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PubMed1987

Identification with monoclonal antibodies of hemolysin produced by clinical isolates of Escherichia coli.

Murine monoclonal antibodies were generated against the 107,000-dalton hemolysin encoded by the hemolytic determinant from Escherichia coli LE 2001, and colony blotting was used to assay for production of the hemolysin by 35 hemolytic strains of E. coli and other hemolytic members of the family Enterobacteriaceae of clinical origin. All hemolytic E. coli strains gave positive reactions with two monoclonal antibodies. In contrast, none of the hemolytic, non-E. coli isolates yielded positive colony blots. In addition, Western blotting showed that the hemolysins produced by all clinical E. coli isolates had a similar molecular weight of about 107,000. Discrete antigenic variation may occur in the molecule, since a third monoclonal antibody did not react with the hemolysin from a number of wild-type E. coli strains. Western blot analysis was used to assess the presence of immunoglobulin G (IgG), IgA, and IgM antibodies to E. coli hemolysin in human sera. All 20 of the tested sera from healthy adults contained antibodies to the toxin, with various constellations among the antibody classes. In contrast, sera from five of eight infants aged 8 to 36 months contained no antihemolysin antibodies. We conclude that the 107,000-dalton hemolysin of E. coli is a widespread immunogen that is produced by most or all hemolytic E. coli strains in the human host.

Animals↗

PubMed1987

Characterization of hemolysin in extracellular products of Pseudomonas cepacia.

Pseudomonas cepacia is recognized as an opportunistic pathogen in immunocompromised patients. We screened 120 strains of P. cepacia isolated from clinical specimens for production of extracellular products. About 70% of these strains produced lipase, protease, and lecithinase, but only 4% produced hemolysin. A hemolysin produced by P. cepacia JN106 was characterized. The hemolysin was most active against human erythrocytes. Horse, sheep, chicken, and rabbit erythrocytes were also susceptible. The hemolysin was heat labile and was inhibited by sterols but was not activated by 2-mercaptoethanol and dithiothreitol. Four hemolysin-negative mutants obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment produced the other extracellular products. A 58-kilobase-pair plasmid found in the parent strain was also found in the mutant strains, suggesting that the hemolysin gene resides on the chromosome.

Hemolysin Proteins↗

PubMed2003

Detection and characterization of the hemolysin genes in Aeromonas hydrophila and Aeromonas sobria by multiplex PCR.

A multiplex PCR assay was designed to amplify the Aeromonas hydrophila and A. veronii bv. sobria hemolysin and aerolysin genes. The assay was evaluated by using 121 clinical isolates and 7 reference strains of Aeromonas spp., and these were divided into five genotypes on the basis of the results of the multiplex PCR. The five genotypes were characterized as type 1 for those carrying the ahh1 gene only (36% of isolates), type 2 for those carrying the asa1 gene only (8.5% of isolates), type 3 for those carrying both the ahh1 and the asa1 genes (4% of isolates), type 4 for those carrying the ahh1 gene and the A. hydrophila aerA (aerolysin) gene (37.5% of isolates), and type 5 for those in which no hemolysin genes were detected (14% of isolates). The most common single hemolysin gene carried among all the Aeromonas isolates examined was ahh1, with 99 of 128 (77%) of isolates testing positive for this gene either alone or in combination with other hemolysin genes. Phenotypic expression of toxins was evaluated in a Vero cell culture cytotoxicity assay. These results indicated that there is a statistically significant correlation between the cytotoxin titers and the hemolysin genotype. Isolates belonging to genotype 4 (carrying both the ahh1 gene and the aerolysin and hemolysin aerA genes) expressed higher cytotoxin titers than isolates of the other genotypes (P < 0.001). These isolates were more cytotoxic in cell culture and may have greater clinical significance.

Aeromonas hydrophila↗

PubMed1977

Effect of hemolysin produced by Vibrio parahaemolyticus on membrane conductance and mechanical tension of rabbit myocardium.

The mechanisms of the action of hemolysin extracted from Vibrio parahaemolyticus in the S-A node and right atrium cells of rabbit were studied by means of the single sucrose gap and isometric tension recording methods. Hemolysin caused the membrane to depolarize reversibly without affecting the action potential generating mechanism. Lowering of [Na+]o inhibited membrane depolarization in the presence of hemolysin while the readmission of normal Tyrode solution induced depolarization. Tetrodotoxin (TTX) barely antagonized the depolarizing action of hemolysin but slowed the rate of development of depolarization. Therefore, this depolarization is considered to be primarily due to the increase in conductance to Na which TTX may not block. The dose-response relationship was obtained by measuring a change in membrane resistance. The concentration necessary to yield one-half of the maximum reduction of the membrane was determined to by 7.5 micrograms/ml. Accumulation of Na within the cell may be responsible for an increase of twitch tension observed during the action of a low concentration of hemolysin. On the other hand, a higher concentration of hemolysin seemed to promote exchange of intracellar Na with extracellular Ca, especially when the Na concentration of the perfusing solution was reduced, and led to stronger contracture.

Animals↗

PubMed1991

The hemolytic and cytolytic activity of group B streptococcal hemolysin and its possible role in early onset group B streptococcal disease.

The in vitro and cytolytic properties of the hemolysin of group B streptococcus (GBS) were investigated using sheep erythrocytes and McCoy cells adapted for growth in a serum-deficient medium. The relationship between the hemolysin, various carrier molecules and phospholipids was examined. Starch-based carriers interfered with the inhibitory activity of phospholipids and solvents for the phospholipids reduced the activity of the hemolysin. These technical problems were resolved by use of an albumin-based carrier, a strain producing large amounts of hemolysin and sonication of the phospholipid. The hemolysin was cytolytic for McCoy cells and this activity and its hemolytic action on sheep erythrocytes were inhibited by a number of phospholipid components of surfactant. It is possible that GBS hemolysin has a direct or indirect role in the pathogenesis of the pneumonitis of early onset GBS infection.

Animals↗

PubMed1999

Cytotoxic activity and cytokine gene induction of Asp-hemolysin to murine macrophages.

We examined the effects of Asp-hemolysin from Aspergillus fumigatus Fresenius-Muramatsu strain on the viability and cytokine gene expression of mouse peritoneal macrophages. The cytotoxic activity of Asp-hemolysin to macrophages cultured in FCS-RPMI medium was increased in a dose-dependent manner. Treatment of Asp-hemolysin with N-ethylmaleimide or sulfo-N-hydroxy-sulfosuccinimide-acetate caused a remarkable loss of the cytotoxic activity, however, the cytotoxic activity of Asp-hemolysin to macrophages cultured in serum-free medium was significantly increased as compared with that in FCS-RPMI medium. As other biological activities of Asp-hemolysin, tumor necrosis factor-alfa (TNF-alfa), interleukin-1 beta(IL-l beta) and interleukin-1 alfa (IL-l alfa) mRNA expression were observed in macrophages cultured with 1 micro g/ml of Asp-hemolysin.

Animals↗

PubMedDate not supplied

[The comparative characteristics of Vibrio cholerae hemolysins].

Biochemical and biological properties of in vivo and in vitro hemolysins of cholera germs isolated by different authors as well as hemolysins of Vibrio parahaemolyticus and Escherichia coli have been comparatively characterized. According to the above data hemolysin of cholera germ El Tor is a thermolabile protein with molecular mass of 60-80 thou.; it is cytotoxic, enteropathogenic and lyses all the species of erythrocytes. Hemolysins of V. eltor and germs are immunologically related. Hemolysin of the cholera germ is thermostable and has molecular mass of 20-40 thou., that makes it similar to hemolysin of V. parahaemolyticus.

Escherichia coli↗

PubMed1992

Rapid purification of a 110-kilodalton hemolysin of Actinobacillus pleuropneumoniae by monoclonal antibody-affinity chromatography.

An efficient, single-step method for purification of the 110-kilodalton (kDa) hemolysin of Actinobacillus pleuropneumoniae was developed. An immunoaffinity column was made by cross-linking murine monoclonal antibody 8C2 to the 110-kDa hemolysin of A pleuropneumoniae strain J45 serotype 5 to protein A-agarose beads. Purified hemolysin with high hemolytic activity was obtained after washing the column with phosphate-buffered saline solution, and eluting the hemolysin with 50 mM diethylamine, pH 11.0. The same column was also used to purify the hemolysin from A pleuropneumoniae strain 4074 serotype 1. The purification procedure could be completed within 5 hours, and almost 50% of the total hemolytic activity and hemolysin protein was recovered in pure form.

Actinobacillus pleuropneumoniae↗

PubMed1976

[alpha-Hemolysin of E. coli].

The activity of alpha-hemolysin increased at the log growth phase in the culture of E. coli P678 Hly+ hemolytic strain; this activity diminished with the change into the stationary phase, and then fell sharply. Replacement of the culture medium in the stationary growth phase by fresh one led to restoration of the hemolytic activity of the culture. The culture fluid separated from the cells at the stationary growth phase produced an inhibitory action on the alpha-hemolysin Ca ions activated and stabilized the alpha-hemolysin. Sodium citrate and sucrose served as hemolysis inhibitors. The action of alpha-hemolysin was maximal against human erythrocytes at pH 6.5. Hemolytic activity was characterized in time by a distinct lag-phase and the phase of the greatest rate of reaction. The duration of the lag-phase and also the rate of hemolysis depended on the concentration of alpha-hemolysin (with the increase of the hemolysin concentration lag-phase was shortened and the reaction was accelerated). There proved to be a linear relationship between the amount of erythrocytes taken into the reaction and the rate of hemoglobin release, and also there was noted a temperature activation of the hemolytic reaction.

Animals↗

PubMed1970

Cardiotoxic and Lethal Effects of Listeria monocytogenes Hemolysin.

Cardiotoxic and lethal effects of Listeria monocytogenes hemolysin were studied in CD-1 mice injected with varying doses of hemolysin. Intravenous injection of 100 complete hemolytic units (CHU) caused 100% lethality within 4 to 5 min. Doses ranging from 40 to 50 CHU caused death of approximately 50% of the animals. Adrenergic blocking agents and antihistamine failed to protect mice against lethality and thereby suggested that death was not due to release of vasoactive agents by hemolysin. Plasma levels of creatine phosphokinase increased after intravenous administration of hemolysin and suggested myopathy, possibly of the myocardium. Electrocardiograms from hemolysin-treated mice indicated serious alterations in heart rate and rhythm, suggesting damage to contractile and pacemaker cardiac tissue. In addition, there were indications of increased potassium levels influencing the heart. Presumably, death was due to functional damage to heart muscle and electrical arrest. The cardiotoxic and lethal effects could be prevented by prior incubation of hemolysin with cholesterol, heating, or failure to reactivate the preparation with cysteine.

Journal Article↗

PubMed1962

Production and characterization of Pseudomon as aeruginosa hemolysin.

Berk, Richard S. (Wayne State University, Detroit, Mich.). Production and characterization of Pseudomonas aeruginosa hemolysin. J. Bacteriol. 84:1041-1048. 1962.-Hemolysin from Pseudomonas aeruginosa was obtained by either extraction of blood agar media after bacterial growth or by saline washing of cellophane plate-grown cells, hemolytic activity being greatest in the latter preparations. Except for variations in heat stability, both types of preparations appeared to be similar if not identical. Complete hemolysis of red blood cells was observed over a wide pH range, although the optima appeared to be below 6.0. Acidification of agar extracts resulted in a coarse precipitate which appeared to be a hemolysin-agar complex. Partial purification of hemolysin present in cellophane washings was obtained by fractionation with ammonium sulfate or by gel filtration. Further characterization of hemolysin is described.

Agar↗

PubMed1963

Serology of Listeria monocytogenes. I. Characteristics of the soluble hemolysin.

Girard, Kenneth F. (St. Margaret's Hospital, Boston, Mass.), Anthony J. Sbarra, and Wadi A. Bardawil. Serology of Listeria monocytogenes. I. Characteristics of the soluble hemolysin. J. Bacteriol. 85:349-355. 1963.-Our results clearly demonstrate that Listeria monocytogenes (strain 9-125) produces a soluble hemolysin. Such hemolysin is completely precipitated out of sterile culture filtrates by 60% saturation with ammonium sulfate at 5 C, and virtually all of the hemolytic activity resides in the so-called "euglobulin" fraction. The protein nature of this hemolysin is further indicated by its nondialyzable property, heat lability, susceptibility to proteolysis by trypsin, and antigenicity in the full sense. Paper electrophoresis indicates that the isolated active fraction migrates as a gamma-type globulin. A procedure for determining Listeria antihemolysin levels in sera adapted from a method commonly employed for quantitating antistreptolysin O is described. The relative value of anti-Listeria hemolysin titration as a possible serological aid in diagnosis remains to be determined and is presently under investigation.

Hemolysin Proteins↗

PubMed1965

HEMOLYSIN OF MYCOPLASMA PNEUMONIAE.

Somerson, Norman L. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), Robert H. Purcell, David Taylor-Robinson, and Robert M. Chanock. Hemolysin of Mycoplasma pneumoniae. J. Bacteriol. 89:813-818. 1965.-Discrete colonies of Mycoplasma pneumoniae or M. laidlawii on agar produced complete (beta) lysis of guinea pig erythrocytes. Crowding of colonies on agar plates, omission of yeast extract from the medium, and incubation under reduced oxygen tension inhibited hemolysin production. Colonies which were not viable after ultraviolet radiation or heating at 56 C did not produce hemolysin. In addition, hemolysis was suppressed in old cultures in which viability was diminished, and was eliminated when colonies were removed from the agar surface by micromanipulation. The hemolysin passed through a viscose dialysis membrane. These findings suggest that the hemolysin is labile, continuously released by the colonies, of low molecular weight, and probably nonprotein in nature.

Agar↗

PubMed1993

Cloning of two distinct hemolysin genes from Porphyromonas (Bacteroides) gingivalis in Escherichia coli.

Hemolysin is considered a potent virulence factor in a large number of Gram-positive and Gram-negative bacterial pathogens. The hemolysin produced by the oral pathogen Porphyromonas gingivalis functions to provide the cell with its required heme-containing molecules for growth in the periodontal pocket. Two distinct P. gingivalis genes, each of which confers a hemolytic phenotype in Escherichia coli, were isolated by screening genomic DNA libraries of P. gingivalis on sheep blood agar plates. The results obtained from physical maps and Southern blots indicated a considerable degree of divergence in the nucleotide sequences of these two genes. Maxicell analyses of the recombinant plasmids in E. coli suggested that plasmid pPGH5 encoded a polypeptide of molecular weight 48 kDa, while an 18-kDa polypeptide was obtained with pPGH1 and pPGH7. When E. coli harboring these hemolysin genes were subjected to iron starvation, the levels of hemolysin activity increased. Biochemical characterization of hemolytic activities indicated that the activity of both hemolysins was inhibited by Mg2+ and Ca2+; but not by EDTA. Elevated levels of hemolytic activity were obtained from the E. coli recombinant strains in the presence of glutathione, DTT and 2-mercaptoethanol. Cholesterol inhibited the activity.

Bacterial Proteins↗

PubMed1994

Cloning and expression of hemolysin genes from Treponema denticola strains ATCC 35404 (TD-4) and human clinical isolate GM-1 in Escherichia coli.

The oral spirochete, Treponema denticola is a putative etiologic agent in adult periodontitis, and acute necrotizing ulcerative gingivitis. In vitro, the oral treponeme produces several factors including proteases, hemolysins, hemin-binding proteins, which could potentially be involved in the virulence of this spirochete. Our laboratory has been investigating the pathobiology of T. denticola, and has demonstrated the production of several hemolysins by T. denticola. In this report two hemolysin genes from T. denticola strains ATCC 35404 (TD-4) and GM-1 were isolated by screening genomic DNA libraries of T. denticola on sheep blood agar plates. Physical maps of the insert fragments were not identical. Southern blot analyses suggested some degree of homology in the nucleotide sequence. Maxicell analyses of [35S]-methionine-labeled polypeptides from the recombinant plasmids have suggested the synthesis of an approximately 62.5 kDa polypeptide. Biochemical characterization of the T. denticola hemolysin genes indicated the activity to be inhibited by Mg2+, Ca2+ and Zn2+ but not by EDTA. Dithiothreitol and glutathione moderately enhanced the hemolytic activity of the recombinant plasmids. Iron partially inhibited the hemolytic activities. Addition of 2-2' bipyridyl moderately enhanced the activities, possibly by iron limitation. These results suggest the isolation of an identical hemolysin gene from T. denticola strains TD-4 and GM-1.

Animals↗

PubMed1994

Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HlyB/HlyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA.

Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hylA from the 5' end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E. coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.

Antibodies, Bacterial↗

PubMed1985

Purification of alpha-hemolysin from an overproducing E. coli strain.

The genetic determinant of the alpha-hemolysin encoded by plasmid pHly152 has been cloned in both orientations in plasmid pBR322 giving rise to plasmids pSU157 and pSU158. E. coli strains carrying either of these recombinant Hly plasmids produced about 20 times more hemolysin activity than the parental plasmid pHly152, when grown in minimal medium supplemented with hemoglobin. Thus high hemolytic activity is not lethal to the cells, contrary to previous assumptions. alpha-Hemolysin was purified from culture supernatants of strain SU100 (pSU157) by ammonium sulfate precipitation and gel filtration in Sephacryl S-200 in the presence of 6 M urea. When purified alpha-hemolysin preparations were subjected to electrophoretic analysis in denaturing conditions, a single 107 kdal polypeptide was observed. This probably corresponds to the alpha-hemolysin protein, since an isogenic E. coli strain carrying plasmid pSU161, an Hly- mutant derivative of pSU157, did not synthesize the 107 kdal polypeptide.

Bacterial Proteins↗

PubMed1986

Isolation and some properties of beta-hemolysin produced by Nocardia asteroides.

An intracellular beta-hemolysin capable of lysing human, sheep and cow erythrocytes but not cells from some other animals was isolated from the cell walls of the three developmental cell-forms of Nocardia asteroides and characterised. The spherical cell-forms contained the highest amounts of the hemolysin (100 h.u./mg protein) and the least LD50 for mice suggesting that this may be the cell-form most pathogenic to susceptible animals. The hemolysin has the properties of a protein, was pH stable and sensitive to both catabolite repression and temperature. The activity of the hemolysin was enhanced by Ca++ and Na+ ions. The hemolysin was immunogenic in rabbits.

Animals↗