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PubMed1983

Characterization of the hemolysin produced by haemophilus pleuropneumoniae.

Production and characterization of Haemophilus pleuropneumoniae hemolysin were investigated by using 5 serotype 2 strains. The hemolysin was produced in chicken meat-infusion broth medium both in stationary and in shaking cultures. In stationary culture, hemolytic activity against horse RBC reached maximum at postincubation day 5 (at the late stage of stationary phase), and the activity was maintained at the same level for 2 days thereafter. The hemolytic activity of shaking culture reached a maximum at postincubation hour 9 (at the early stage of logarithmic-growth phase), gradually decreased, and disappeared at postincubation day 2. The hemolysin was shown to be an extracellular product of the bacterial cells. The RBC of horses, rabbits, and sheep were highly susceptible to the hemolysin, and those of pigs and guinea pigs were less susceptible, whereas RBC of 60-day-old chicks were not susceptible. The hemolysin was not inactivated by autoclaving at 121 C for 2 hours, and by treatments with formalin, trypsin, or pronase. The presence of calcium or magnesium ions did not change the activity, whereas iodoacetic acid significantly reduced the activity.

Animals↗
PubMedDate not supplied

[Analysis of Vibrio cholerae hemolysins using monolayer continuous cell cultures].

The comparative study of the preparations of V.cholerae hemolysins of different serovars with the use of continuous cell lines CHO-K1, Vero, Hela, L-929 was carried out. The preparations of hemolysins isolated from such strains as V.cholerae 569B, V.eltor 9949, V.cholerae O-mut 461/67-34 differed in their biological activity on experimental animal models and had different cytotoxic activity. The preparations exhibiting no activity when tested in vivo (V.cholerae and V.eltor hemolysins) were cetotoxins, but in lower doses (1000 and 100 times respectively) than the preparation of V.cholerae O-mut hemolysin, active in vivo. Hemolysins induced the formation of anticytotoxic antibodies in low titers.

Animals↗
PubMed1991

Nucleotide sequence and expression of an extracellular hemolysin gene of Aeromonas hydrophila.

The extracellular hemolysin (AHH1) gene of Aeromonas hydrophila ATCC7966 was cloned into Charomid9-28 in Escherichia coli DH1, and its complete nucleotide sequence determined. Escherichia coli carrying this gene expressed an extracellular heat-labile hemolysin for rabbit red blood cells. The minimum size of the coding region of the 2.6 kilobase-pair BamHI-SphI fragment was subcloned into pUC118 and pUC119, selecting for hemolytic activity. The nucleotide sequence of this region contained a single open reading frame of 1734 base pairs, corresponding to a protein of 577 amino acid residues (63,658 daltons). A consensus promoter sequence was present upstream of the AHH1 open reading frame. Maxicell analysis of [35S]methionine-labelled proteins in E. coli CSR603 carrying the AHH1 plasmid suggested that AHH1 gene codes for an approximately 60,000 dalton polypeptide. By colony DNA-DNA hybridization analysis, the AHH1 gene was detected in 43 of 62 hemolysin-producing strains of A. hydrophila (isolated from various sources and areas) and in all 43 hemolysin-producing strains of A. salmonicida (isolated from fish). Three hemolysin-negative strains of A. hydrophila did not react with the AHH1 probe, whereas three non-hemolytic A. salmonicida strains hybridized with the probe.

Aeromonas hydrophila↗
PubMed1982

Partial purification and characterization of hemolysin from a psychrotrophic kanagawa-positive marine Vibrio.

Psychrotrophic Kanagawa-positive marine vibrios were isolated from soft-shelled clams (Mya arenaria) collected in Yaquina Bay, Oreg. The 235 vibrio isolates obtained were screened for Gram reaction and morphology, Kanagawa reaction on Wagastsuma agar, and response to selected biochemical tests. The vibrio selected for further study was grown in broth, and the hemolysin was precipitated from a cleared supernatant with solid ammonium sulfate. The hemolytic substance was partially purified by DEAE-cellulose and Sephadex G-100 column chromatography. The hemolysin contained protein essential for activity, was thermolabile, and was more active against rabbit erythrocytes at 37 degrees C than at lower temperatures. The molecular weight was estimated at 55,000 by using a Sephadex G-100 column. Hemolytic activity was partially inactivated by gangliosides and lowered against horse erythrocytes. The hemolysin did not react with antibody prepared against vibriolysin from Vibrio parahaemolyticus WP-1 by the Ouchterlony method. The hemolysin was high in aspartic and glutamic acids and low in arginine and histidine. Electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel gave three major bands. The hemolysin from a psychrotrophic vibrio and the hemolytic exotoxin of V. parahaemolyticus had some similar and dissimilar characteristics. The possibility that a Vibrio sp. other than V. parahaemolyticus might serve as the reservoir for the Kanagawa phenotype is discussed.

Journal Article↗
PubMed1983

Spontaneous deletions and flanking regions of the chromosomally inherited hemolysin determinant of an Escherichia coli O6 strain.

The hemolytic Escherichia coli strain 536 (O6) propagates spontaneous hemolysin-negative mutants at relatively high rates (10(-3) to 10(-4)). One type of mutant (type I) lacks both secreted (external) and periplasmic (internal) hemolysin activity (Hlyex-/Hlyin-) and in addition shows no mannose-resistant hemagglutination (Mrh-), whereas the other type (type II) is Hlyex-/Hlyin+ and Mrh+. The genetic determinants for hemolysin production (hly) and for mannose-resistant hemagglutination (mrh) of this strain are located on the chromosome. Hybridization experiments with DNA probes specific for various parts of the hly determinant reveal that mutants of type I have lost the total hly determinant, whereas those of type II lack only part of the hlyB that is essential for transport of hemolysin across the outer membrane. Using a probe that contains the end sequence of the plasmid pHly152-encoded hly determinant (adjacent to hlyB), we determined that a related sequence flanks also the hlyB-distal end of the chromosomal hly determinant of E. coli 536. In addition several other similar or even identical sequences are found in the vicinity of the hlyC- and the hlyB-distal ends of both the chromosomal and the plasmid hly determinants.

Base Sequence↗
PubMed1964

PARTIAL PURIFICATION OF THE EXTRACELLULAR HEMOLYSIN OF PSEUDOMONAS AERUGINOSA.

Berk, Richard S. (Wayne State University, College of Medicine, Detroit, Mich.). Partial purification of the extracellular hemolysin of Pseudomonas aeruginosa. J. Bacteriol. 88:559-565. 1964.-Through a series of chemical fractionation steps, the extracellular hemolysin of Pseudomonas aeruginosa was purified 126-fold with a recovery of 49%. Hemolytic activity of crude preparations was irreversibly lost upon contact with anionic exchange materials such as diethylaminoethyl Sephadex or ECTEOLA-Cellulose, but traveled with the solvent front during passage through Sephadex G-200 and carboxymethyl Sephadex. The hemolysin was soluble in water and ethanol, and was partially extractable with ether, but not with trichlorotrifluoroethane (Freon). Although normal serum and serum albumin blocked hemolytic activity, it was unaffected by trypsin, deoxyribonuclease, or ribonuclease. Partially purified hemolysin was studied in vivo, but did not exert dermonecrotic activity in mice or rabbits in the concentrations tested. Although preparations were toxic to mice, lethality appeared to be more a reflection of the nonhemolytic protein content of the preparations rather than of hemolytic activity.

Animals↗
PubMed1964

TYPES OF HEMOLYSINS PRODUCED BY STAPHYLOCOCCUS AUREUS, AS DETERMINED BY THE REPLICA PLATING TECHNIQUE.

Haque, Riaz-Ul (The Ohio State University, Columbus), and Jack N. Baldwin. Types of hemolysins produced by Staphylococcus aureus as determined by the replica plating technique. J. Bacteriol. 88:1442-1447. 1964.-The type of hemolysins produced by a representative number of cells of a culture of Staphylococcus aureus was determined by the replica plating technique, with the use of sheep, rabbit, human, and horse blood agar plates. Unless horse and human red blood cells were used, a distinction between alpha-and delta-hemolysins was difficult to make. Strains producing alpha-beta-, beta-delta-, and alpha-delta-hemolysins were recognized by the replica plating technique. A few colonies produced lysis of rabbit, horse, and human cells without lysis of sheep red blood cells. This pattern of lysis was not detected by the radial streak or the tube titration procedures when mixed populations were examined, and may be due to a lytic factor not hitherto described.

Animals↗
PubMed1985

Escherichia coli and human neutrophils. Effect of bacterial supernatant with hemolysin activity upon chemotaxin receptors.

A greater proportion of Escherichia coli strains isolated from clinical extraintestinal infections produce alpha-hemolysin than do strains isolated from normal fecal flora. Proposed mechanisms to explain this observation have stressed the fact that hemolysis liberates hemoglobin, which may provide a nutritional boost for E coli growth. Alternatively, a cytolytic effect of hemolysin upon host neutrophils has been postulated. Our previous studies have suggested a third possibility: Human neutrophils incubated in the supernatant of an alpha-hemolysin-producing E coli strain produced a selective inhibition of chemotaxis toward C5a. Bacteria-free supernatants from 14 clinical strains of E coli were therefore evaluated for an ability to lyse sheep erythrocytes, alter human polymorphonuclear neutrophil (PMN) chemotaxin receptors, and affect release of PMN enzymes. Supernatants possessing hemolytic activity decreased the C5a receptor activity of human PMNs and increased the number of peptide receptors. A stimulation of secondary granule release, as evidenced by the release of PMN lysozyme, may account for the increased expression of peptide receptors. Perturbation of host defenses through a loss of neutrophil migratory function and secondary granule contents may allow for enhanced survival of E coli, which produce alpha-hemolysin.

Bacterial Proteins↗
PubMed1990

Kinetics of hemolysin production in bovine group B streptococci.

Thirteen strains of group B streptococci etiologically related to bovine infections were investigated in order to observe the effects of non-hemoglobin iron and glucose on growth and hemolysin production; and to determine the necessity to stabilize the hemolysin with a carrier-stabilizer agent. Ferric citrate was diluted to give final concentrations of 1-11 micrograms/ml, added to iron-reduced (by CaCl2 precipitation) brain heart infusion media, inoculated and incubated at 37 degrees C. Parallel experiments were carried out with glucose. A variety of agents was employed to act as hemolysin stabilizers. Hemolysin production was detected by lysis of sheep erythrocytes. Both iron and glucose were crucial in concentrations 5-7 micrograms/ml and 0.5-1% respectively. Bovine serum albumin-starch mixture acted as an excellent stabilizer.

Animals↗
PubMed1993

Lack of association between hemolysin production and acute inflammation in human urinary tract infection.

Hemolysins are cytolytic proteins which have been extensively characterized at the molecular level, however, their in vivo functions remain unclear. This study analyzed the association of hemolysin production with the inflammatory response in patients with urinary tract infection (UTI). Infants and children with their first episode of UTI (n = 644) were followed prospectively. The body temperature, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), urinary leucocyte count and renal concentrating capacity were used as measures of the inflammatory response. The hemolytic genotype (hly) of the Escherichia coli strain from each UTI episode was defined by DNA-DNA hybridization, and the phenotype by hemolysis in blood agar. There was no significant increase in the level of fever, CRP, ESR, or decrease in renal concentrating capacity during UTI episodes caused by hly positive compared to hly negative E. coli. Multiple regression analysis did not demonstrate significant associations of hly with elevated fever, CRP, ESR or reduced renal concentrating capacity. In contrast, patients infected with P fimbriated E. coli strains had higher fever, CRP, ESR and lower renal concentrating capacity than those infected with other strains. This association was not influenced by the hly genotype of the P fimbriated strains. The frequency of hly+ strains was not significantly higher in the subset of patients assigned a diagnosis of acute pyelonephritis compared to asymptomatic bacteriuria. This was in contrast to P fimbriae, which were accumulated in acute pyelonephritis. The results suggested that the acute inflammatory response to E. coli UTI is independent of hemolysin production. The inflammatogenic potential of uropathogenic E. coli clones was better described by the presence or absence of P-fimbriae than by hemolysin.

Child, Preschool↗
PubMed1988

The hemolysin of Escherichia coli.

Many strains of E. coli elaborate a hemolysin which is responsible for the zone of beta-hemolysis surrounding bacterial colonies on blood agar. The significance of this cytolysin as a determinant of bacterial pathogenicity has been established in animal models with the use of genetically engineered, isogenic bacterial strains. An analogous role in human infections has been inferred from the high association of hemolysin production with disease. Studies at a molecular genetical level have defined 4 genes that are required for the synthesis, post-translational modification and secretion of the hemolysin. The structural gene hlyA encodes for a 107-110,000 polypeptide, which must be modified in an unknown manner to its active form by the product of the neighboring hlyC gene. Genes hlyB and hlyD encode for proteins that export the molecule to the extracellular medium. The signal for secretion is contained in the C-terminal portion of the toxin molecule. The secreted hemolysin attacks plasma membranes of target mammalian cells by inserting as a monomer into the bilayer and generating hydrophilic transmembrane pores of approximately 2 nm effective diameter. The pores display a marked selectivity for cations over anions and pore-opening is dependent on the presence of a correct transmembrane potential. Binding to a membrane target does not require the presence of a specific receptor, and pores may be generated in planar lipid membranes consisting solely of phosphatidylcholine. Pore formation in nucleated cells can trigger secondary reactions such as stimulation of arachidonate metabolism with release of lipid mediators, probably initiated by passive influx of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Characterization of a sequence (hlyR) which enhances synthesis and secretion of hemolysin in Escherichia coli.

A sequence (hlyR) of about 600 bp which enhances the expression of hemolysin (HlyA) more than 50-fold was identified in the plasmid pHly152-specific hemolysin (hly) determinant. Deletion of this entire hlyR sequence led to the same low level of hemolysin synthesis and excretion as that expressed by the recombinant plasmid pANN202-312. HlyR was active in cis but its activity was orientation-dependent. The enhancing sequence, hlyR, is separated from the promoter phlyI transcribing hlyC, hlyA and possibly hlyB by more than 1.5 kb including an IS2 element. Stepwise removal of the hlyR sequence from its 5' end by exonuclease III (ExoIII) digestion yielded several types of deletion mutants which expressed decreasing amounts of hemolysin. A similar observation was made when hlyR was shortened by ExoIII from its 3' end, which suggests that more than one functional region may be present in the hlyR sequence. A deletion of 717 bp within the adjacent IS2 element reduced the activity of hlyR only slightly, indicating that IS2 is not directly involved in the enhancement mechanism but that it may support an optimal positioning in hlyR relative to the hly promoter. The nucleotide sequence of hlyR is rich in A + T and does not contain an extended open reading frame, but exhibits several sequence motives that may represent sites for protein binding and DNA bending.

Base Sequence↗
PubMed1991

Altered hemolysin production in urine-grown uroisolates of Escherichia coli.

Fifteen uroisolates of E. coli were studied for both cell-free and cell-bound hemolysin production. Estimations were done in Trypticase soy broth (TSB, providing iron-replete medium) TSB + 2,2'-bipyridine (providing experimentally created iron-depleted conditions) and pooled normal human urine (providing natural iron-depleted growth medium). In TSB 40% of strains showed no detectable cell-free hemolysin, they were able to produce it in the presence of 2,2'-bipyridine and more so when grown in urine. The cell-bound hemolysin was produced by all the strains in TSB, but in the presence of 2,2'-bipyridine and urine an insignificant increase was observed. All the strains when given 2nd and 3rd passage in urine, were found to elaborate significantly more cell-free as well as cell-bound hemolysin.

Bacterial Proteins↗
PubMed1988

Production and purification of Escherichia coli hemolysin.

Eight strains of Escherichia coli, isolated from patients with a urinary tract infection were investigated for production of hemolysin. Six of these produced hemolysin and one revealed maximum hemolytic activity. Three urinary and two faecal isolates were positive for mannose-resistant hemagglutination. One isolate positive for hemagglutination and giving maximum hemolytic activity was then used. Hemolysin was present in the supernatant broth and the medium of choice to obtain the optimum yield was the alkaline meat extract broth followed by brain heart infusion broth. The highest yield appeared in the exponential phase of growth. Hemolysin is a heat-labile protein, being produced optimally at pH 8. A three-stage procedure was the best method for its purification.

Bacterial Proteins↗
PubMed1996

Permeability increase induced by Escherichia coli hemolysin A in human macrophages is due to the formation of ionic pores: a patch clamp characterization.

Escherichia coli hemolysin is known to cause hemolysis of red blood cells by forming hydrophilic pores in their cell membrane. Hemolysin-induced pores have been directly visualized in model systems such as planar lipid membranes and unilamellar vesicles. However this hemolysin, like all the members of a related family of toxins called Repeat Toxins, is a potent leukotoxin. To investigate whether the formation of channels is involved also in its leukotoxic activity, we used patch-clamped human macrophages as targets. Indeed, when exposed to the hemolysin, these cells developed additional pores into their membrane. Such exogenous pores had properties very different from the endogenous channels already present in the cell membrane (primarily K+ channels), but very similar to the pores formed by the toxin in purely lipidic model membranes. Observed properties were: large single channel conductance, cation over anion selectivity but weak discrimination among different cations, quasilinear current-voltage characteristic and the existence of a flickering pre-open state of small conductance. The selectivity properties of the toxin channels appearing in phospholipid vesicles were also investigated, using a specially adapted polarization/depolarization assay, and were found to be completely consistent with that of the current fluctuations observed in excised macrophage patches.

Bacterial Proteins↗
PubMed2000

Expression of the hemolysin operon in Escherichia coli is modulated by a nucleoid-protein complex that includes the proteins Hha and H-NS.

The Escherichia coli protein Hha is a temperature- and osmolarity-dependent modulator of the expression of the hemolysin operon. The Hha protein was purified and its DNA-binding properties analyzed. Hha binds in a non-specific manner throughout the upstream regulatory region of the hemolysin operon in the recombinant hemolytic plasmid pANN202-312. A search for interacting proteins revealed that Hha interacts with H-NS. DNA-binding studies showed that, in vitro, Hha and H-NS together form a complex with DNA that differs from those formed with either protein alone. These data, together with the effects of hha and hns mutations on the expression of the hemolysin genes, suggest that in vivo H-NS and Hha form a nucleoid-protein complex that accounts for the thermo-osmotic regulation of the hemolysin operon in E. coli.

Bacterial Proteins↗
PubMed1996

Vibrio vulnificus hemolysin dilates rat thoracic aorta by activating guanylate cyclase.

Hemolysin produced by Vibrio vulnificus caused hypotension and tachycardia in rats and dilated rat thoracic aorta. Hemolysin-induced vasodilatation of the aorta was not affected by N omega-nitro-L-arginine methyl ester and aminoguanidine, NO synthase inhibitors, whereas the vasodilatation was inhibited by LY 83,583, a guanylate cyclase inhibitor. Hemolysin elevated cGMP levels, and the elevation was abolished by LY 83,583. These results suggest that V. vulnificus hemolysin activates guanylate cyclase independently of NO synthase, and the subsequent increase in cGMP levels results in vasodilatation.

Animals↗
PubMed1985

Isolation and characterization of a lethal hemolysin in the sea anemone Parasicyonis actinostoloides.

Four species of sea anemones in the coastal waters of Japan were surveyed for hemolysins. Powerful hemolysins were detected in Parasicyonis actinostoloides and Anthopleura japonica, whereas extracts of A. fuscoviridis showed much weaker hemolytic activity and those of of Haliplanella luciae no activity. Among the animal erythrocytes tested sheep were most sensitive to the hemolysins of the three positive species. The major hemolysin (parasitoxin) in whole bodies of P. actinostoloides was isolated by ion exchange chromatography, gel filtration and chromatofocusing. In addition to hemolytic activity (119900 HU/mg) it exhibited lethal activity in mice (LD50 65 micrograms/kg, i.v.) and fish Oryzias latipes (approximate minimum lethal concentration 1.5 micrograms/ml). Parasitoxin was slightly basic (pI 7.9) in nature and its amino acid composition was characterized by the absence of half-cystine. The molecular weight was 19,000 by SDS-polyacrylamide gel electrophoresis or 17,000 by sedimentation equilibrium, indicating that parasitoxin has no subunit structure.

Amino Acids↗