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

Poor = delinquent, lhe equation of a system that cannon see beyond it’s prejudices: about the case of Cristina Vázquez

En la presente ponencia se intentó analizar ciertos estereotipos que se fueron construyendo e instalando para criminalizar a un sector preciso de la población: los marginales, respecto del poder central. Para ello, se hizo alusión al Documental Fragmentos de una amiga desconocida, sobre el caso de Cristina Vázquez, una joven de 19 años, que en el 2001 fue injustamente acusada y privada de su libertad por un crimen que no cometió: el asesinato de quien era su vecina. En este documental se pudo observar las operaciones de los medios de comunicación y del sistema penal para justificar tal acusación, a través de discursos positivistas y sexistas que parten de la idea de que “peligroso se nace”: por la condición social, el aspecto físico, los hábitos, entre otros calificativos. De esta manera, se estigmatiza, criminaliza, margina y vulnera a ciertos sectores de la población por portar determinados estereotipos, como “pobre” y “chorro”. Estudiar los recorridos sociohistóricos de estos significantes, entendidos como estigmas, pretende promover una reflexión para comenzar a pensar cómo entendemos la realidad en la que vivimos y que ello nos invite a posicionarnos críticamente frente a lo instituido (Lourau, 1970) con la posibilidad de construir un contra-discurso.In this paper we will try to analize certain steriotypes built and installed to criminalize a specific segment of the population: the marginal, in relation to status quo. For that purpose, we will refer to the documentary Fragmentos de una amiga desconocida, which shows Cristina Vazquez’s life, a nineteen-year-old woman unfairly accused and deprived of freedom for a crime she had not commited: her neighbour’s murder. In the mentioned documentary we can easily observe the manipulation of the media and the criminal justice system to justify such accusation. Through a positivist and sexist discourse with the underlying idea that the social and economic factors determine who is criminal and who is not, there is a tendendency to put the blame always in the poor. By this manner, the most vulnerable population groups are stigmatized and criminalized for bearing certain steriotypes, such as “pobre” and “chorro”. By studying the sociohistorical backgroud of theses concepts “pobre” y “chorro”, we intend to promote reflection on our understanding of the reality we live in, thus enouraging critical thinking against the stablished order (Lourau, 1970) and creating the possibility of a new and more humanistic discourse.Facultad de Psicologí

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

Mecanismo de acción de la 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 toxinasEscherichia 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

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

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. © 2011 Sanchez et al

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