Hemolysin of <em>Vibrio</em> Species

Hemolysin is one of the major pathogenic factors among Vibrio species, which shows hemolytic activity against erythrocytes. It is associated with different Vibrio spp. that manifest either wound infection or intestinal infection as their clinical symptom. V. vulnificus and V. alginolyticus are well-known causative organisms for wound infection, whereas the gastrointestinal infection is caused by V. cholerae, V. mimicus, and V. parahaemolyticus. There are two major groups of hemolysins in Vibrio spp.: the thermostable direct hemolysin (TDH) from V. parahaemolyticus and the HlyA (El Tor hemolysin) from V. cholerae. These hemolysins have homology in certain degrees; however, the essential amino acids for the activity are variable depending on the species. This chapter summarizes the functions and features of hemolysins from Vibrio species, which has been reported so far

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

Genomic Analysis of Immune Response against Vibrio Cholerae Hemolysin in Caenorhabditis elegans

Vibrio cholerae cytolysin (VCC) is among the accessory V. cholerae virulence factors that may contribute to disease pathogenesis in humans. VCC, encoded by hlyA gene, belongs to the most common class of bacterial toxins, known as poreforming toxins (PFTs). V. cholerae infects and kills Caenorhabditis elegans via cholerae toxin independent manner. VCC is required for the lethality, growth retardation and intestinal cell vacuolation during the infection. However, little is known about the host gene expression responses against VCC. To address this question we performed a microarray study in C. elegans exposed to V. cholerae strains with intact and deleted hlyA genes. Many of the VCC regulated genes identified, including C-type lectins, Prion-like (glutamine [Q]/asparagine [N]-rich)-domain containing genes, genes regulated by insulin/ IGF-1-mediated signaling (IIS) pathway, were previously reported as mediators of innate immune response against other bacteria in C. elegans. Protective function of the subset of the genes up-regulated by VCC was confirmed using RNAi. By means of a machine learning algorithm called FastMEDUSA, we identified several putative VCC induced immune regulatory transcriptional factors and transcription factor binding motifs. Our results suggest that VCC is a major virulence factor, which induces a wide variety of immune response- related genes during V. cholerae infection in C. elegans

Der katalytische Zyklus der Nukleotid-bindenden Domäne des ABC-Transporters HlyB

Nucleotide-binding domains (NBDs), roughly 27 kDa in size, are conservative components of the large family of ABC (ATP-binding cassette) transporters, which includes importers, exporters, and receptors. NBDs or ABC-ATPases supply energy for the translocation of a vast variety of substrates across biological membranes. Despite their hydrophilic sequence, many NBDs tend to aggregate and precipitate in solution upon isolation from the complete transporter. The conditions stabilizing an extremely labile NBD component of the E.coli HlyA transporter, HlyB-NBD, were developed. As a result, the pure highly concentrated enzyme was protected from precipitation for months that allowed screening of the unlimited crystallization conditions in the presence of different substrates and performance of the reproducible functional assays. HlyB-NBD was characterized in regard to its uncoupled ATPase activity, oligomeric state, and stability in solution. Comparative analysis of protein stability and ATPase activity in various buffers suggested an inverse relationship between the two. Kinetic analysis of ATPase activity revealed ATP-induced protein dimerization. Gel-filtration experiments with the wild type protein and H662A-mutant of HlyB-NBD provided further evidence of protein dimerization in the presence of ATP. The crystal structures in post- and pre-hydrolysis nucleotide-bound states of HlyB-NBD were determined at 1.6Å and 2.5Å resolution, respectively. While the hydrolytically deficient H662A mutant of HlyB-NBD was crystallized as a stable dimer in the presence of ATP or ATP-Mg2+, with two nucleotide molecules sandwiched between the two monomers, the same protein was shown to be a monomer in the ADP-loaded state. The wild type protein failed to develop crystals with bound ATP, yet formed ADP-bound crystals identical to those of the H662A-mutant. The X-ray structures of HlyB-NBD in various states of the hydrolytic cycle and the functional studies of the enzyme have provided an opportunity to characterize enzyme-substrate complexes and protein-protein interactions between the NBD subunits in great detail. Comparison of the nucleotide-free, the ADP-, and the ATP-loaded states revealed oligomeric and conformational changes of the protein upon substrate binding and resulted in a molecular picture of the catalytic cycle. The correlated results of the structural and functional investigations of HlyB-NBD are discussed with relation to the mechanism of action of ABC transporters.Nukleotid-bindende Domänen (NBDs) sind konservierte Komponenten der großen Familie der ABC (ATP-Bindungskassette) Transporter, die ein Molekulargewicht von ungefähr 27 kDa besitzen. Diese Transporter umfassen Proteine, die als Importer, Exporter oder Rezeptoren fungieren. NBDs oder ABC-ATPasen stellen die Energie für den Transport einer großen Vielzahl unterschiedlicher Substrate über biologische Membranen zur Verfügung. Trotz ihrer hydrophilen Natur besitzen viele isolierte NBDs die Eigenschaft, in Abwesenheit der anderen Komponenten des Transporters in Lösung zu aggregieren und zu präzipitieren. In der hier vorliegenden Arbeit wurden Bedingungen etabliert, die die labile NBD des HlyB Transporters aus E. coli stabilisierten. Die homogene und konzentrierte Enzymlösung wurde dadurch über Monate hinweg gegen Präzipitation geschützt. Dies ermöglichte es, eine große Vielzahl unterschiedlicher Kristallisationsbedingungen in Gegenwart verschiedener Substrate zu analysieren und funktionelle Assays durchzuführen. Die HlyB-NBD wurde bezüglich ihrer basalen ATPase Aktivität, ihres oligomeren Zustandes und ihrer Stabilität in Lösung analysiert, die eine inverse Beziehung zwischen Aktivität und Stabilität implizierte. Kinetische Untersuchungen belegten darüber hinaus eine ATP-induzierte Dimerisierung der NBD. Größenausschlusschromatographie mit dem Wildtyp und der H662A Mutante ergaben weitere Indizien für die ATP-vermittelte Dimerisierung. Die Kristallstrukturen des prä- und post-hydrolytischen Zustandes wurden mit einer Auflösung von 2.5 Å und 1.6 Å bestimmt. Während die H662A Mutante, die keinerlei ATPase Aktivität aufwies, im ATP und ATP/Mg2+ gebundenen Zustand als Dimer mit zwei Nukleotiden an der Interaktionsfläche der beiden Proteinmonomere kristallisiert wurde, bildet die H662A Mutante in Gegenwart von ADP nur Monomer. Für den Wildtyp konnten keine Kristalle in Gegenwart von ATP erhalten werden. Im Gegensatz hierzu war die Struktur im ADP-gebundenen Zustand identisch zur Struktur der H662A Mutante. Die Kristallstrukturen der HlyB-NBD in den unterschiedlichen Zuständen des hydrolytischen Zyklus in Kombination mit funktionalen Untersuchungen in Lösung ermöglichten es, Protein-Substrat und Protein-Protein Wechselwirkungen mit großer Genauigkeit zu bestimmen. Ein Vergleich der Nukleotid-freien, der ADP- und ATP-gebundenen Zustände verdeutlichte, dass sowohl Dimerisierung als auch konformationelle Änderungen durch die Substratbindung erfolgten. Diese Analysen ergaben ein molekulares Bild der essentiellen Schritte des katalytischen Zyklus der HlyB-NBD, die im Zusammenhang mit einem allgemeinen Transportmechanismus von ABC-Transportern diskutiert wurden

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

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

ABC transporter architecture and regulatory roles of accessory domains

AbstractWe present an overview of the architecture of ATP-binding cassette (ABC) transporters and dissect the systems in core and accessory domains. The ABC transporter core is formed by the transmembrane domains (TMDs) and the nucleotide binding domains (NBDs) that constitute the actual translocator. The accessory domains include the substrate-binding proteins, that function as high affinity receptors in ABC type uptake systems, and regulatory or catalytic domains that can be fused to either the TMDs or NBDs. The regulatory domains add unique functions to the transporters allowing the systems to act as channel conductance regulators, osmosensors/regulators, and assemble into macromolecular complexes with specific properties

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

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