a small protein expressed along the small intestine that bind long-chain fatty acids and other hydrophobic ligands. Several lines of evidence suggest that, once in the nucleus, it interacts with nuclear receptors, activating them and thus transferring the bound ligand into the nucleus. Previous work by our group suggests that FABP2 would participate in the cytoplasm-nucleus translocation of fatty acids. Because the consensus NLS is absent in the sequence of FABP2, we propose that a 3D signal could be responsible for its nuclear translocation. The results obtained by transfection assays of recombinant wild type and mutated forms of Danio rerio Fabp2 in Caco-2 cell cultures, showed that lysine 17, arginine 29 and lysine 30 residues, which are located in the helix-turn-helix region, would constitute a functional non-classical three-dimensional NLS.

Bioinformatic analysis of a novel Echinococcus granulosus nuclear receptor with two DNA binding domains.

Nuclear receptors are ligand-activated transcription factors capable of regulating the expression of complex gene networks. The family includes seven subfamilies of proteins with a wide phylogenetic distribution. A novel subfamily with two DNA-binding domains (2DBDs) has been reported in Schistosoma mansoni (Platyhelminth, Trematoda). This work describes the cDNA cloning and bioinformatics analysis of Eg2DBDα, a 2DBD nuclear receptor isoform from the parasite Echinococcus granulosus (Platyhelminth, Cestoda). The Eg2DBDα gene coding domain structure was analysed. Although two additional 2DBD nuclear receptors are reported in the parasite database GeneDB, they are unlikely to be expressed in the larval stage. Phylogenetic relationships between these atypical proteins from different cestodes are also analysed including S. mansoni 2DBD nuclear receptors. The presence of two DNA binding domains confers particular interest to these nuclear receptors, not only concerning their function but to the development of new antihelminthic drugs.

Two novel Mesocestoides vogae fatty acid binding proteins--functional and evolutionary implications.

This work describes two new fatty acid binding proteins (FABPs) identified in the parasite platyhelminth Mesocestoides vogae (syn. corti). The corresponding polypeptide chains share 62% identical residues and overall 90% similarity according to CLUSTALX default conditions. Compared with Cestoda FABPs, these proteins share the highest similarity score with the Taenia solium protein. M. vogae FABPs are also phylogenetically related to the FABP3/FABP4 mammalian FABP subfamilies. The native proteins were purified by chromatographical procedures, and apparent molecular mass and isoelectric point were determined. Immunolocalization studies determined the localization of the expression of these proteins in the larval form of the parasite. The genomic exon-intron organization of both genes is also reported, and supports new insights on intron evolution. Consensus motifs involved in splicing were identified.

Identification of novel CAP superfamily protein members of Echinococcus granulosus protoscoleces.

Echinoccocus granulosus is the causative agent of Cyst Echinococcosis, a zoonotic infection affecting humans and livestock representing a public health and an economic burden for several countries. Despite decades of investigation an effective vaccine still remains to be found. Parasitic cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins (CAPs) have been proposed as vaccine candidates against helmith's infection. In this work we have identified two novel proteins of this superfamily expressed at the protoescoleces larval stage named EgVAL1 and EgVAL2. The open reading frame sequences were deduced. The aminoacidic sequence was analyzed and confronted against already known vertebrate' and helminth's proteins sequences in order to infer putative functions. Immunolocalization studies were also performed. The obtained data supported by immunolocalization studies and homology models suggest that these proteins could be involved in protease activity inhibition.

Genomic structure and expression of a gene coding for a new fatty acid binding protein from Echinococcus granulosus.

This work describes a new gene coding for a fatty acid binding protein (FABP) in the parasite Echinococcus granulosus, named EgFABP2. The complete gene structure, including the promoter sequence, is reported. The genomic coding domain organisation of the previously reported E. granulosus FABP gene (EgFABP1) has been also determined. The corresponding polypeptide chains share 76% of identical residues and an overall 96% of similarity. The two EgFABPs present the highest amino acid homologies with the mammalian FABP subfamily containing heart-FABPs (H-FABPs). The coding sequences of both genes are interrupted by a single intron located in the position of the third intron reported for vertebrate FABP genes. Both genes are expressed in the protoscolex stage of the parasite. The promoter region of EgFABP2 presents several consensus putative cis-acting elements found in other members of the family, suggesting interesting possible mechanisms involved in the host-parasite adaptation.

The crystal structure of Echinococcus granulosus fatty-acid-binding protein 1.

We describe the 1.6 A crystal structure of the fatty-acid-binding protein EgFABP1 from the parasitic platyhelminth Echinococcus granulosus. E. granulosus causes hydatid disease, which is a major zoonosis. EgFABP1 has been implicated in the acquisition, storage, and transport of lipids, and may be important to the organism since it is incapable of synthesising most of its lipids de novo. Moreover, EgFABP1 is a promising candidate for a vaccine against hydatid disease. The crystal structure reveals that EgFABP1 has the expected 10-stranded beta-barrel fold typical of the family of intracellular lipid-binding proteins, and that it is structurally most similar to P2 myelin protein. We describe the comparison of the crystal structure of EgFABP1 with these proteins and with an older homology model for EgFABP1. The electron density reveals the presence of a bound ligand inside the cavity, which we have interpreted as palmitic acid. The carboxylate group of the fatty acid interacts with the protein's P2 motif, consisting of a conserved triad R em leader R-x-Y. The hydrophobic tail of the ligand assumes a fairly flat, U-shaped conformation and has relatively few interactions with the protein.We discuss some of the structural implications of the crystal structure of EgFABP1 for related platyhelminthic FABPs.

Towards an understanding of Mesocestoides vogae fatty acid binding proteins' roles.

Two fatty acid binding proteins, MvFABPa and MvFABPb were identified in the parasite Mesocestoides vogae (Platyhelmithes, Cestoda). Fatty acid binding proteins are small intracellular proteins whose members exhibit great diversity. Proteins of this family have been identified in many organisms, of which Platyhelminthes are among the most primitive. These proteins have particular relevance in flatworms since de novo synthesis of fatty acids is absent. Fatty acids should be captured from the media needing an efficient transport system to uptake and distribute these molecules. While HLBPs could be involved in the shuttle of fatty acids to the surrounding host tissues and convey them into the parasite, FABPs could be responsible for the intracellular trafficking. In an effort to understand the role of MvFABPs in fatty acid transport of M. vogae larvae, we analysed the intracellular localization of both MvFABPs and the co-localization with in vivo uptake of fatty acid analogue BODIPY FL C16. Immunohistochemical studies on larvae sections using specific antibodies, showed a diffuse cytoplasmic distribution of each protein with some expression in nuclei and mitochondria. MvFABPs distribution was confirmed by mass spectrometry identification from 2D-electrophoresis of larvae subcellular fractions. This work is the first report showing intracellular distribution of MvFABPs as well as the co-localization of these proteins with the BODIPY FL C16 incorporated from the media. Our results suggest that fatty acid binding proteins could target fatty acids to cellular compartments including nuclei. In this sense, M. vogae FABPs could participate in several cellular processes fulfilling most of the functions attributed to vertebrate's counterparts.

In silico studies of Echinococcus granulosus FABPs.

Fatty acid (FA) binding proteins are small intracellular proteins whose members exhibit great diversity and low similarity at the primary structure level, but a highly conserved three-dimensional structure. Characterised by a high-affinity non-covalent binding of hydrophobic ligands, these proteins have a molecular mass of 14-15 kDa with a characteristic ß-barrel structure. Members of this family have been identified along the zoological scale, with Platyhelminthes being the more primitive organisms where they have been reported. Two FA binding proteins (FABPs), EgFABP1 and EgFABP2, with 88% similarity have been identified in Echinococcus granulosus. In an effort to understand why two such similar proteins are expressed by this organism, we performed an in silico analysis of the binding capabilities of both proteins. The crystallographic structure of EgFABP1 was utilised as a template to model EgFABP2, and both were docked against palmitate, oleate, linoleate and arachidonate. The docked structures were submitted to 4 ns molecular dynamics simulations, and their protein-ligand interaction energies were measured. The collected data demonstrated that linoleate and arachidonate had the higher interaction energies when bound to EgFABP1 and that palmitate and linoleate had the higher interaction energies when bound to EgFABP2. External and internal binding surfaces were analysed, showing differences at both levels. Internal surface compositions suggested that both proteins could have preferences for certain FAs. Comparisons of the holo and apo forms of each protein indicated that the ligand imposed subtle, but specific modifications that could trigger surface signals. The differences found between the proteins under study suggest that they could have functional uniqueness in the parasite's metabolism.

Lipid binding proteins from parasitic platyhelminthes.

TWO MAIN FAMILIES OF LIPID BINDING PROTEINS HAVE BEEN IDENTIFIED IN PARASITIC PLATYHELMINTHES: hydrophobic ligand binding proteins (HLBPs) and fatty acid binding proteins (FABPs). Members of the former family of proteins are specific to the Cestoda class, while FABPs are conserved across a wide range of animal species. Because Platyhelminthes are unable to synthesize their own lipids, these lipid-binding proteins are important molecules in these organisms. HLBPs are a high molecular mass complex of proteins and lipids. They are composed of subunits of low molecular mass proteins and a wide array of lipid molecules ranging from CoA esters to cholesterol. These proteins are excretory-secretory molecules and are key serological tools for diagnosis of diseases caused by cestodes. FABPs are mainly intracellular proteins of low molecular weight. They are also vaccine candidates. Despite that the knowledge of their function is scarce, the differences in their molecular organization, ligand preferences, intra/extracellular localization, evolution, and phylogenetic distribution, suggest that platyhelminths HLBPs and FABPs should play different functions. FABPs might be involved in the removal of fatty acids from the inner surface of the cell membrane and in their subsequent targeting to specific cellular destinations. In contrast, HLBPs might be involved in fatty acid uptake from the host environment.

Direct interaction between EgFABP1, a fatty acid binding protein from Echinococcus granulosus, and phospholipid membranes.

BACKGROUND: Growth and maintenance of hydatid cysts produced by Echinococcus granulosus have a high requirement for host lipids for biosynthetic processes, membrane building and possibly cellular and developmental signalling. This requires a high degree of lipid trafficking facilitated by lipid transporter proteins. Members of the fatty acid binding protein (FABP) family have been identified in Echinococcus granulosus, one of which, EgFABP1 is expressed at the tegumental level in the protoscoleces, but it has also been described in both hydatid cyst fluid and secretions of protoscoleces. In spite of a considerable amount of structural and biophysical information on the FABPs in general, their specific functions remain mysterious. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the way in which EgFABP1 may interact with membranes using a variety of fluorescence-based techniques and artificial small unilamellar vesicles. We first found that bacterial recombinant EgFABP1 is loaded with fatty acids from the synthesising bacteria, and that fatty acid binding increases its resistance to proteinases, possibly due to subtle conformational changes induced on EgFABP1. By manipulating the composition of lipid vesicles and the ionic environment, we found that EgFABP1 interacts with membranes in a direct contact, collisional, manner to exchange ligand, involving both ionic and hydrophobic interactions. Moreover, we observed that the protein can compete with cytochrome c for association with the surface of small unilamellar vesicles (SUVs). CONCLUSIONS/SIGNIFICANCE: This work constitutes a first approach to the understanding of protein-membrane interactions of EgFABP1. The results suggest that this protein may be actively involved in the exchange and transport of fatty acids between different membranes and cellular compartments within the parasite.