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At least 19 recordsLinked to original sources
PubMed1992

Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction.

Polymerase chain reaction (PCR) protocols were established for specific detection of the tdh and trh genes, the virulence marker genes of Vibrio parahaemolyticus encoding two related hemolysins. The tdh and trh genes are known to have sequence divergence of up to 3.3% and 16%, respectively. Attempts were made to find suitable primer pairs and annealing temperatures to detect each gene without fail. DNAs extracted from 36 representative strains of V. parahaemolyticus were used in the initial screening with various combinations of primer pairs and annealing temperatures. The combinations of primer pairs and annealing temperatures selected were then tested with DNAs extracted from 227 more strains of V. parahaemolyticus and from 133 bacterial strains belonging to 40 species other than V. parahaemolyticus. PCR protocols (primer pairs and annealing temperatures) were established that gave identical results to those obtained with the tdh- and trh-specific polynucleotide probes. These protocols established for the tdh and trh genes could detect 400 fg (100 cells) of cellular DNA carrying the respective gene. Spike experiments demonstrated that the sensitivities of the established PCRs were reduced by a factor of 10(4)-10(5) by an inhibitor(s) present in a normal faecal sample, indicating the need for either DNA extraction or enrichment of the faecal sample in alkaline peptone water for 4 h before the PCR of faecal samples.

Base Sequence↗
PubMed2001

[Maturation pathway of hemolysin of Aeromonas sobria and the mechanism of action of the hemolysin].

Aeromonas sobria has been recognized as pathogens associated with acute gastroenteritis in both adults and children. The major virulence factor has been proposed to be hemolysin which possesses both hemolytic and enterotoxic activities. Mature (bio-active) hemolysin secreted out of cells binds to the target cells of the host and injure the cells. However, hemolysin remained in bacteria can not express such toxicity. It means that the maturation and secretion pathway of hemolysin is closely related to the pathogenicity of bacteria. Therefore, I examined the pathway and clarified the following events. Hemolysin synthesized in cytoplasm translocates across the inner membrane and appears in a periplasmic space. Hemolysins appeared in the space associates to form dimer in the space. The C-terminal region of hemolysin functions as a trigger in the association. Dimerized hemolysin crosses the outer membrane and emerges in milieu, but monomer can not cross it. Therefore, the C-terminal region of hemolysin attributes not only to the formation of the dimer but also to its secretion into milieu. Hemolysin emerged in milieu is inactive. Inactive hemolysin is converted to bio-Active hemolysin by deleting its carboxyl-terminal 42-amino-acid peptide. Active hemolysin generated binds to the receptor of the target cell and stimulates the production of cyclic AMP by the cell. I assume that this stimulation closely relates to the induction of diarrhea by hemolysin.

Aeromonas↗
PubMed1963

OXYGEN-STABLE HEMOLYSINS OF GROUP A STREPTOCOCCI. I. THE ROLE OF VARIOUS AGENTS IN THE PRODUCTION OF THE HEMOLYSINS.

The production of oxygen-stable hemolysin in growing and resting Group A streptococci has been induced by RNA, by detergents, and by mammalian blood serum proteins, in the presence of glucose, Mg(++), and cysteine. Of the serum proteins, albumin and alpha lipoprotein could act as inducers. In the case of both these serum proteins treatment with trypsin did not affect the capacity to induce hemolysin production, but removal of the bound lipids by alcohol-ether or chloroform-methanol destroyed this property. In comparisons of the conditions of production and of activity between the hemolysin produced by RNA on one hand and albumin and detergents on the other, some data indicated similarities among the hemolysins, and others, differences. The similarities included similar degrees of temperature dependence for production and equal degrees of inhibition by serum beta lipoprotein. Differences found among these hemolysins included differences between, the rate of production of the RNA hemolysin from that of albumin or detergent hemolysin by both resting and growing streptococci, and the failure of utilization of glucosamine as an energy source for the production of albumin hemolysin, in contrast with that of RNA hemolysin. The fact that the data have in some cases indicated similarities and in other cases differences among the hemolysins raises the question of whether these are different molecular species, or a single hemolysin synthesized by the streptococci via different pathways of metabolism, or complexes of a single hemolytic moiety with various molecular carriers.

Animals↗
PubMed1985

Cloning and expression in Escherichia coli of Vibrio parahaemolyticus thermostable direct hemolysin and thermolabile hemolysin genes.

Two hemolysin genes of Vibrio parahaemolyticus WP1, a thermostable direct (TSD) hemolysin gene and a thermolabile hemolysin gene, were cloned into the pBR322 vector in Escherichia coli K-12 C600. A large amount of the TSD hemolysin produced in E. coli K-12 accumulated in the periplasmic space. The TSD hemolysin gene was localized on a 0.9-kilobase HindIII-BamHI fragment by identifying qualitatively the production of the TSD hemolysin by a reverse passive hemagglutination assay in the osmotic shock fluid. The thermolabile hemolysin gene was isolated on a 1.3-kilobase HindIII-PstI fragment by selection with the hemolysin on blood agar. Southern blot hybridization and colony hybridization experiments indicated that the TSD hemolysin gene was present in the chromosomal DNA of 15 Kanagawa phenomenon-positive strains but not in 14 negative strains, whereas the thermolabile hemolysin gene was detected in all strains. No homologous DNA sequences to TSD and thermolabile hemolysin genes were detected in the chromosomes of Vibrio cholerae, Vibrio vulnificus, non-O1 V. cholerae, and Vibrio anguillarum.

Cloning, Molecular↗
PubMed1986

Comparison of the nucleotide sequences of the genes for the thermostable direct hemolysin and the thermolabile hemolysin from Vibrio parahaemolyticus.

The nucleotide sequences of genes encoding the thermostable direct (TSD) hemolysin and the thermolabile (TL) hemolysin of Vibrio parahaemolyticus were determined. From the nucleotide sequence of the TSD hemolysin gene, it was revealed that the preprotein and the mature protein consisted of 189 amino acids and 165 amino acids, and that the molecular weights were 21.1 kDa or 18.5 kDa, respectively. Our data regarding TSD hemolysin were in complete agreement with previously published data. From the nucleotide sequence of the TL hemolysin gene, it was revealed that the preprotein and the mature protein consisted of 418 amino acids and 398 amino acids, and that the molecular weights were 47.5 kDa and 45.3 kDa, respectively. The GC content of the TSD hemolysin gene was 35.6%, while that of the TL hemolysin gene was 47.6% which is almost the same as that of V. parahaemolyticus genome. Maxicell analysis revealed that the molecular weights of the proteins encoded by the TSD hemolysin gene were 22.0 and 19.5 kDa, and that of the protein encoded by the TL hemolysin gene was 45.5 kDa, and that the promoters of these two hemolysin genes of V. parahaemolyticus were functional in Escherichia coli.

Amino Acid Sequence↗
PubMed1989

Cloning and nucleotide sequence of the gene (trh) encoding the hemolysin related to the thermostable direct hemolysin of Vibrio parahaemolyticus.

Vibrio parahaemolyticus isolates derived from an outbreak of gastroenteritis in the Republic of Maldives did not have the genetic potential to produce the thermostable direct hemolysin, but one such isolate produced a hemolysin immunologically related to the thermostable direct hemolysin (T. Honda, Y. Ni, and T. Miwatani, Infect. Immun. 56:61-965, 1988). The Maldives isolates hybridized with the DNA probe for the gene encoding the thermostable direct hemolysin (the tdh gene) under reduced stringencies. A DNA fragment containing the probe-reactive nucleotide sequence was isolated from a selected strain and cloned into pBR322 in Escherichia coli. A clone producing the thermostable direct hemolysin-related hemolysin was obtained by screening with hemolysis assays and by an immunological assay. Nucleotide sequence analysis of the cloned DNA fragment revealed that the gene encoding the thermostable direct hemolysin-related hemolysin (the trh gene), like the tdh gene, encoded the hemolysin subunit composed of 189 amino acid residues. The trh gene had significant nucleotide sequence homology with the tdh gene (68.4% with the tdh1 gene copy and 68.6% with the tdh2 gene copy). The amino acid sequences of the hemolysin subunits deduced from the nucleotide sequences of the trh gene and tdh gene were homologous (61.9% homology with the tdh1-encoded subunit and 63.0% homology with the tdh2-encoded subunit) and contained the two cysteine residues to form an intrachain bond at the same positions, and their possible conformations appeared to be similar as determined by hydrophobicity-hydrophilicity analysis and a secondary structure prediction. The trh and tdh genes may have had a common ancestor and may have evolved by single-base changes so that they maintained the fundamental architecture of the molecules.

Amino Acid Sequence↗
PubMed1964

PURIFICATION AND PROPERTIES OF STAPHYLOCOCCAL BETA-HEMOLYSIN. I. PRODUCTION OF BETA-HEMOLYSIN.

Haque, Riaz-ul (Ohio State University, Columbus), and Jack N. Baldwin. Purification and properties of staphylococcal beta-hemolysin. I. Production of beta-hemolysin. J. Bacteriol. 88:1304-1309. 1964.-Highest activity of beta-hemolysin was observed when buffered saline (pH 7.0) containing 0.001 m magnesium sulfate was used as a diluent, and the tubes were incubated at 37 C for 80 min and then refrigerated for 30 min. Either Heart Infusion semisolid agar or a dialysate of Heart Infusion containing 0.3% agar was suitable for the production of large quantities of beta-hemolysin. The concentration of beta-hemolysin in semisolid and broth cultures was greatest after incubation for 24 hr. Continued incubation resulted in a loss of active hemolysin in broth cultures but not in semisolid agar cultures. Incubation in atmospheres containing 20% carbon dioxide greatly enhanced the production of beta-hemolysin. The presence of fermentable sugars inhibited the production of beta-hemolysin. Highest yields of beta-hemolysin were obtained when the initial pH of the medium was 5.5 to 5.8.

Agar↗
PubMed1997

alpha-Hemolysin, gamma-hemolysin, and leukocidin from Staphylococcus aureus: distant in sequence but similar in structure.

alpha-Hemolysin from Staphylococcus aureus assembles from a water-soluble, monomeric species to a membrane-bound heptamer on the surface of target cells, creating water-filled channels that lead to cell death and lysis. Staphylococcus aureus also produces the gamma-hemolysin and leukocidin toxins, which function as two component toxins in the disruption and lysis of erythrocytes and leukocytes. Analysis of the aligned sequences of alpha-hemolysin, gamma-hemolysin, and leukocidin in the context of the alpha-hemolysin heptamer structure supports the conclusion that even though the level of sequence identity between alpha-hemolysin and the gamma-hemolysin and leukocidin toxins is in the so-called twilight zone, the three-dimensional structures of the protomers are probably conserved. By analogy with alpha-hemolysin, gamma-hemolysin and leukocidin may also form oligomeric, transmembrane channels in which an antiparallel beta-barrel constitutes the primary membrane-embedded domain.

Amino Acid Sequence↗
PubMed1989

Characterization of Aeromonas sobria hemolysin by use of monoclonal antibodies against Aeromonas hydrophila hemolysins.

Aeromonas sobria produces hemolysin in a form activable with trypsin under defined cultural conditions. In immunoblotting analyses with the culture supernatant of A. sobria, the monoclonal antibody reacting specifically to Aeromonas hydrophila CA-11 hemolysin bound to the 53,000- and 49,000-dalton bands before and after trypsinization, respectively. The monoclonal antibody reacting to A. hydrophila AH-1 hemolysin did not bind either band. A. sobria hemolysin is, therefore, related antigenically to CA-11 hemolysin, while the molecular weights before and after activation differ from those of A. hydrophila hemolysins, being 54,000 and 51,000, respectively. The hemolytic and enterotoxigenic activities of A. sobria hemolysin were both neutralized by the monoclonal antibody against CA-11 hemolysin. It seems, therefore, that the same site on A. sobria hemolysin is responsible for both biological activities.

Aeromonas↗
PubMed1984

Non-O1 Vibrio cholerae hemolysin: purification, partial characterization, and immunological relatedness to El Tor hemolysin.

Hemolysin of a non-O1 Vibrio cholerae strain was purified and characterized. The purified hemolysin gave a single protein band on conventional and sodium dodecyl sulfate-gel electrophoresis. Its molecular weight was estimated as 60,000 by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. It had a pI of 5.7. The purified hemolysin caused increased vascular permeability of rabbit skin and rapid death of mice on intravenous injection and also lysed erythrocytes of various animal species. An Ouchterlony double gel diffusion test using antiserum against the purified hemolysin indicated that the hemolysin from non-O1 V. cholerae was immunologically related, but not identical, to the hemolysin from El Tor V. cholerae. Antiserum against the purified hemolysin neutralized the hemolytic activity of the hemolysins from not only non-O1 but also El Tor V. cholerae.

Animals↗
PubMed1986

Hemolysin production and cloning of two hemolysin determinants from classical Vibrio cholerae.

The hemolytic activity of 20 classical and 3 El Tor strains of V. cholerae O1 was examined phenotypically and genetically. The El Tor strains lysed bovine, chicken, human, rabbit, and sheep erythrocytes (RBCs), while the classical strains lysed only chicken and rabbit RBCs. The assay was done with RBCs in Tris-NaCl buffer, since phosphate-buffered saline was found to inhibit hemolytic activity. Hemolytic activity in culture supernatants from El Tor strains was more sensitive to heat inactivation than that in supernatants from the classical strain 395. A gene library of strain 395 was examined for hemolytic activity, and two distinct hemolytic clones were identified. One clone appeared identical to the previously cloned hemolysin structural gene from El Tor V. cholerae, while the other did not hybridize to the El Tor hemolysin probe, had a unique restriction enzyme digestion pattern, and encoded a hemolysin whose activity differed from that of the El Tor hemolysin clones. We suggest that the hemolysin specified by the determinant originally cloned from an El Tor vibrio be designated hemolysin I and the second hemolysin, cloned from the classical vibrio, be designated hemolysin II.

Animals↗
PubMed1969

Purification and properties of staphylococcal beta hemolysin. II. Purification of beta hemolysin.

Staphylococcal beta hemolysin from the 681 strain of Staphylococcus aureus grown in a Heart Infusion dialysate semisolid medium under 10% carbon dioxide was obtained in an immunoelectrophoretically pure form by a combination of procedures of precipitation with 2 volumes of acetone followed by chromatography on diethylaminoethyl cellulose at pH 6.0. The acetone precipitation procedure did not show any deleterious effect on the hemolytic activity of the beta hemolysin unless the precipitate was left in contact with the acetone for at least 4 hr. The crude preparations contained two types of beta hemolysin. One of these represented the major portion of the total activity of beta hemolysin and behaved as a cation. The other represented a minor (1/5,000) portion of the total beta hemolysin activity and behaved as an anion. These active principles were designated as cationic and anionic beta hemolysins, respectively. An unexpected increase in the total beta hemolysin activity of the crude preparations was noted when these were concentrated by dialysis against polyethylene glycol (20 m). This effect was probably due to polyethylene glycol. A further unexpected increase in the titer of the acetone-precipitated preparations occurred when these were lyophilized. The reason for this incremental increase is not known. It may be due to fragmentation of the beta hemolysin.

Acetone↗
PubMed1963

MECHANISM OF ACTION OF STAPHYLOCOCCAL ALPHA-HEMOLYSIN. I. SOME FACTORS INFLUENCING THE MEASUREMENT OF ALPHA-HEMOLYSIN.

Marucci, Americo A. (Upstate Medical Center, Syracuse, N.Y.). Mechanism of action of staphylococcal alpha-hemolysin. I. Some factors influencing the measurement of alpha-hemolysin. J. Bacteriol. 86:1182-1188. 1963.-A kinetic method for the accurate and reproducible measurement of the action of staphylococcal alpha-hemolysin on rabbit erythrocytes is described. The activity of the alpha-hemolysin depends upon the temperature used for measurement. At 37 C the hemolysin lyses cells faster, but it in turn is rather quickly inactivated. At 0 C there is no inactivation, but the rate of lysis is greatly decreased. There is no change in the activity with change in total reaction volume, providing that the concentrations of cells and hemolysin are kept constant. The fraction of rabbit red cells lysed by a given amount of hemolysin in a given time is constant and independent of the total number of cells in the reaction mixture.

Animals↗
PubMed1987

The secreted hemolysins of Proteus mirabilis, Proteus vulgaris, and Morganella morganii are genetically related to each other and to the alpha-hemolysin of Escherichia coli.

Secreted hemolysins were extremely common among clinical isolates of Proteus mirabilis, Proteus vulgaris, and Morganella morganii, and hemolytic activity was either cell associated or cell free. Southern hybridization of total DNA from hemolytic isolates to cloned regions of the Escherichia coli alpha-hemolysin (hly) determinant showed clear but incomplete homology between genes encoding production of hemolysins in the four species. One of the two E. coli secretion genes, hlyD, hybridized only with DNA from P. vulgaris and M. morganii, which produced cell-free hemolysis, but not with that from P. mirabilis, which showed only cell-associated activity. Molecular cloning of the genetic determinants of cell-free hemolytic activity from P. vulgaris and M. morganii chromosomal DNA allowed their functional analysis via inactivation with the transposons Tn1000 and Tn5. Both hemolysin determinants were about 7.5 kilobase pairs and comprised contiguous regions directing regulation, synthesis, and specific secretion out of the cell. Transposon mutations which eliminated secretion of the Proteus and Morganella hemolysins could be complemented specifically by the E. coli hemolysin secretion genes hlyB or hlyD. Alignment of the physically and functionally defined hly determinants from P. vulgaris and M. morganii with that of the E. coli alpha-hemolysin confirmed a close genetic relationship but also indicated extensive evolutionary divergence.

Bacterial Proteins↗
PubMed1965

OXYGEN-STABLE HEMOLYSINS OF GROUP A STREPTOCCI. IV. STUDIES ON THE MECHANISM OF LYSIS BY CELL-BOUND HEMOLYSIN OF RED BLOOD CELLS AND EHRLICH ASCITES TUMOR CELLS.

In studies of the mechanism of lysis of red blood cells by washed streptococci with hemolytic activity (cell-bound hemolysin, CBH) no components released spontaneously by RBC or streptococci, or by interaction between these cells, could be found to induce the formation of soluble hemolysin by the streptococci. It was also found that separation of RBC from streptococci even by Millipore filter or a very thin layer of agar could prevent their hemolysis. By means of cellulose columns it was possible to separate RBC from streptococci after a short incubation. RBC thus separated from streptococci with which they had been incubated underwent hemolysis on subsequent incubation at 37 degrees C. By varying the period of incubation prior to separation it was possible to demonstrate the transfer of increasing amounts of hemolysin from streptococci to RBC with increasing periods of incubation. A considerable part of this appeared to be at a constant rate. A theory is presented on the relationship between the streptococcal cell-bound hemolysin and the group of oxygen-stable streptococcal hemolysins, in terms of a transferable hemolytic moiety and binding sites for this moiety on the streptococcal cell, on various molecular species which can act as inducers of the oxygen-stable hemolysins, and on the RBC, with the affinity of the respective binding sites for the hemolytic moiety increasing in that order.

Animals↗
PubMed1985

Studies on toxin of Aspergillus fumigatus. XXII. Fashion of binding of Asp-hemolysin to human erythrocytes and Asp-hemolysin-binding proteins of erythrocyte membranes.

The fashion of binding of Asp-hemolysin to human erythrocytes and the isolation of Asp-hemolysin-binding proteins from erythrocyte membranes were investigated by the immunocytochemical technique and affinity chromatography. Asp-hemolysin bound best at a pH range from 5 to 7. The erythrocytes treated with Asp-hemolysin showed diffuse, ring-like or cap-like staining by the peroxidase-labeled antibody method under the light microscope. The distribution of Asp-hemolysin on the erythrocyte surface was clearly observed as patches or caps in the scanning electron microscope. The erythrocyte ghosts were extracted with 1% sodium deoxycholate-0.1 M Tris-HC1 buffer (pH 7.5) containing 0.2 M NaCl and 1 mM EDTA, and the extract was chromatographed on an affinity column consisting of Asp-hemolysin attached to activated thiol-Sepharose 4B. Four proteins present in the membrane extract were retained by activated thiol-Sepharose 4B and eluted with 50 mM cysteine as toxin-membrane components. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the polypeptides correspond to band 2.1, one protein of the 2 region, band 3 and band 7 in the Steck nomenclature system.

Aspergillus fumigatus↗
PubMed1991

Alpha-hemolysin contributes to the pathogenicity of piliated digalactoside-binding Escherichia coli in the kidney: efficacy of an alpha-hemolysin vaccine in preventing renal injury in the BALB/c mouse model of pyelonephritis.

Digalactoside-binding (Gal-Gal) pili and alpha-hemolysin of Escherichia coli have been implicated as important virulence determinants in the pathogenesis of human ascending, nonobstructive pyelonephritis. The pathogenic significance of these determinants was evaluated in vitro and in the BALB/c mouse pyelonephritis model by employing wild-type, avirulent laboratory, and genetically defined cosmids, transformants, and recombinant strains. In vitro data suggest that the cytolytic activity of hemolysin is significantly (P less than 0.05) enhanced among digalactoside-binding strains which agglutinate erythrocytes. The basis of increased hemolysis is related presumably to more efficient delivery of the toxin to target lipid substrate in the host plasma membrane. Intravesicular administration of bacteria that express both digalactoside binding and hemolysin generally resulted in greater mortality and renal parenchymal injury in mice than strains that expressed none or only one of these determinants. Analyses convincingly demonstrate that digalactoside-binding pili are correlated with upper urinary tract colonization and that hemolysin is correlated with septicemia and renal parenchymal damage. These determinants collectively constitute the minimal virulence factors to produce disease in this model. Their efficacy as vaccines for the prevention of pyelonephritis was also assessed. A purified Gal-Gal pilus vaccine prevented (P less than 0.05) subsequent colonization by a challenge wild-type strain that exhibited homologous pili. The hemolysin vaccine did not abrogate subsequent bacterial renal colonization on challenge, but it did protect (P less than 0.05) mice which survived challenge from subsequent renal injury compared with those in the saline control group. The combination of these determinants was also protective. The combination of Gal-Gal pili and hemolysin in a vaccine preparation represents a potentially worthwhile strategy for human immunoprophylaxis against pyelonephritis by interdicting several steps in the pathogenesis of a bacterial mucosal infection.

Animals↗