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PubMed1989

Purification and characterization of Aeromonas hydrophila beta-hemolysin.

Aeromonas hydrophila hemolysin was excreted in our culture conditions during the stationary growth phase. The toxin was purified to homogeneity by a three-step method: ultrafiltration, acid precipitation in the presence of RNA and anion exchange chromatography with FPLC apparatus. Beta-hemolysin is a protein not associated with lipids, carbohydrates or nucleic acids whose subunit mol. wt is 51,000. The mol. wt determined by polyacrylamide gel electrophoresis suggests that the molecule is in a trimeric form. The toxin is thermolabile and inactivated by proteolytic enzymes such as trypsin, chymotrypsin, pronase, subtilisin and proteinase K. Antibodies raised against the beta-hemolysin neutralize both hemolytic and cytotoxic activities. When injected at high dose, this purified hemolytic protein causes a positive rabbit ileal loop test, thus indicating that beta-hemolysin could be the main virulence factor involved in intestinal symptoms.

Aeromonas↗
PubMed1990

Purification and some properties of a hemolysin from the poisonous mushroom Rhodophyllus rhodopolius.

A hemolytic protein which causes diarrhea and death to mice was purified from the fruit bodies of a poisonous mushroom species Rhodophyllus rhodopolius (Fries) by DEAE-cellulose column chromatography, ammonium sulfate precipitation, gel filtration on Sephadex G-200, and DEAE-Sephadex A-25 column chromatography. The mol. wt of the purified hemolysin was estimated to be 40,000 by SDS-polyacrylamide slab gel electrophoresis. The hemolytic activity of the purified hemolysin was destroyed by heating at 60 degrees C for 10 min, and partially reduced by pepsin, papain and 2-mercaptoethanol. Cholesterol and phosphatidylcholine did not inhibit the activity. The hemolysin was unstable below pH 7.0 but stable at pH 8.0. The optimal pH for hemolysis was 6.0. Hemolysis did not occur below 4 degrees C even though the hemolysin bound to the erythrocyte. Mouse, chicken, rat, horse and human erythrocytes were sensitive in this order, but sheep and cow erythrocytes were not lysed by the toxin.

Animals↗
PubMed1988

Studies on Holothuria polii (Echinodermata) coelomocyte lysate. II. Isolation of coelomocyte hemolysins.

The lytic activity of the Holothuria polii coelomocyte lysate resides in two electrophoretically distinct hemolysins identified as He1 and He2. He1 represents the calcium dependent, heat-labile component whereas He2 is calcium independent and heat-stable. The two hemolysins share serological identity. Both hemolysins appear as single protein molecules of 80KDa molecular weight by SDS-PAGE and transblotting analysis under non-reducing conditions. However under reducing conditions, they are doublets of 76 and 80KDa molecular weight. The hypothesis that the two hemolysins could be isoforms is discussed.

Animals↗
PubMed1989

Effect of temperature on hemolysin production in Aeromonas spp. isolated from warm and cold environments.

The effect of growth temperature on the hemolysin production in 97 Aeromonas isolates was studied. Only 9% of the isolates obtained from low-temperature sources such as refrigerated foods, drinking water and trout aquaculture were able to produce high hemolysin titers (titer greater than 128) at 37 degrees C compared with 65% of the isolates from warm sources such as human clinical cases and warm water aquaculture (p less than 0.001). In contrast, 61% of the low-temperature isolates were producing high hemolysin titers at 10 degrees C while only 24% of the isolates from high temperature sources produced high titers at this temperature (p less than 0.05). Hemolytic activity could decrease markedly during longer (48 h) incubation at 37 degrees C. Some of the filtrates from growth at 37 degrees C and 10 degrees C were tested for enterotoxin-like activity in the suckling mice test. Of the culture filtrates produced at 37 degrees C, 40% of A. hydrophila were positive for enterotoxin-like activity, irrespective of hemolysin titer. Of A. sobria 88% of the filtrates with high hemolytin titers and none of the low-titer filtrates were enterotoxigenic. Due to a high mortality rate of mice injected with filtrates produced at 10 degrees C the enterotoxic effect of these filtrates could not be established. Autoagglutination after boiling was observed in 31% of the clinical isolates and 2% of the strains of environmental origin.

Aeromonas↗
PubMed1988

The interaction of delta-hemolysin with calmodulin.

Delta-Hemolysin forms a 1:1 complex with Ca2+ -liganded calmodulin. Probably because of the pronounced tendency of delta-hemolysin to self-associate, the apparent binding affinity is much less than that for melittin. Complex formation is reflected by an increase in quantum yield of Trp-15 of delta-hemolysin and by increased shielding from acrylamide quenching. There is, however, no indication of a change in peptide molecular ellipticity. The binding of 2-toluidinyl-naphthalene-6-sulfonate is reduced by complex formation, suggesting the involvement of a hydrophobic region. Complex formation also blocks the proteolysis by trypsin of the bond between residues 77 and 78. The time decays of fluorescence intensity and anisotropy for tryptophan are multiexponential for both free and complexed delta-hemolysin; the average decay time for intensity is substantially increased for the complex. The localized mobility of tryptophan is greatly reduced in the complex. Complex formation appears to involve both the C-terminal lobe and the connecting strand of calmodulin.

Animals↗
PubMed1990

Nucleotide sequence of the thermostable direct hemolysin gene (tdh gene) of Vibrio mimicus and its evolutionary relationship with the tdh genes of Vibrio parahaemolyticus.

The gene encoding a hemolysin similar to the thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus was previously cloned from the chromosome of Vibrio mimicus. The nucleotide sequence of the hemolysin gene was determined in this study. The gene proved to be a variant of the thermostable direct hemolysin gene (tdh gene) and was designated as Vm-tdh because the sequence divergences between the Vm-tdh gene and four tdh genes of V. parahaemolyticus were 2.1-3.0%, while the sequence divergences among the four tdh genes of V. parahaemolyticus ranged between 1.4 and 3.3%. Analysis of these five tdh genes revealed that they evolved from a common ancestor in discrete and understandable order by sporadic base substitutions.

Base Sequence↗
PubMed1996

The effects of physical and chemical agents on the secretion and stability of a Fusobacterium necrophorum hemolysin.

The effect of various agents as enhancers or inhibitors of hemolysin secretion by Fusobacterium necrophorum was investigated. The hemolysin secreted in phosphate buffered saline (PBS) alone was inactivated shortly after secretion. Tween-80 or albumin preserved the hemolytic activity in PBS in which cultures of F. necrophorum had been suspended. Hemoglobin was found to enhance hemolysin secretion. However, higher concentrations diminished secretion. Chloramphenicol, an inhibitor of protein synthesis, exhibited no effect on the hemolytic activity. However, the addition of sodium azide, an energy metabolic inhibitor, significantly reduced the hemolytic activity. Lower temperatures and pH above 9 and below 6 all yielded a low hemolytic activity. Cells suspended in Tween-80 prior to sonication yielded a substantial amount of extracellular hemolytic activity with low intracellular activity detected. However, cells suspended in PBS alone yielded a low extracellular activity but rather a high intracellular activity. The same spectrum of red blood cells of different species were found to be sensitive to both the extracellular and intracellular hemolysins.

Albumins↗
PubMed1991

Characterization of a non-hemolytic mutant of Actinobacillus pleuropneumoniae serotype 5: role of the 110 kilodalton hemolysin in virulence and immunoprotection.

To determine the role of hemolysin(s) in virulence and immunoprotection, non-hemolytic mutants of Actinobacillus pleuropneumoniae serotype 5, strain J45, were isolated following chemical mutagenesis. One mutant was selected for extensive characterization. Differences in capsule content, or in lipopolysaccharide or membrane protein electrophoretic profiles of the parent and mutant were not detected. A predominant, calcium-inducible protein of 110 kDa was present in culture supernatant of the parent, but absent from the mutant. Two-dimensional (2-D) gel electrophoresis confirmed that the 110 kDa protein was absent in culture supernatant of the mutant, but few, if any, minor differences could be detected in whole-cell proteins between the parent and mutant. The mutant totally lacked extracellular hemolytic and cytotoxic activity. Lysates of whole cells of the mutant contained weak hemolytic activity, and the 110 kDa protein could be detected by immunoblotting. Neutralization titers were negative in pigs immunized with the mutant or purified, denatured hemolysin, although enzyme-immunoassay titers were detected. Four additional independently isolated non-hemolytic mutants were avirulent in pigs and mice at doses greater than 10 times the lethal dose of the parent. Neither pigs nor mice were protected against lethal infection following immunization with the non-hemolytic mutant. We conclude that the 110 kDa hemolysin plays an important role in bacterial virulence and the pathogenesis of pleuropneumonia, and that sufficiently high levels of neutralizing antibodies to the 110 kDa hemolysin may be required for protection of pigs against disease.

Actinobacillus↗
PubMed2005

Placental and intestinal alkaline phosphatases are receptors for Aeromonas sobria hemolysin.

The Aeromonas sobria hemolysin causes diarrhea following infection by this enteropathogenic bacterium. We previously identified the putative receptor for A. sobria hemolysin as a p66 protein on Intestine 407 cells (Microb. Pathog. 27 (1999) 215-221). Here, we have partially purified and obtained a peptide mass fingerprint of p66 which revealed its identity with placental alkaline phosphatase (PLAP). Recombinant PLAP expressed in 293T cells was also found to bind to hemolysin and the binding was found not to be dependent on the N-linked glycosylation of PLAP. By immunohistochemical analysis, PLAP expression was detected in human intestinal mucosa, the target tissue in disease. In addition to PLAP, hemolysin also binds to intestinal alkaline phosphatase (IAP), an enzyme that is also abundantly expressed in intestine. Thus, both PLAP and IAP are very likely involved in the pathogenesis of diarrhea caused by this bacterial toxin.

Aeromonas↗
PubMedDate not supplied

In vitro activation of the hemolysin in Prevotella nigrescens ATCC 33563 and Prevotella intermedia ATCC 25611.

Hemolytic activity was evaluated in the putative periodontopathogens Prevotella intermedia and Prevotella nigrescens. Whole cells of both species present weak hemolytic activity evidenced only by solid media assays after 48 h of bacterial growth or after 5 h of interaction with erythrocytes at 37 degrees C in liquid assays. In this work we show that the use of crude extract allowed the detection of a higher hemolytic activity for P. intermedia, but surprisingly not for P. nigrescens. Incubation at 37 degrees C for 9 h, or treatment with trypsin or proteinase K, increased or exposed the hemolytic activity of P. intermedia and P. nigrescens crude extract, respectively. The activation process was inhibited by TLCK and PMSF but not by EDTA, E-64 or pepstatin A, indicating the serino-protease nature of the factor involved in activation of P. intermedia and P. nigrescens hemolysins. Both the buffer and the pH employed for cell fractionation influenced the activation of hemolysin, and the best results were obtained with Universal buffer at pH 8.0. The activated hemolysins acted optimally at pH 6.5 at 37 degrees C and the maximum hemolytic activity was detected at the early log phase of growth. The results of this study show for the first time a strong hemolytic activity for P. nigrescens and evidence of proteolytic activation of hemolysins produced by periodontopathogens.

Animals↗
PubMed1998

Immunoglobulin A response against Gardnerella vaginalis hemolysin and sialidase activity in bacterial vaginosis.

OBJECTIVE: The aim of this study was to investigate the correlation between the immunoglobulin A immune response to Gardnerella vaginalis hemolysin and sialidase activity in vaginal fluids from patients with bacterial vaginosis. STUDY DESIGN: Nonpregnant women who were examined at a gynecologic clinic, in an age range of 18 to 62 years, were enrolled. The study population comprised 131 healthy volunteers, 32 women with bacterial vaginosis that was positive for immunoglobulin A to Gardnerella vaginalis hemolysin, 40 women with bacterial vaginosis that was negative for immunoglobulin A to Gardnerella vaginalis hemolysin, and 19 women with Candida vaginitis. Bacterial vaginosis was diagnosed by clinical criteria and Gram stain. RESULTS: Sialidase activity was present in 75% (54/72) of patients with bacterial vaginosis. Women having bacterial vaginosis and lacking a specific immunoglobulin A response had a significantly higher level of sialidase activity than patients who had an immune response against Gardnerella vaginalis hemolysin. Sialidase activity was detected in 87% (35/40) of the former subgroup of patients with bacterial vaginosis and in 59% (19/32) of women of the latter subgroup. No sialidase activity was measured in patients with candidiasis. Specificity of the assay for healthy controls was 95% (124/131 women without sialidase activity). CONCLUSIONS: Sialidases produced by Prevotella bivia and other microorganisms present in the microflora of patients with bacterial vaginosis are very likely a virulence factor not only by destroying the mucins and enhancing adherence of bacteria but also by impairing a specific immunoglobulin A immune response against other virulence factors such as cytotoxin from Gardnerella vaginalis.

Adolescent↗
PubMed1998

Self-assembled alpha-hemolysin pores in an S-layer-supported lipid bilayer.

The effects of a supporting proteinaceous surface-layer (S-layer) from Bacillus coagulans E38-66 on a 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC) bilayer were investigated. Comparative voltage clamp studies on plain and S-layer supported DPhPC bilayers revealed no significant difference in the capacitance. The conductance of the composite membrane decreased slightly upon recrystallization of the S-layer. Thus, the attached S-layer lattice did not interpenetrate or rupture the DPhPC bilayer. The self-assembly of a pore-forming protein into the S-layer supported lipid bilayer was examined. Staphylococcal alpha-hemolysin formed lytic pores when added to the lipid-exposed side. The assembly was slow compared to unsupported membranes, perhaps due to an altered fluidity of the lipid bilayer. No assembly could be detected upon adding alpha-hemolysin monomers to the S-layer-faced side of the composite membrane. Therefore, the intrinsic molecular sieving properties of the S-layer lattice do not allow passage of alpha-hemolysin monomers through the S-layer pores to the lipid bilayer. In comparison to plain lipid bilayers, the S-layer supported lipid membrane had a decreased tendency to rupture in the presence of alpha-hemolysin.

Bacillus↗
PubMed1999

Water transport by the bacterial channel alpha-hemolysin.

This study is an investigation of the ability of the bacterial channel alpha-hemolysin to facilitate water permeation across biological membranes. alpha-Hemolysin channels were incorporated into rabbit erythrocyte ghosts at varying concentrations, and water permeation was induced by mixing the ghosts with hypertonic sucrose solutions. The resulting volume decrease of the ghosts was followed by time-resolved optical absorption at pH 5, 6, and 7. The average single-channel permeability coefficient of alpha-hemolysin for water ranged between 1.3x10-12 cm/s and 1.5x10-12 cm/s, depending on pH. The slightly increased single-channel permeability coefficient at lower pH-values was attributed to an increase in the effective pore size. The activation energy of water transport through the channel was low (Ea=5.4 kcal/mol), suggesting that the properties of water inside the alpha-hemolysin channel resemble those of bulk water. This conclusion was supported by calculations based on macroscopic hydrodynamic laws of laminar water flow. Using the known three-dimensional structure of the channel, the calculations accurately predicted the rate of water flow through the channel. The latter finding also indicated that water permeation data can provide a good estimate of the pore size for large channels.

Animals↗
PubMed1996

Reversible adsorption and nonreversible insertion of Escherichia coli alpha-hemolysin into lipid bilayers.

Alpha-Hemolysin is an extracellular protein toxin (107 kDa) produced by some pathogenic strains of Escherichia coli. Although stable in aqueous medium, it can bind to lipid bilayers and produce membrane disruption in model and cell membranes. Previous studies had shown that toxin binding to the bilayer did not always lead to membrane lysis. In this paper, we find that alpha-hemolysin may bind the membranes in at least two ways, a reversible adsorption and an irreversible insertion. Reversibility is detected by the ability of liposome-bound toxin to induce hemolysis of added horse erythrocytes; insertion is accompanied by an increase in the protein intrinsic fluorescence. Toxin insertion does not necessarily lead to membrane lysis. Studies of alpha-hemolysin insertion into bilayers formed from a variety of single phospholipids, or binary mixtures of phospholipids, or of phospholipid and cholesterol, reveal that irreversible insertion is favored by fluid over gel states, by low over high cholesterol concentrations, by disordered liquid phases over gel or ordered liquid phases, and by gel over ordered liquid phases. These results are relevant to the mechanism of action of alpha-hemolysin and provide new insights into the membrane insertion of large proteins.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

Assembly of Staphylococcus aureus gamma-hemolysin into a pore-forming ring-shaped complex on the surface of human erythrocytes.

Staphylococcal gamma-hemolysin consists of Hlg1 (or Luk F) of 34 kDa and Hlg2 of 32 kDa, which cooperatively lyse human erythrocytes. Since gamma-hemolysin caused swelling of human erythrocytes prior to lysis, we studied pore-forming nature of the toxin by use of polyethylene glycols as osmotic protectants and determined the functional diameter of the pore. To elucidate the molecular architecture of the membrane pore formed by gamma-hemolysin, we solubilized the pore complex with 2% sodium dodecyl sulfate, separated it from erythrocyte membrane proteins by sucrose gradient ultracentrifugation, and observed the isolated complex under an electron microscope. Our data showed that Hlg1 and Hlg2 of gamma-hemolysin assemble into a ring-shaped 195 kDa complex in a molar ratio of 1 : 1, which may form a membrane pore with a functional diameter of 2.1-2.4 nm.

Bacterial Proteins↗
PubMed1999

Vitronectin and its fragments purified as serum inhibitors of Staphylococcus aureus gamma-hemolysin and leukocidin, and their specific binding to the hlg2 and the LukS components of the toxins.

Staphylococcal gamma-hemolysin and leukocidin are bi-component cytolysins, consisting of LukF (or Hlg1)/Hlg2 and LukF/LukS, respectively. Here, we purified serum inhibitors of gamma-hemolysin and leukocidin from human plasma. Protein sequencing showed that the purified inhibitors of 62, 57, 50 and 38 kDa were the vitronectin fragments with truncation(s) of the C-terminal or both N- and C-terminal regions. The purified vitronectin fragments specifically bound to the Hlg2 component of gamma-hemolysin and the LukS component of leukocidin to form high-molecular-weight complexes with them, leading to inhibition of the toxin-induced lysis of human erythrocytes and human polymorphonuclear leukocytes, respectively. Intact vitronectin also showed inhibitory activity to the toxins. The ability of gamma-hemolysin and leukocidin to bind vitronectin and its fragments is a novel function of the pore-forming cytolysins.

Amino Acid Sequence↗
PubMed2001

Mode of action of beta-barrel pore-forming toxins of the staphylococcal alpha-hemolysin family.

Staphylococcal alpha-hemolysin is the prototype of a family of bacterial exotoxins with membrane-damaging function, which share sequence and structure homology. These toxins are secreted in a soluble form which finally converts into a transmembrane pore by assembling an oligomeric beta-barrel, with hydrophobic residues facing the lipids and hydrophilic residues facing the lumen of the channel. Besides alpha-hemolysin the family includes other single chain toxins forming homo-oligomers, e.g. beta-toxin of Clostridium perfringens, hemolysin II and cytotoxin K of Bacillus cereus, but also the staphylococcal bi-component toxins, like gamma-hemolysins and leucocidins, which are only active as the combination of two similar proteins which form hetero-oligomers. The molecular basis of membrane insertion has become clearer after the determination of the crystal structure of both the oligomeric pore and the soluble monomer. Studies on this family of beta-barrel pore-forming toxins are important for many aspects: (i) they are involved in serious pathologies of humans and farmed animals, (ii) they are a good model system to investigate protein-membrane interaction and (iii) they are the basic elements for the construction of nanopores with biotechnological applications in various fields.

Amino Acid Sequence↗
PubMed1999

Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh.

Vibrio parahaemolyticus is an important human pathogen which can cause gastroenteritis when consumed in raw or partially-cooked seafood. A multiplex PCR amplification-based detection of total and virulent strains of V. parahaemolyticus was developed by targeting thermolabile hemolysin encoded by tl, thermostable direct hemolysin encoded by tdh, and thermostable direct hemolysin-related trh genes. Following optimization using oligonucleotide primers targeting tl, tdh and trh genes, the multiplex PCR was applied to V. parahaemolyticus from 27 clinical, 43 seafood, 15 environmental, 7 strains obtained from various laboratories and 19 from oyster plants. All 111 V. parahaemolyticus isolates showed PCR amplification of the tl gene; however, only 60 isolates showed amplification of tdh, and 43 isolates showed amplification of the trh gene. Also, 18 strains showed amplification of the tdh gene, but these strains did not show amplification of the trh gene. However, one strain exhibited amplification for the trh but not the tdh gene, suggesting both genes need to be targeted in a PCR amplification reaction to detect all hemolysin-producing strains of this pathogen. The multiplex PCR approach was successfully used to detect various strains of V parahaemolyticus in seeded oyster tissue homogenate. Sensitivity of detection for all three target gene segments was at least between 10(1)-10(2) cfu per 10 g of alkaline peptone water enriched seeded oyster tissue homogenate. This high level of sensitivity of detection of this pathogen within 8 h of pre-enrichment is well within the action level (10(4) cfu per 1 g of shell stock) suggested by the National Seafood Sanitation Program guideline. Compared to conventional microbiological culture methods, this multiplex PCR approach is rapid and reliable for accomplishing a comprehensive detection of V. parahaemolyticus in shellfish.

Animals↗