Estudio de proteínas que unen ácidos grasos (FABPs) de Echinococcus spp.

Los cestodos del género Echinococcus, en particular su estadio larval o metacestodo, son los agentes patógenos responsables de las enfermedades denominadas hidatidosis o echinococcosis, de importancia tanto en la salud humana como de animales domésticos. Las especies más representativas, por su amplia distribución geográfica y el impacto que representan en la economía y la salud pública, son E. granulosus y E. multilocularis. La Organización Mundial de la Salud ha incluido a estas enfermedades dentro de un grupo prioritario de enfermedades tropicales desatendidas. Estos cestodos, al igual que muchos otros platelmintos parásitos, son incapaces de sintetizar la gran mayoría de sus lípidos de novo, por lo que deben adquirirlos a partir de sus hospedadores. En ese contexto, se cree que las proteínas que unen ácidos grasos (FABPs) podrían tener un rol importante en la adquisición de dichos nutrientes y la distribución de los mismos entre los diferentes tejidos, estadios y/o vías metabólicas de estos cestodos. Hasta el momento, sólo una FABP de E. granulosus, denominada EgFABP1, ha sido parcialmente caracterizada. Sin embargo, para poder determinar si esta proteína participa en el transporte e intercambio de lípidos, es necesario avanzar en el análisis de los ligandos que EgFABP1 es capaz de unir y en su capacidad de intercambiar estos ligandos. Para ello, en la presente tesis se aplicaron diversas técnicas biofísicas y bioquímicas para la caracterización de la interacción de EgFABP1 con ligandos y membranas fosfolipídicas. Mediante análisis por cromatografía en capa fina y cromatografía gas-líquido de los lípidos que copurifican con EgFABP1 recombinante expresada en Escherichia coli, se logró determinar que en un ambiente celular complejo, como es el citoplasma de una bacteria, EgFABP1 sólo uniría ácidos grasos libres y no lípidos más complejos, como por ejemplo, fosfolípidos. Asimismo, mediante técnicas de proteólisis parcial y dicroísmo circular se estableció que la unión de EgFABP1 a ligandos es capaz de inducir diferentes cambios conformacionales en la proteína, dependiendo del tipo de ácido graso al que se una. Por otro lado, el estudio de los mecanismos de transferencia de ácidos grasos fluorescentes a membranas fosfolipídicas artificiales mostró que EgFABP1 emplearía un mecanismo de transferencia de tipo colisional, lo cual implica que la proteína debe interactuar con la membrana para entregar sus ligandos. Esto es similar a lo observado para FABPs de mamíferos, ampliamente estudiadas, que se asemejan a EgFABP1 tanto a nivel de secuencia como de estructura terciaria, lo que puede facilitar el enfoque de futuros estudios sobre esta proteína de E. granulosus. Por otra parte, la exploración de los genomas de E. granulosus y E. multilocularis recientemente publicados, permitió establecer la existencia de un total de seis genes de FABPs en cada una de las dos especies, destacándose en el caso de E. multilocularis el hecho de que dos de esos genes codifican para una misma proteína. En el presente trabajo, se clonaron las cinco secuencias codificantes de E. multilocularis, por lo que se pudo establecer experimentalmente parte de la estructura génica de dichos genes. Asimismo, ensayos preliminares sugieren que estas FABPs se expresan diferencialmente en distintos tejidos del parásito. Por otro lado, se realizó una caracterización bioinformática de los genes y proteínas FABPs predichos en ambos cestodos, comparándolos con información existente sobre proteínas de esta familia en otros organismos. En conjunto, los resultados obtenidos en esta tesis amplían el conocimiento relativo a las FABPs de Echinococcus spp., revelando la existencia de toda una familia de FABPs que podrían cumplir roles específicos en diferentes tejidos y/o estadios de estos parásitos. A diferencia de lo que se creía previamente, esto plantea un panorama bastante más complejo en el transporte y metabolismo de ácidos grasos, y posiblemente otros ligandos hidrofóbicos, en parásitos cestodos. Asimismo, la capacidad de EgFABP1 de interactuar con membranas y transferir ácidos grasos podría implicar su relación con el transporte y derivación de ligandos hacia diferentes compartimentos y/o vías metabólicas en las células de E. granulosus. Los cambios conformacionales inducidos por ciertos ligandos, podrían tener implicancias desde el punto de vista de la señalización y regulación génica, análogamente a lo descripto para otras FABPs.Facultad de Ciencias Exacta

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

Direct Interaction between EgFABP1, a Fatty Acid Binding Protein from <i>Echinococcus granulosus</i>, 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.Facultad de Ciencias Médica

Direct Interaction between EgFABP1, a Fatty Acid Binding Protein from <i>Echinococcus granulosus</i>, 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.Facultad de Ciencias Médica

Soluble lipid binding proteins from relevant parasitic helminths: form structure to function

Parasitic helminths cause serious and difficult to treat diseases in humans, animals and plants. A high percentage of the earth’s population, mainly in developing countries, suffers from helminth infections. Helminth parasites have a restricted lipid metabolism and must acquire simple undo complex lipids from their hosts for energy metabolism, membrane construction, and lipid-based signaling, the latter possibly also encompassing modifications of the host’s immune and inflammatory defense systems. These organisms produce and release an unexpectedly wide range of lipid binding proteins (LBPs) types structurally distinct from those of their hosts. Some will be associated with specialized external functions, including acquisition and distribution of nutrients. Yet others will be involved in modulation of the host’s local tissue environment, and its innate and acquired immune systems by secreted lipids and carrier proteins. It is important to note that antihelmintic drugs are partially hydrophobic and may require parasite’s own carrier proteins for reaching their site of action.Facultad de Ciencias Médica

Removal of co-purifying hydrophobic ligands from rEgAgB8 by RP-HPLC.

<p>The lipid composition of rEgAgB8 subunits prior to delipidation (Pre), as well as rEgAgB8 subjected to RP-HPLC (Post) were analysed by TLC, in parallel with standards for neutral and polar lipids. Lipid bands were visualised using CuSO₄/H₃PO₄ and identified by comparison with the standards. <b>(A)</b> The lipids extracted from <i>E</i>. <i>coli</i> grown under the same culture conditions is shown for comparison. TLCs from neutral and polar lipids were undertaken separately. <b>(B)</b> The lipid moiety of recombinant subunits were analysed by TLC using double development. The lipid fraction of rEgAgB8 pre-HPLC contained mainly polar lipids (PE and CL), which were successfully removed by the RP-HPLC method. PC: phosphatidylcholine; PS: phosphatidylserine; PI: phosphatidylinositol; CL: cardiolipin; PE: phosphatidylethanolamine; Cho: cholesterol; FA: free fatty acids; DAG: diacylglycerols; TAG: triacylglycerols; SE: sterol esters.</p

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

Fluorimetric titration of 12-AS with EgAgB8 subunits.

<p>Changes in relative 12-AS fluorescence were monitored from 400 to 500 nm after excitation at 383 nm upon incremental additions of EgAgB8/2 or EgAgB8/3 to a cuvette initially containing 2 mL of 0.5 μM 12-AS in TBS buffer. <b>(A)</b> Emission spectra of 12AS in TBS or upon adding EgAgB8/2 (0.5 μM). <b>(B)</b> Changes in relative 12-AS fluorescence at 440 nm were used to build the binding isotherm of the complex EgAgB8/2–12AS. <b>(C)</b> Emission spectra of 12AS in TBS or upon adding EgAgB8/3 (0.7 μM). <b>(D)</b> Changes in relative 12-AS fluorescence at 440 nm were used to build the binding isotherm of the complex EgAgB8/2–12AS. For both proteins, 12-AS spectra showed a blue shift in emission spectrum that accompanies a strong increase in fluorescence emission. The data were consistent with one binding site per monomer unit of protein and K_d values of 0.16 ± 0.09 μM for EgAgB8/2 and 0.34 ± 0.02 μM for EgAgB8/3 were obtained using SigmaPlot software. The solid line is the theoretical binding curve for complex formation. One representative experiment of three is shown for both EgAgB8 subunits.</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