Purification and characterization of an Aeromonas hydrophila hemolysin.

A hemolysin produced by Aeromonas hydrophila CA-11, isolated from an environmental source, was purified by sulfopropyl-Sephadex C-25 chromatography at pH 5.0. This hemolysin caused fluid accumulation in infant mouse intestines and rabbit intestinal loops and killed Vero cells, as did the hemolysin produced by strain AH-1, isolated from a diarrheal case. In polyacrylamide gel electrophoreses at pHs 4.0 and 9.4 and in thin-layer isoelectric focusing, CA-11 hemolysin migrated as a single band to a position different from that of AH-1 hemolysin. Immunodiffusion tests indicated that CA-11 hemolysin was immunologically related to AH-1 hemolysin but possessed unique antigenic determinants. Neutralization tests with antihemolysin sera also demonstrated immunological cross-reactivity between AH-1 and CA-11 hemolysins. These results apparently indicate that the hemolysins produced by the two strains of A. hydrophila are immunologically and physicochemically different from each other.     

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.     

Domains of Escherichia coli hemolysin (HlyA) involved in binding of calcium and erythrocyte membranes.

The primary structure of Escherichia coli hemolysin (HlyA) contains a 9-amino-acid sequence which is tandemly repeated 13 times near the C terminus and which is essential for hemolytic activity. Hemolysin also requires an unknown modification by an accessory protein, HlyC, for hemolytic activity. The role of calcium in the interaction of HlyA with erythrocytes was investigated by using recombinant strains which produced inactive hemolysins unmodified by HlyC or deleted of the repeat sequences. 45Ca2+ autoradiography of the recombinant hemolysins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose showed that full-length, active hemolysin bound calcium. The domain involved in binding calcium was identified as the tandemly repeated sequences, since the deletion derivative missing 11 of the 13 repeats did not bind calcium. Inactive hemolysin, unmodified by HlyC, contained the repeated sequences and bound calcium as efficiently as the active, full-length toxin. The binding of the inactive toxins to erythrocytes was investigated by immunoblotting saline-washed, toxin-treated cells with monoclonal antibodies after sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of membrane proteins. The binding of full-length, active hemolysin to erythrocytes was calcium dependent. Inactive hemolysin deleted of the repeat units did not bind to cells. The inactive hemolysin, unmodified by HlyC, bound calcium but did not bind to erythrocytes. These results highlight the importance of calcium in the binding of hemolysin to erythrocytes and suggest that the binding of hemolysin to cells requires an interaction between the calcium-binding repeat domain and the modification produced by the HlyC protein.     

Biological and Immunological Properties of Encapsulated Strains of Staphylococcus aureus from Human Sources

Of 875 strains of Staphylococcus aureus isolated from human source clinical specimens, 37 (4.2%) were encapsulated strains. These were all negative for clumping factor and could not be typed with bacteriophages or by serology. Twenty-one of these did not produce any hemolysins, 15 produced alpha hemolysin, 1 produced beta hemolysin, and 1 produced both beta and delta hemolysins. After one or two subcultures, 27 of the encapsulated strains converted to the compact variant form, all became positive for clumping factor, 12 became phage-typable, and 24 became sero-typable. In addition, 7 strains converted from negative to alpha hemolysin production. Comparison of phage- and sero-types did not reveal any relationships. Immunologically, mice challenged with heat-killed encapsulated strains were protected against a challenge infection with the Smith diffuse strain. Protective antibodies in rabbit anti-Smith diffuse strain antisera were removed by adsorption using the encapsulated organisms isolated in this study. The adsorbed sera no longer protected against challenge infection in mice with the Smith diffuse strain. From these results, it appears that the encapsulated strains isolated were immunologically and biologically similar to the classical Smith diffuse strain.     

Identity of hemolysins produced by Vibrio cholerae non-O1 and V. cholerae O1, biotype El Tor.

Hemolysins purified from non-O1 Vibrio cholerae (non-O1 hemolysin) and a Vibrio cholerae O1, biotype El Tor (El Tor hemolysin) were investigated for their homology. The hemolysins were isolated from the culture supernatant fluids by ammonium sulfate precipitation and gel filtration on Sephadex G-100 columns. The purified hemolysins gave single bands with an identical mobility on conventional polyacrylamide gel disc electrophoresis. The molecular weights of the non-O1 and El Tor hemolysins were estimated to be about 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the amino acid compositions of the hemolysins were very similar. The specific activities of the hemolysins were identical, and both hemolysins were neutralized to the same extent with antisera against the homologous and heterologous hemolysins. Ouchterlony double immunodiffusion tests with both hemolysins and antihemolysin serum gave a common (fused) precipitin line. These data indicate that the non-O1 hemolysin is biologically, physicochemically, and immunologically indistinguishable from the El Tor hemolysin.     

Lysis of erythrocytes by a hemolysin produced by a group B Streptococcus sp.

An improved procedure for the isolation and purification of the hemolysin produced by a group B streptococcus was developed, and the inactivation of partially purified hemolysin by several enzymes was studied. Hemolysin obtained in buffer containing starch and Tween 80 was inactivated by subtilisin and alpha-amylase, suggesting that the hemolysin may consist of a protein hemolytic moiety complexed to starch which acts as a carrier or stabilizer. Properties of the hemolytic reaction were studied by using sheep erythrocytes as target cells. Experiments to examine the kinetics of hemolysis at different hemolysin concentrations resulted in a family of sigmoidal curves characterized by a short prelytic lag phase followed by a period of rapid release of hemoglobin. The binding of the group B hemolysin at 37 degrees C was rapid; within 3 min, most of the cells had bound sufficient hemolysin to produce lysis. In contrast, the hemolysin did not bind to erythrocytes at 0 degrees C. The length of the prelytic lag period and the rate of hemolysis were also temperature dependent. A decrease in total hemolysis was observed when the target cell/hemolysin ratio was increased, suggesting that a multihit response is required for lysis. Intracellular 86Rb and hemoglobin were released at the same rate from hemolysin-treated cells, indicating that a colloid-osmotic process is not involved in the lytic mechanism.     

Existence of Two Distinct Hemolysins in Vibrio parahaemolyticus

Two distinct hemolysins were demonstrated in Vibrio parahaemolyticus. A thermostable direct hemolysin purified from V. parahemolyticus WP-1, a Kanagawa phenomenon (KP)-positive strain, is antigenically different from a thermolabile hemolysin produced by V. parahaemolyticus T-3454, a KP-negative strain. The thermostable direct hemolysin was found in KP-positive strains but not in KP-negative strains. On the other hand, the thermolabile hemolysins were found in both KP-positive and -negative strains, although some KP-positive strains did not produce this hemolysin.     

Quantitative study of the binding and hemolytic efficiency of Escherichia coli hemolysin.

Mono- and polyclonal antibodies were used to construct a sandwich enzyme-linked immunosorbent assay that permitted quantitation of Escherichia coli hemolysin in soluble and membrane-bound forms. Toxin concentrations of 4 to 14 micrograms/ml were measured in culture supernatants of E. coli LE 2001 at times of peak hemolytic activity. Quantitative studies on the binding of E. coli hemolysin to rabbit erythrocytes were conducted at 0 and 37 degrees C. At 37 degrees C, 85 to 95% of bindable toxin was cell bound after 60 min, and no saturability of binding was observed in the studied range of concentrations, which resulted in deposition of approximately 100 to 50,000 toxin molecules per cell. Binding was slower and less effective at 0 degrees C; however, hemolysis did occur at low temperature. The number of cell-bound toxin molecules required to generate a hemolytic lesion within 60 min was estimated to be approximately 100 molecules per cell at 37 degrees C and 800 to 1,000 molecules per cell at 0 degrees C. Upon prolonged incubation (5 to 20 h, 37 degrees C), the number of molecules evoking a functional lesion decreased to approximately 5 to 20 per cell. These results are compatible with the concept that E. coli hemolysin first adsorbs to the cell surface, with membrane insertion and pore formation following in a second step that may be temporally dissociated from that of binding. The data support the pore concept of toxin action by showing that attachment of a low and finite number of toxin molecules to an erythrocyte will ultimately generate a cytolytic lesion.     

Purification and characterization of a hemolysin produced by Vibrio mimicus.

Vibrio mimicus is a causative agent of human gastroenteritis. This pathogen secretes a pore-forming toxin, V. mimicus hemolysin (VMH), which causes hemolysis by three sequential steps: binding to an erythrocyte membrane, formation of a transmembrane pore, and disruption of the cell membrane. VMH with a molecular mass of 63 kDa was purified by ammonium sulfate precipitation and column chromatography with phenyl Sepharose HP and Superose 6 HR. The hemolytic reaction induced by VMH continued up to disruption of all erythrocytes in the assay system. Moreover, VMH that bound preliminarily to erythrocyte ghosts showed a sufficient ability to attack intact erythrocytes. These results suggest reversible binding of the toxin molecule to the membrane. The final cell-disrupting stage was effectively inhibited by various divalent cations. Additionally, some cations, such as Zn2+ and Cu2+, blocked the pore-forming stage at high concentrations. Although VMH could disrupt all kinds of mammalian erythrocytes tested, those from horses were most sensitive to the hemolysin. Horse erythrocytes were found to have the most toxin-binding sites and to be hemolyzed by the least amount of membrane-bound toxin molecules, suggesting that toxin binding to and pore formation on erythrocytes are more effective in horses than in other mammals. Purified VMH induced fluid accumulation in a ligated rabbit ileal loop in a dose-dependent manner. In addition, the antibody against the hemolysin obviously reduced enteropathogenicity of living V. mimicus cells. These findings clearly demonstrate that VMH is probably involved in the virulence of this human pathogen.     

The HpmA hemolysin is more common than HlyA among Proteus isolates.

Two different hemolysins, HpmA and HlyA, have been reported in Proteus spp. To study the distribution of these hemolysins among Proteus strains, isolates from various infections and normal feces were screened for hemolysin production. All 63 Proteus mirabilis strains and 23 of the 24 Proteus vulgaris strains produced a calcium-independent hemolytic activity detectable in cell-free supernatants. The calcium-independent activity was due to HpmA; this activity correlated with the presence of hpmA sequences and the production of an extracellular 166-kilodalton (kDa) protein that reacted with anti-HpmA antiserum. HpmA- mutants, constructed by deletion of the central portion of the hpmA gene, did not produce the 166-kDa protein and were no longer hemolytic when compared with their respective parent strains. Among the 87 P. mirabilis and P. vulgaris isolates examined, calcium-dependent hemolytic activity was produced by only two P. vulgaris strains. These strains produced a 110-kDa protein which comigrated with the Escherichia coli hemolysin (HlyA) antibodies in immunoblots. These studies show that Proteus spp. produce two distinct extracellular hemolysins, with nearly all strains producing the calcium-independent hemolysin, HpmA, but only an occasional P. vulgaris isolate producing HlyA.     

Mechanism of membrane damage by El Tor hemolysin of Vibrio cholerae O1.

El Tor hemolysin (ETH; molecular mass, 65 kDa) derived from Vibrio cholerae O1 spontaneously assembled oligomeric aggregates on the membranes of rabbit erythrocyte ghosts and liposomes. Membrane-associated oligomers were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting into two to nine bands with apparent molecular masses of 170 to 350 kDa. ETH assembled oligomers on a liposomal membrane consisting of phosphatidylcholine and cholesterol, but not on a membrane of phosphatidylcholine alone. Cholesterol could be replaced with diosgenin or ergosterol but not with 5alpha-cholestane-3-one, suggesting that sterol is essential for the oligomerization. The treatment of carboxyfluorescein-encapsulated liposomes with ETH caused a rapid release of carboxyfluorescein into the medium. Because dextrin 20 (molecular mass, 900 Da) osmotically protected ETH-mediated hemolysis, this hemolysis is likely to be caused by pore formation on the membrane. The pore size(s) estimated from osmotic protection assays was in the range of 1.2 to 1.6 nm. The pore formed on a rabbit erythrocyte membrane was confirmed morphologically by electron microscopy. Thus, we provide evidence that ETH damages the target by the assembly of hemolysin oligomers and pore formation on the membrane.     

Purification and characterization of a hemolysin produced by Vibrio cholerae biotype El Tor: another toxic substance produced by cholera vibrios.

A thermolabile direct hemolysin from an El Tor cholera vibrio strain has been isolated and partially characterized as a simple protein of ca. 20,000 molecular weight. In addition to its hemolytic activity, the hemolysin is cytotoxic, cardiotoxic, and rapidly lethal. In these respects it resembles the thermostable direct hemolysin/cytotoxin/cardiotoxin/lethal toxin of Vibrio parahaemolyticus and certain other bacterial hemolysins, although there are other significant differences. Because identical diseases are produced by both hemolytic and nonhemolytic cholera vibrios, the El Tor hemolysin may be presumed to be pathogenetically irrelevant. These observations raise the question of "When is a toxic substance also a toxin?"     

Serotype-related differences in production and type of heat-labile hemolysin and heat-labile cytotoxin of Actinobacillus (Haemophilus) pleuropneumoniae.

Reference strains of serotypes 1 to 12 of Actinobacillus (Haemophilus) pleuropneumoniae were cultured in Eagle minimal essential medium with 10% Serum Plus. Culture supernatants were examined for cytotoxicity to alveolar macrophages and for the ability to hemolyze sheep erythrocytes. All strains except the reference strain of serotype 6 produced cytotoxin, whereas only serotypes 1, 5, 9, 10, and 11 produced hemolysin. Both cytotoxin and hemolysin appeared to be heat labile. Antisera were raised against cytotoxin- and hemolysin-containing culture supernatants of serotypes 1 to 11. Cross-neutralization studies revealed that the hemolysins were serologically homogeneous. In contrast, four serologically different cytotoxins were distinguished. One cytotoxin was produced by serotypes 1, 5, 9, and 11, and a second was produced by serotypes 2, 3, 4, and 8. A third cytotoxin was produced by serotypes 7 and 12; this cytotoxin was related to the cytotoxins of serotypes 1, 2, 4, 5, 9, and 11. A fourth cytotoxin, produced by serotype 10, was related to the cytotoxin of serotypes 1, 5, 9, and 11. Seventy field strains belonging to serotypes 2, 3, 7, 8, 9, and 11 were also tested for production of cytotoxin and hemolysin. All strains belonging to serotypes 9 and 11 produced hemolysin and cytotoxin, whereas all strains of serotypes 2, 3, 7, and 8 produced only cytotoxin. Hemolysins and cytotoxins of both the field strains and the corresponding serotype reference strains were comparably neutralized. These findings strongly suggest that the observed differences in production and type of hemolysin and cytotoxin were related to serotype and not to strain.     

Quantitative analysis of the binding and oligomerization of staphylococcal alpha-toxin in target erythrocyte membranes.

The binding of staphylococcal alpha-toxin to rabbit and human erythrocytes was quantitated over a wide range of toxin concentrations (3 x 10(-11) to 3 x 10(-6) M) with the use of an enzyme-linked immunosorbent assay that permitted simultaneous quantitation of monomeric and oligomeric toxin forms. Three basic observations were made. First, in no range of concentrations did the binding of alpha-toxin to rabbit erythrocytes display characteristics of a receptor-ligand interaction. Net binding to rabbit cells was nil at sublytic concentrations (10(-10) M or 3 ng/ml). The onset of binding occurred at around 10 ng/ml and remained fairly constant and ineffective (5 to 8% of toxin offered) over a wide concentration range (up to 10 micrograms/ml). Second, hemolysis of rabbit and human erythrocytes at 37 degrees C was always accompanied by the formation of toxin oligomers in the membrane. Third, overall toxin binding at 0 degree C followed a pattern similar to that at 37 degrees C. However, oligomer formation and cell lysis were retarded (but not totally inhibited) at 0 degree C. When rabbit erythrocytes were incubated with low levels of toxin at 0 degree C (0.5 microgram/ml) for 30 min, the toxin became bound exclusively in monomer form, and no lysis occurred. When cells thus treated were washed and suspended at 37 degrees C, lysis rapidly ensued, and native monomeric toxin was replaced by oligomeric toxin. The collective results directly support the oligomer pore concept of toxin action and also indicate that toxin oligomers form by lateral aggregation of bound monomers in the bilayer. They speak against the existence of specific binding sites for alpha-toxin on rabbit erythrocytes.     

Properties of a Hemolysin Produced by Group B Streptococci

A hemolysin that appears to be responsible for the zones of beta-hemolysis surrounding colonies of group B streptococci (Streptococcus agalactiae) on blood agar plates has been isolated and partially purified. No soluble hemolysin was detectable in the supernatants of streptococcal cultures grown in several types of media. However, hemolytic activity was detected when streptococci were incubated with erythrocytes, and soluble hemolysin was isolated when bacterial suspensions were incubated in the presence of a variety of agents, including calf serum, albumin, Tween 80, and starch. Glucose and other fermentable carbohydrates stimulated hemolysin production, and metabolic inhibitors greatly reduced the titer of hemolysin that could be recovered, suggesting that cellular metabolism is necessary for hemolysin production or release. The soluble hemolysin was concentrated by ammonium sulfate precipitation and partially purified by gel filtration. Agents known to inhibit other streptococcal hemolysins, including phospholipids, trypan blue, proteases, and cholesterol, were tested for their effect on the group B hemolysin. Only the phospholipids inhibited hemolysin activity. The group B streptococcal hemolysin appears to be similar to, but distinct from, streptolysin S produced by Streptococcus pyogenes.     

Purification and some properties of Aeromonas hydrophila hemolysin

A hemolysin produced by a strain of Aeromonas hydrophila isolated from a patient with diarrhea was purified by acid precipitation and quarternary aminoethyl-Sephadex chromatography. The molecular weight of the hemolysin was estimated at 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at 48,000 by Sephadex G-100 gel filtration. In polyacrylamide gel electrophoresis at pH 4.0 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the hemolysin migrated as a single band, whereas electrophoresis at pH 9.4 and thin-layer isoelectric focusing demonstrated multiple bands. The results may indicate charge isomers of the hemolysin. The purified hemolysin had a hemolytic activity of 134 hemolytic units per microgram of protein on rabbit erythrocytes. It caused fluid accumulation in infant mouse intestines and rabbit ligated ileal loops. Purified hemolysin also elicited cytotoxicity to Vero cells and lethal toxicity to mice. All these biological activities were lost on heating for 5 min at 56 degrees C. These findings support the notion that A. hydrophila hemolysin is a cytotoxic enterotoxin.     

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.     

Analysis of the lytic activity of the Serpulina hyodysenteriae hemolysin.

The hemolysins of Serpulina hyodysenteriae are active at 27 to 40 degrees C and pH 3 to 9 and are unaffected by enzymatic inhibitors. Pore formation was demonstrated by the inhibition of hemolysis with molecules of 2.0 to 2.3 nm in diameter and the release of 86rubidium from erythrocytes without hemoglobin release after exposure to native hemolysin.     

Hemolysin patterns of Actinobacillus pleuropneumoniae.

The secreted hemolytic activities produced by the reference strains and field isolates of the 12 serotypes and 2 subtypes of Actinobacillus pleuropneumoniae were analyzed. Serotype 1 produced a Ca2(+)-inducible hemolysin, which was previously characterized as a 105-kilodalton protein and was named hemolysin I (HlyI). Serotypes 2, 4, 6, 7, and 8 produced a different hemolytic activity that was not inducible by Ca2+ but required this ion for its activity. The hemolytic activity produced by these serotypes was much weaker than that found in serotype 1 and was not neutralized by rabbit antibodies against HlyI. It was, however, neutralized by serum from pigs that were experimentally infected with a serotype 2 strain and was called hemolysin II (HlyII). Serotypes 5a, 5b, 9, 10, and 11 produced both HlyI and HlyII. In these strains, HlyI was the major contributor to the hemolytic activity. The remaining serotypes, 3 and 12, produced a very weak hemolytic activity, which was not further analyzed. Immunoblot analysis of the culture supernatants from all 12 serotypes with rabbit polyclonal antibodies directed against HlyI revealed reactions with a protein in the 105-kilodalton size range for all serotypes, indicating that HlyI and HlyII might be serologically related. Strains producing active HlyI seem to belong to serotypes that are generally considered to be virulent types and that are frequently isolated from pigs in severe pleuropneumonia outbreaks.     

High-molecular-weight hemolysin of Clostridium tetani.

Clostridium tetani excretes hemolysins of two size classes, a high-molecular-weight hemolysin (HMH), which was eluted near void volume of a Sepharose 6B column, and conventional tetanolysin (molecular weight, approximately 50,000). The total hemolysin activity in the culture supernatant increased sharply with growth of bacteria and remained at a high level during autolysis. The content of HMH, however, decreased from 41% at 4 h of culture to 0.4% at the early stage of autolysis. The cell bodies also exhibited hemolytic activity, 70% of which could be solubilized and separated into HMH and the 50,000 Mr tetanolysin as extracellular hemolysins. The activity ratio of HMH to the total solubilized hemolysins was 0.45, on the average, at 6 h of culture but was 0.23 at the middle of logarithmic growth. Partially purified HMH from both sources appeared as broken pieces of cytoplasmic membranes under an electron microscope. The ratio of proteins to phospholipids in HMH was found to 3.26, a value similar to that in cell membrane. The total cell hemolytic activity decreased by 90 or 75% upon addition of chloramphenicol or anti-tetanolysin serum, respectively, into a 6-h-old culture of bacteria. It is suggested that HMH is a complex of tetanolysin with a membrane fragment and releases the conventional tetanolysin during bacterial culture.