Functional Study Of CRAC And CARC Peptides Derived From E. Coli Alpha Hemolysin

Escherichia coli alpha hemolysin (HlyA) is a pore-forming protein which belongs to the family of 'Repeat in toxins'(RTX). The CRAC domain refers to the Cholesterol Recognition/interaction Aminoacid Consensus sequence. The CARC domain is similar to the CRAC sequence, but exhibits the opposite orientation along the polypeptide chain. The aims of this work were to study the participation of CRAC and CARC in the stabilization of HlyA monomers in membranes by their interaction with cholesterol, to evaluate the role of Y347 in the interaction with membrane, and finally to find a cytotoxic peptide for the construction of an immunotoxin. On the basis of experimental data and structural predictions, six peptides derived from HlyA were synthesized: PEP 1: transmembrane domain described as hemolytically active; PEP 2: also a transmembrane domain which sequence corresponds to a cholesterol binding domain (CARC); PEP3: similar to PEP2 but with residue Y347 substituted by A; PEP4: similar to PEP2 but with a CRAC sequence; PEP5 and PEP6 correspond to CARC sequences located near the acylation sites. Peptides were synthesized by the solid phase peptide synthesis method (Fmoc strategy), purified by HPLC (C-18 column), the molecular mass was determined by mass spectrometry and peptide structure by circular dichroism. The hemolytic activity of peptides was measured using human erythrocytes and inhibition of hemolytic activity assays were performed pre-incubating erythrocytes with peptides and then adding them to wild type toxin. Results describe PEP2 as hemolytic, which is promising and encourage us to use it in the design of immunotoxins. PEP3 was found not to be hemolytic suggesting residue Y347 is fundamental for the interaction of HlyA with lipidic membranes. PEP4 was found not to be hemolytic, which implicates the CRAC sequence added was unfavorable for peptide activity. PEP 6 competes with HlyA for binding sites in erythrocytes.Universidad Nacional de La Plat

Contribution of the C-terminal end of apolipoprotein AI to neutralization of lipopolysaccharide endotoxic effect

It is well known that high density lipoprotein (HDL) binds bacterial lipopolysaccharide (LPS) and neutralizes its toxicity. The aim of this work was to study changes in the apolipoprotein (apo) AI structure after its interaction with LPS as well as to determine the protein domain involved in that interaction. The presented data indicate that LPS does not lead to major changes in the structure of apoAI, judging from Trp fluorescence spectra. However, analysis of denaturation behavior and binding of ANS show that LPS induces a loosened protein conformation. Further evidence for an apoAI-LPS specific interaction was obtained by incubation of the protein with 125I-ASD- LPS. The results show that multiple regions of the protein were able to interact with LPS, according to its amphiphatic nature. Finally, the contribution of the purified C-terminal fragment of the protein in the endotoxin neutralization was evaluated in comparison with the effect of apoAI. In both cases, the same decrease in tumor necrosis factor-α released was observed. This result suggests that the C-terminal half of apoAI is the main domain responsible of the neutralization effect of this protein. Our data may provide innovative pharmacological tools in endotoxin neutralization therapies.Instituto de Investigaciones Bioquímicas de La Plat

Contribution of the C-terminal end of apolipoprotein AI to neutralization of lipopolysaccharide endotoxic effect

It is well known that high density lipoprotein (HDL) binds bacterial lipopolysaccharide (LPS) and neutralizes its toxicity. The aim of this work was to study changes in the apolipoprotein (apo) AI structure after its interaction with LPS as well as to determine the protein domain involved in that interaction. The presented data indicate that LPS does not lead to major changes in the structure of apoAI, judging from Trp fluorescence spectra. However, analysis of denaturation behavior and binding of ANS show that LPS induces a loosened protein conformation. Further evidence for an apoAI-LPS specific interaction was obtained by incubation of the protein with 125I-ASD- LPS. The results show that multiple regions of the protein were able to interact with LPS, according to its amphiphatic nature. Finally, the contribution of the purified C-terminal fragment of the protein in the endotoxin neutralization was evaluated in comparison with the effect of apoAI. In both cases, the same decrease in tumor necrosis factor-α released was observed. This result suggests that the C-terminal half of apoAI is the main domain responsible of the neutralization effect of this protein. Our data may provide innovative pharmacological tools in endotoxin neutralization therapies.Instituto de Investigaciones Bioquímicas de La Plat

Paradoxical lipid dependence of pores formed by the Escherichia coli α-hemolysin in planar phospholipid bilayer membranes

α-Hemolysin (HlyA) is an extracellular protein toxin (117 kDa) secreted by Escherichia coli that targets the plasma membranes of eukaryotic cells. We studied the interaction of this toxin with membranes using planar phospholipid bilayers. For all lipid mixtures tested, addition of nanomolar concentrations of toxin resulted in an increase of membrane conductance and a decrease in membrane stability. HlyA decreased membrane lifetime up to three orders of magnitude in a voltage-dependent manner. Using a theory for lipidic pore formation, we analyzed these data to quantify how HlyA diminished the line tension of the membrane (i.e., the energy required to form the edge of a new pore). However, in contrast to the expectation that adding the positive curvature agent lysophosphatidylcholine would synergistically lower line tension, its addition significantly stabilized HlyA-treated membranes. HlyA also appeared to thicken bilayers to which it was added. We discuss these results in terms of models for proteolipidic pores.Facultad de Ciencias ExactasInstituto de Investigaciones Bioquímicas de La Plat

Alpha hemolysin induces an increase of erythrocytes calcium: a FLIM 2-photon phasor analysis approach

α-Hemolysin (HlyA) from Escherichia coli is considered as the prototype of a family of toxins called RTX (repeat in toxin), a group of proteins that share genetic and structural features. HlyA is an important virulence factor in E. coli extraintestinal infections, such as meningitis, septicemia and urinary infections. High concentrations of the toxin cause the lysis of several cells such as erythrocytes, granulocytes, monocytes, endothelial and renal epithelial cells of different species. At low concentrations it induces the production of cytokines and apoptosis. Since many of the subcytolytic effects in other cells have been reported to be triggered by the increase of intracellular calcium, we followed the calcium concentration inside the erythrocytes while incubating with sublytic concentrations of HlyA. Calcium concentration was monitored using the calcium indicator Green 1, 2-photon excitation, and fluorescence lifetime imaging microscopy (FLIM). Data were analyzed using the phasor representation. In this report, we present evidence that, at sublytic concentrations, HlyA induces an increase of calcium concentration in rabbit erythrocytes in the first 10 s. Results are discussed in relation to the difficulties of measuring calcium concentrations in erythrocytes where hemoglobin is present, the contribution of the background and the heterogeneity of the response observed in individual cells.Facultad de Ciencias Médica

Liberación de microvesículas de eritrocitos tratados con alfa hemolisina de <i>E. coli</i>

Alfa hemolisina (HlyA) pertenece a una familia de toxinas denominadas RTX (Repeat in toxins), y es uno de los factor de virulencia de las cepas uropatogénicas de E.coli. HlyA, al igual que muchas de las toxinas de esta familia, debe ser acilada en dos residuos internos de Lys en el interior de la bacteria antes de ser secretada al medio. Esta modificación postraduccional hace que la toxina pase de la forma inactiva (ProHlyA), sin acilar, a una proteína activa acilada (HlyA). En los últimos años hemos estudiados los efectos que produce esta toxina a los eritrocitos a concentraciones sublíticas. Hemos encontrado que induce un aumento bifásico de calcio, que produce la activación de calpaínas que degradan proteínas del citoesqueleto; siendo responsables de los cambios morfológicos que sufren los eritrocitos previos a la lisis. Además, HlyA activa esfingomielinasas endógenas, produciendo un aumento de ceramida en la membrana, hecho que cambia las propiedades físicas de la membrana. Por otro lado, estudios más detallados de los cambios morfológicos observados, demostraron que estos cambios son independientes del ingreso de calcio y que HlyA sufre un proceso de transformación de discocito a esferocito.Facultad de Ciencias Médica

Caracterización proteica y funcional de microrovesículas liberadas por eritrocitos tratados con alfa hemolisina de E.coli

Alfa hemolisina (HlyA) pertenece a una familia de toxinas denominadas RTX (Repeat in toxins), y es uno de los factor de virulencia de las cepas uropatogénicas de E.coli. HlyA, al igual que muchas de las toxinas de esta familia, debe ser acilada en dos residuos internos de Lys en el interior de la bacteria antes de ser secretada al medio. Esta modificación postraduccional hace que la toxina pase de la forma inactiva (ProHlyA), sin acilar, a una proteína activa acilada (HlyA). En los últimos años hemos estudiados los efectos que produce esta toxina a los eritrocitos a concentraciones sublíticas. Hemos encontrado que induce un aumento bifásico de calcio, que produce la activación de calpaínas que degradan proteínas del citoesqueleto; siendo responsables de los cambios morfológicos que sufren los eritrocitos previos a la lisis. Además, HlyA activa esfingomielinasas endógenas, produciendo un aumento de ceramida en la membrana, hecho que cambia las propiedades físicas de la membrana. Por otro lado, estudios más detallados de los cambios morfológicos observados, demostraron que estos cambios son independientes del ingreso de calcio y que HlyA sufre un proceso de transformación de discocito a esferocito. Todos los eventos anteriormente descriptos ocurren en aquellas células que secretan microvesículas al medio como un proceso de remodelación de la membrana. Por otro lado se ha visto que estas microvesículas participan en proceso de intercomunicación celular. or lo anteriormente mencionado es que recientemente hemos logrado purificar microvesículas secretadas por eritrocitos tratados con HlyA y es el objetivo del presente trabajo caracterizar la composición proteica de estas microvesiculas y determinar si la liberación de microvesículas puede actuar como una forma de diseminación de la toxina.Facultad de Ciencias Médica

Propiedades de membrana que determinan la susceptibilidad de los eritrocitos frente a la toxina HlyA de <i>E. coli</i>

El objetivo del presente trabajo es estudiar las propiedades de membrana que determinan las diferentes susceptibilidades de eritrocitos de caballo, carnero y conejo frente a HlyA. Dichos eritrocitos presentan diferencias concernientes tanto a la composición lipídica como proteica de su membrana.Facultad de Ciencias Médica

Induction of eryptosis by low concentrations of E. coli alpha-hemolysin

Uropathogenic strains of Escherichia coli deliver the toxin alpha-hemolysin (HlyA) to optimize the host environment for the spread of infection. It was reported that at high concentrations, the toxin forms pores in eukaryotic membranes, leading to cell lysis, while lower concentrations have appeared to interfere with host-cell-signaling pathways causing cell death by apoptosis. Nevertheless, what is not clear is how often HlyA reaches levels that are high enough to lyse host target cells during the course of an infection. In the present investigation, we demonstrate that a low toxin concentration induces the suicidal death of erythrocytes (eryptosis), the major cell type present in blood. Eryptosis is triggered both by an increment in intracellular calcium and by ceramide. Since we have previously demonstrated that a low concentration of HlyA induces an increase in intraerythrocyte calcium, in the present experiments we have shown that this ion activates calpains, which hydrolyze skeleton proteins such as spectrin, ankyrin, protein 4.1 and the electrophoretic Band-3 species, thus resulting in morphologic changes in the erythrocytes. We furthermore observed that a low toxin concentration induced the activation of endogenous sphingomyelinases that in turn increased the amount of ceramide in erythrocyte membranes. Both spectrin proteolysis and ceramide formation may cause the exposure of phosphatidylserine on the membrane so as to trigger a macrophage engulfment of the erythrocyte. By this means eryptosis may be an advantageous mechanism for removing defective erythrocytes before hemolysis.Instituto de Investigaciones Bioquímicas de La PlataFacultad de Ciencias Exacta

Mecanismo de ação da toxina alfa-hemolisina de Escherichia coli

Escherichia coli es una de las bacterias anaerobias facultativas más predominantes en el intestino, siendo, en la mayoría de los casos, inocua para el huésped. Existen cepas que traslocan al torrente sanguíneo causando enfermedades extraintestinales como infecciones urinarias, septicemia y meningitis. Dentro de éstas se encuentran las cepas uropatogénicas (Uropathogenic Escherichia coli: UPEC), que secretan varios factores de virulencia. Estos últimos incluyen: toxinas, sistemas de adquisición de hierro, adhesinas y antígenos capsulares. Las principales toxinas secretadas son: alfa-hemolisina (HlyA) y el factor necrotizante citotóxico 1 (CNF-1). En esta revisión se presenta una descripción exhaustiva de HlyA, incluyendo su síntesis, maduración y exportación desde la bacteria. La acilación de la proteína en dos residuos internos de lisina la convierte en una toxina muy virulenta al exponer regiones intrínsecamente desordenadas que son esenciales en diferentes pasos del mecanismo de acción de la misma. Específicamente, la exposición de estas regiones está involucrada en interacciones proteína-proteína dentro del proceso de oligomerización. La formación del oligómero es responsable de la permeabilidad inducida en las células blanco. Finalmente, basado en los conocimientos acerca de las características estructurales y funcionales de HlyA, se presentan potenciales usos de HlyA en terapias basadas en toxinas.Escherichia coli is one of the predominant species of facultative anaerobes in the human gut, and in the majority of the cases it is harmless to the host. Some strains of this species can translocate to blood and cause infection such as urinary infection, septicemia and meningitis. These are the uropathogenic E. coli strains (UPEC) that secrete a number of virulence factors. The latter include a number of secreted toxins, iron-acquisition systems, adhesins, and capsular antigens. Secreted toxins include HlyA, the cytotoxic necrotizing factor-1 (CNF-1). In this review an exhaustive description of the toxin has been delineated, including its synthesis, maturation, and export from the bacteria. The acylation of the protein at two internal lysine residues gives the toxin its virulence, by exposing intrinsic disordered regions that are essential in different steps of the toxin’s mechanism of action. The further exposure of regions involved in the protein-protein interaction within the oligomerization process is responsG-ible for the permeability induced in all the target cells. Based on the already known structural and functional characteristics of HlyA, the potential use in toxin-based therapy is presented.Escherichia coli é uma das bactérias anaérobias facultativas mais predominantes no intestino, sendo na maioria dos casos inócua para o hóspede. Há cepas que passam ao torrente sanguíneo causando doenças extraintestinais como infecção urinária, septicemia e meningite. Dentro destas se encontram as cepas uropatogênicas (Uropathogenic Escherichia coli: UPEC) que secretam varios fatores de virulência. Estos últimos incluem: toxinas, sistemas de aquisição de ferro, adesinas e antígenos capsulares. As principais toxinas secretadas são: alfa hemolisina (HlyA) e o fator necrotizante citotóxico 1 (CNF-1). Nesta revisão apresenta- -se uma descrição exaustiva de HlyA incluindo sua sintese, seu amadurecimento e exportação a partir da bactéria. A acilação da proteína em dois residuos internos de lisina a transforma numa toxina muito virulenta ao expor regiões intrinsecamente desordenadas que são essenciais em diferentes passos do mecanismo de ação da mesma. Especificamente, a exposição destas regiões esta envolvida em interações proteína-proteína dentro do processo de oligomerização. A formação do oligômero é responsável pela permeabilidade induzida nas células alvo. Finalmente, com base nos conhecimentos acerca das características estruturais e funcionais de HlyA, apresentam-se potenciais usos de HlyA em terapias baseadas em toxinas.Fil: Bakas, Laura Susana. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentina; Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina;Fil: Maté, Sabina María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Investigaciones Bioquímicas de la Plata; Argentina; Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina;Fil: Vazquez, Romina Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Investigaciones Bioquímicas de la Plata; Argentina;Fil: Herlax, Vanesa Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Investigaciones Bioquímicas de la Plata; Argentina