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

Effect of Vanadium Compounds on the Lipid Organization of Liposomes and Cell Membranes

The influence of vanadate on the adsorption properties of Merocyanine 540 (MC540) to UMR cells was studied by means of specrofluorometry. An increment in the fluorescence was observed in the osteoblasts incubated with 0.1 mM vanadate. This effect could be interpreted in terms of vanadate inhibitory effects on aminotraslocase activity. However, vanadate promotes a similar behavior to that found in UMR 106 cells when it was added to lipid vesicles composed of phosphatidylcholine. The effect of vanadium in different oxidation states, such as vanadate(V) and vanadyl(IV) on lipid membrane properties was examined in large unilamellar vesicles by means of spectrofluorometry employing different probes. Merocyanine 540 and 1,6-diphenylhexatriene were used in order to sense the changes at interfacial and hydrophobic core of membranes, respectively. In contrast to vanadate, vanadyl decreased the fluorescence of MC540. Both vanadium compounds slightly perturbed the hydrocarbon core. The results can be interpreted by the specific adsorption of both compounds on the polar head groups of phospholipid and suggest a possible influence of vanadium compounds on the lipid organization of cell membranes

Effect of Vanadium Compounds on the Lipid Organization of Liposomes and Cell Membranes

The influence of vanadate on the adsorption properties of Merocyanine 540 (MC540) to UMR cells was studied by means of specrofluorometry. An increment in the fluorescence was observed in the osteoblasts incubated with 0.1 mM vanadate. This effect could be interpreted in terms of vanadate inhibitory effects on aminotraslocase activity. However, vanadate promotes a similar behavior to that found in UMR 106 cells when it was added to lipid vesicles composed of phosphatidylcholine. The effect of vanadium in different oxidation states, such as vanadate(V) and vanadyl(IV) on lipid membrane properties was examined in large unilamellar vesicles by means of spectrofluorometry employing different probes. Merocyanine 540 and 1,6-diphenylhexatriene were used in order to sense the changes at interfacial and hydrophobic core of membranes, respectively. In contrast to vanadate, vanadyl decreased the fluorescence of MC540. Both vanadium compounds slightly perturbed the hydrocarbon core. The results can be interpreted by the specific adsorption of both compounds on the polar head groups of phospholipid and suggest a possible influence of vanadium compounds on the lipid organization of cell membranes.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.Facultad de Ciencias Médica

Effect of Vanadium Compounds on the Lipid Organization of Liposomes and Cell Membranes

The influence of vanadate on the adsorption properties of Merocyanine 540 (MC540) to UMR cells was studied by means of specrofluorometry. An increment in the fluorescence was observed in the osteoblasts incubated with 0.1 mM vanadate. This effect could be interpreted in terms of vanadate inhibitory effects on aminotraslocase activity. However, vanadate promotes a similar behavior to that found in UMR 106 cells when it was added to lipid vesicles composed of phosphatidylcholine. The effect of vanadium in different oxidation states, such as vanadate(V) and vanadyl(IV) on lipid membrane properties was examined in large unilamellar vesicles by means of spectrofluorometry employing different probes. Merocyanine 540 and 1,6-diphenylhexatriene were used in order to sense the changes at interfacial and hydrophobic core of membranes, respectively. In contrast to vanadate, vanadyl decreased the fluorescence of MC540. Both vanadium compounds slightly perturbed the hydrocarbon core. The results can be interpreted by the specific adsorption of both compounds on the polar head groups of phospholipid and suggest a possible influence of vanadium compounds on the lipid organization of cell membranes.Facultad de Ciencias Médica

SURFACTANTES DERIVADOS DE AMINOÁCIDOS COMO POTENCIALES POTENCIADORES DE PENETRACIÓN DE MOLÉCULAS TERAPÉUTICAS A TRAVÉS DE LA PIEL. CARACTERIZACIÓN DE LA INTERACCIÓN CON MEMBRANAS MODELO

La administración tópica de moléculas terapéuticas presenta numerosas ventajas frente a métodos convencionales, pero se ve dificultada por la naturaleza de barrera de la piel otorgada principalmente por el estrato córneo (SC). Los potenciadores de penetración (PP) son sustancias que pueden alterar de forma reversible la organización de los lípidos del SC, aumentando su permeabilidad. Sin embargo, la toxicidad asociada a la mayoría de ellos ha estimulado la búsqueda de compuestos más seguros. Los surfactantes, especialmente los derivados de aminoácidos resultan una opción interesante ya que mimetizan compuestos naturales, poseen baja irritabilidad y toxicidad, y alto biodegradabilidad. Además, al ser obtenidos mediante calálisis enzimatica empleando peptidasas, se usan tecnologías de bajo impacto ambiental en su producción. El objetivo general del plan es lograr una mayor comprensión de la interacción de surfactantes derivados de arginina con diferentes sistemas modelo de membrana a fin de evaluar su potencial efecto como PP. En nuestro laboratorio se han sintetizado surfactantes catiónicos derivados de arginina empleando papaina adsorbida sobre poliamida como biocatalizador. Se caracterizaron algunas propiedades fisicoquímicas de los mismos, así como la interacción de uno de ellos, Bz-Arg-NHC10, con diferentes sistemas modelo de membrana. La concentración micelar crítica (CMC), tanto en agua como en buffer, fue determinada mediante medidas de tensión superficial. Se obtuvieron menores valores de CMC a mayor largo de cadena hidrocarbonada y además, para cada surfactante el valor medido en agua fue mayor que el medido en buffer. A través distintas metodologías se estudió la interacción de Bz-Arg-NHC10 con varios sistemas de membrana: vesículas multilamelares, monocapas y bicapas soportadas, compuestos por dipalmitoilfosfatidilcolina (DPPC). Mediante el uso de monocapas de Langmuir se vio que Bz-Arg-NHC10 fue capaz de insertarse hasta una presión crítica de 59,6 mN/m, con lo cual podría insertase en membranas biológicas. Los ensayos de DSC revelaron que el mismo puede alterar ligeramente la fluidez de bicapas de DPPC. Por medio de AFM se observó el efecto de Bz-Arg-NHC10 sobre bicapas soportadas resultando en una remoción parcial de lípidos, principalmente en defectos y/o bordes de la bicapa, a bajas concentraciones y una remoción total de la bicapa al incrementar la concentración de surfactante. En los experimentos de SPR se observó adsorción de Bz-Arg-NHC10 sobre la bicapa o remoción de lípidos, a bajas o altas concentraciones de DPPC respectivamente, consistente con las observaciones en imágenes de AFM. Los efectos fueron mayores a mayor concentración de surfactante, esto sugeriría que no solo los monómeros, a través de la inserción y saturación de la membrana con consecuente liberación de micelas mixtas, sino que también los agregados de surfactante, al colisionar con la membrana y extraer lípidos, son responsables de la ruptura de la membrana

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

Caracterización de las propiedades fisicoquímicas y del mecanismo de interacción con membranas biológicas de surfactantes derivados de arginina

Se caracterizaron algunas propiedades fisicoquímicas de dos surfactantes derivados de arginina, Bz-Arg-NHC10 y Bz-Arg-NHC12, así como su interacción con membranas biológicas utilizando glóbulos rojos humanos (GRH) como sistema modelo. Dichos compuestos fueron capaces de reducir la tensión superficial del agua a un valor constante, y mostraron además concentraciones micelares críticas (CMC) definidas. La observación de agregados cilíndricos de Bz-Arg-NHCn a través de microscopía de fuerza atómica corroboró las predicciones basadas en los valores del parámetro de empaquetamiento. Los estudios de la actividad hemolítica evidenciaron que la solubilización de la membrana de GRH es inducida por los agregados de los surfactantes, ya que solo se observó lisis celular a concentraciones mayores que sus respectivas CMC. Los cambios morfológicos observados en los GRH expuestos a los surfactantes demostraron la existencia de dos fenómenos involucrados en el mecanismo hemolítico: la incorporación de monómeros de surfactante en la capa externa de la membrana de los GRH, que provoca su desestabilización con la consiguiente formación de equinocitos y liberación de microvesículas, y la extracción de los componentes de la membrana, resultante de colisiones con los agregados de los surfactantes, lo que provoca una disminución del área relativa de la capa externa y favorece la aparición de estomatocitos.Trabajo presentado por el Centro de Investigación de Proteínas Vegetales (CIProVe

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