Some properties of the alpha-hemolysin produced by a hemolytic strain of E. coli]

It was found that alpha-hemolysin of E. coli P 678 HIy+ was maximally active against human erythrocytes at pH 6.5. The hemolytic activity is characterized in time by a distinct lag-phase and a phase of the greatest velocity of the reaction immediately following it. The duration of the lag-phase and also the rate of hemolysis depends on alpha-hemolysin concentration, whose increase is accompanied by a decrease of the lag-phase and acceleration of hemolysis. There is a definite limit below which the duration of the lag-phase remains unchanged with further increase of hemolysin concentration. There was noted a linear relationship between the amount of erythrocytes taken for the test and the rate of hemoglobin release and also a temperature activation of the hemolytic reaction.     

Evidence for the production of a novel proteinaceous hemolytic exotoxin by dinoflagellate Alexandrium taylori

We found that a whole cell suspension of Alexandrium taylori, which is toxic to Artemia, causes species-specific hemolysis against mammalian erythrocytes. Among the erythrocytes tested, rabbit and guinea-pig erythrocytes were highly sensitive, but human, sheep, and cattle erythrocytes were insensitive. The cell-free culture supernatant also showed potent hemolytic activity toward rabbit erythrocytes as seen in whole cell suspension. The hemolytic activity in the culture medium gradually increased with increase in cell number during exponential growth phase, and relatively high activity was maintained even after reaching the death phase. These results suggest that the hemolytic substance is actively released into the medium from A. taylori cells rather than simple leakage from ruptured or dead cells, and a part of them are steadily accumulated in the medium during the algal growth. Chemical characterization with ultrafiltration and trypsin-treatment suggested that the hemolytic substance released into the medium is protein-like compound with molecular weight more than 10,000 Da. The ammonium sulfate precipitated fraction obtained from the cell-free supernatant of A. taylori showed cytotoxic effect on HeLa cells as well as the hemolytic activity in a similar concentration range on a protein content basis. Our results suggest that A. taylori produces a novel proteinaceous hemolytic exotoxin.     

Lysis of erythrocytes byTrichomonas vaginalis

The in vitro hemolytic activity of 4 isolates ofTrichomonas vaginalis was investigated. Repetitive hemolysis assays of any one isolate showed cyclical fluctuations in hemolytic activity, varying over 24 hr of continuous culture. Maximal hemolytic activity was detected using trichomonads in the lag phase of the growth cycle. Investigations showed that hemolysis was a contact-dependent phenomenon and microscopic investigation of samples showed a significant correlation between hemolysis and attachment of erythrocytes to the trichomonad surface. Quantitative data from cytoadherence assays using [⁵¹Cr]-labeled erythrocytes were consistent with these observations. It is suggested that hemolytic activity is dependent upon adherence of red blood cells to the surface ofT. vaginalis.     

Studies on the factors affecting the growth and hemolytic activity of <Emphasis Type="Italic">Anabaena variabilis</Emphasis>

The hemolytic activity of the cell-free culture supernatant of Anabaena variabilis OL S1 was investigated using the hemolysis of rabbit erythrocytes as an assay. The culture medium of A. variabilis started to exhibit hemolytic activity at the late exponential growth phase, and maximized at the stationary phase. The hemolytic toxin is heat-stable and can be extracted in dichloromethane. The hemolytic activities under different temperature, light intensity and pH showed a high correlation with the cell densities (r=0.965, 0.951, 0.865, respectively), and the optimum condition is 28~30°C, pH 7.5~8.0, light intensity 120 μmol photons m⁻²s⁻¹. The addition of 10~20 μg mL⁻¹ chloramphenicol, an inhibitor of protein synthesis, exhibited no marked suppression on the hemolytic activity. The supplement of 1~20 μg mL⁻¹ glycerol increased the hemolytic activity significantly, suggesting that synthesis of hemolysin was dependent on carbohydrate and lipid metabolism. The spectrum of erythrocyte sensitivity to the hemolysin indicated that rabbit erythrocytes were more sensitive to the hemolysin than were rat and human erythrocytes. Goldfish and cat erythrocytes were, however, insensitive to the hemolytic toxin of A. variabilis.     

Evidence for the production of a proteinaceous hemolytic exotoxin by wild-type strain of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 (Cyanobacteria)

A proteinaceous hemolysin produced by a wild-type strain of Synechocystis sp. PCC 6803 was purified from cell-free culture supernatants by successive column chromatography on DEAE-Sepharose Fast Flow and Sephacryl S-300 High Resolution. The molecular mass of the hemolysin, determined by SDS-PAGE, was approximately 81 kDa. The hemolysin was heat labile and showed potent hemolytic activity against rabbit and sheep erythrocytes. The hemolysin started to be secreted during the exponential growth phase and accumulated maximally at the stationary phase. The production of hemolysin varied with the amount of calcium present in modified BG-11 culture medium. Hemolysin production decreased in calcium-free medium, whereas it increased in medium containing 0.48 mM calcium. In contrast, the potency of hemolysin, as shown by hemolysis assay, was enhanced by deprivation of calcium (EDTA treatment) but decreased in the presence of calcium. Our results show that calcium stimulated production and secretion of hemolysin, but inhibited hemolytic potency.     

Partial characterization of a cell-free hemolytic factor produced by Helicobacter pylori

Abstract Maximum cell-free hemolytic activity of Helicobacter pylori cultured in broth containing 10% horse serum occurred only after the stationary phase of growth was reached, unlike many hemolysins produced by Gram-negative bacteria which are active during exponential growth. This characteristic of the H. pylori hemolytic factor suggested that it might also possess protease activity. However, because no evidence of albumin degradation was found, the hemolysis by cell-free concentrates of H. pylori appears to be due to a unique factor derived from the organism. Because variable hemolysis results were obtained with culture broths lacking albumin or serum, these proteins may act as carriers or stabilizers of the putative hemolysin.     

Hemolysis by aliphatic alcohols and saponin measured by the coil planet centrifugation method

Using the coil planet centrifugation method, the mechanism of hemolysis by alcohols and saponin was investigated. With this technique, erythrocytes are introduced into a gradient of hemolytic agents in saline, which is prepared in a long coiled polyethylene tube. The tube is centrifuged so that the cells move from a low to a high concentration of hemolytic agent. When the cells lyse, they release hemoglobin which remains stationary, and therefore hemolytic potency can be determined spectrophotometrically by the distance the cells move before lysing. We found that alcohols caused hemolysis at a particular concentration, whereas saponin-induced hemolysis was dependent on the amount of saponin accumulated in the environment of the cell. In addition, alcohols with longer carbon chains were more potent hemolytic agents than those with shorter chains, but each additional carbon group produced less of an increase in hemolysis per mole of alcohol. This chain-length dependency is consistent with a previous study on in vivo alcohol-induced hemolysis. The coil planet centrifugation method is also adaptable to comparative studies on the mechanism of other types of hemolysis, such as immune or drug-induced lysis, and to toxicological studies.     

Adenylate cyclase toxin from Bordetella pertussis. The relationship between induction of cAMP and hemolysis.

Bordetella pertussis produces a calmodulin-activated adenylate cyclase (AC) that exists in several forms. Only one form of AC, of apparent 200 kDa, is a toxin that penetrates eukaryotic cells and generates uncontrolled levels of intracellular cAMP. Recombination studies in transposon Tn5-insertion mutants of B. pertussis and amino acid sequence homology with alpha-hemolysin of Escherichia coli suggested that AC toxin may also have a hemolytic activity. Here, we demonstrate that only the toxic form of B. pertussis AC possesses hemolytic activity. Immunoblotting of membranes from sheep erythrocytes throughout the process of cell lysis detects the presence and accumulation of only the 200-kDa form of B. pertussis AC. cAMP generation induced by AC toxin in sheep erythrocytes is immediate whereas appearance of hemolysis is delayed by about 1 h and requires a higher level of AC toxin activity. Addition of exogenous calmodulin to sheep erythrocyte incubation medium potentiates the hemolytic activity of AC toxin but blocks cAMP generation. Extracellular Ca2+ at mM concentrations is absolutely required for cAMP generation but not for hemolysis. However, binding of AC toxin to sheep erythrocytes in the absence of exogenous Ca2+ followed by reincubation of cells in a toxin-free buffer containing Ca2+ leads to an immediate rise in intracellular cAMP. Human erythrocytes bind AC toxin and generate cAMP but are resistant to lysis. These results show that binding of AC toxin to erythrocytes can cause both cAMP generation and hemolysis or only one of these depending on conditions applied and cell type used.     

Hemolysis of phosphatidylcholine-containing erythrocytes by serratamic acid from Serratia marcescens.

1. The hemolysis by serratamic acid, "N-(D-3-hydroxydecanoyl)-L-serine and N-(D-3-hydroxydodecanoyl)-L-serine", was investigated with human and animal erythrocytes using serratamic acid-containing liposomes. 2. The hemolytic activity was found to depend on the incubation temperature and the concentration of the liposomes. 3. The concentration of serratamic acid for 50% hemolysis was 0.17 mM at 37 degrees C for 0.2% human erythrocyte suspension in the liposomes which composed of phosphatidylserine, cholesteryl nervonate and serratamic acid (1:0.50:0.37 by mol). 4. The hemolysis was shown specifically in human, horse and rabbit erythrocytes containing phosphatidylcholine, but not in sheep or bovine erythrocytes lacking phosphatidylcholine. 5. The hemolytic activity was strongly inhibited by the exogenous addition of phosphatidylcholine. It was suggested that the hemolysis by serratamic acid-containing liposomes was specific for phosphatidylcholine-containing erythrocyte membranes.     

Bacteriocin (hemolysin) of Streptococcus zymogenes

The sensitivity of Streptococcus faecalis (ATTC 8043) to S. zymogenes X-14 bacteriocin depends greatly on its physiological age. Sensitivity decreases from the mid-log phase on and is completely lost in the stationary phase. The sensitivity of erythrocytes to the hemolytic capacity of the bacteriocin showed considerable species variation. The order of increasing sensitivity was goose < sheep < dog < horse < human < rabbit. However, when red cell stromata were used as inhibitors of hemolysis in a standard system employing rabbit erythrocytes the order of increasing effectiveness was sheep < rabbit < human < horse < goose. When rabbit cells were used in varying concentrations with a constant hemolysin concentration, there was a lag of about 30 min, which for a given hemolysin preparation was constant for all red cell concentrations. Furthermore, the rate of hemolysis increased with increasing red cell concentration. If red cells are held constant and lysin varied, the time to reach half-maximal lysis varies directly with lysin but is not strictly proportional. Bacterial membranes were one to three orders of magnitude more effective than red cell stromata as inhibitors. The order of increasing effectiveness seems to be Escherichia coli < Bacillus megaterium < S. faecalis < Micrococcus lysodeikticus. In addition to membranes, a d-alanine containing glycerol teichoic acid, trypsin in high concentration, and deoxyribonuclease also inhibited hemolysis. Ribonuclease, d-alanine, l-alanine, dl-alanyl-dl-alanine, N-acetyl-d-alanine, N-acetyl-l-alanine did not inhibit hemolysis.     

Ceramidase enhances phospholipase C-induced hemolysis by Pseudomonas aeruginosa

We previously reported the purification, molecular cloning, and characterization of a neutral ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M. (1998) J. Biol. Chem. 273, 14368-14373; Okino, N., Ichinose, S., Omori, A., Imayama, S., Nakamura, T., and Ito, M. (1999) J. Biol. Chem. 274, 36616-36622). Interestingly, the gene encoding the enzyme is adjacent to that encoding hemolytic phospholipase C (plcH) in the genome of Pseudomonas aeruginosa, which is a well known pathogen for opportunistic infections. We report here that simultaneous production of PlcH and ceramidase was induced by several lipids and PlcH-induced hemolysis was significantly enhanced by the action of the ceramidase. When the strain was cultured with sphingomyelin or phosphatidylcholine, production of both enzymes drastically increased, causing the increase of hemolytic activity in the cell-free culture supernatant. Ceramide and sphingosine were also effective in promoting the production of ceramidase but not that of PlcH. Furthermore, we found that the hemolytic activity of a Bacillus cereus sphingomyelinase was significantly enhanced by addition of a recombinant Pseudomonas ceramidase. TLC analysis of the erythrocytes showed that ceramide produced from sphingomyelin by the sphingomyelinase was partly converted to sphingosine by the ceramidase. A ceramidase-null mutant strain caused much less hemolysis of sheep erythrocytes than did the wild-type strain. Sphingosine was detected in the erythrocytes co-cultured with the wild-type strain but not the mutant strain. Finally, we found that the enhancement of PlcH-induced hemolysis by the ceramidase occurred in not only sheep but also human erythrocytes. These results may indicate that the ceramidase enhances the PlcH-induced cytotoxicity and provide new insights into the role of sphingolipid-degrading enzymes in the pathogenicity of P. aeruginosa.     

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.     

The hemolytic activity of deoxynivalenol and T-2 toxin.

The hemolytic effects of deoxynivalenol (DON) and T-2 toxin (T-2) individually on rat erythrocytes were studied at different concentrations. Sodium azide was used as an enzyme inhibitor to prevent T-2 toxin metabolism. The concentration of T-2 was controlled by GC-MS and no decrease of the toxin was found during the time of the experiment. In spite of the much higher toxicity of T-2 toxin to eucaryotic cells, DON and T-2 showed similar lytic activity toward erythrocytes at high and low concentrations. Neither of these toxins at a concentration of 130 micrograms/ml, produced significant hemolysis even after 11 hr incubation. This finding suggests that there is a threshold level for both T-2 and DON, below which the lytic reaction does not occur. An additional hemolysis test was conducted in the presence of mannitol, glutathione, ascorbic acid, alfa-tocopherol, and histidine. The assay demonstrated that all the compounds inhibited to some extent the hemolytic reaction of the toxins. It is suggested that DON and T-2 exert their toxicity on procaryotic cells in three different ways: by penetrating the phospholipid bilayer and acting at the subcellular level, by interacting with the cellular membranes, and by free radical mediated phospholipid peroxidation. Most probably, more than one mechanism operates at the same time.     

Biosynthesis of endochitinase of Serratia marcescens.

The growth of a mutant strain of Serratia marcescens with high chitinase activity and the biosynthesis of endochitinase by this strain were investigated. The study was carried out using semisynthetic culture medium without inducers and culture medium containing colloidal chitin as a sole nitrogen and carbon source, with and without mitomycin C. The mutant strain, unlike the native one, was shown to produce endochitinase and to secrete the enzyme into the medium during the growth on culture medium without the inducers, chitin and mitomycin C. During growth on the medium with chitin the mutant strain differed from the native one with a short lag-phase of growth, the early appearance of endochitinase in the culture liquid and a high level of endochitinase activity. The difference between the strains disappeared after the addition of mitomycin C, an inducer of the cell SOS-response, to the culture medium containing chitin. Specific endochitinase activity of S. marcescens mutant strain grown on various culture media had two maxima, namely at the beginning and at the end of the stationary phase. Mitomycin C increased the specific activity in a second peak of endochitinase activity during the growth of the mutant strain.     

Growth and hemolysin production by Haemophilus pleuropneumoniae cultivated in a chemically defined medium

A chemically defined medium (CDM) has been developed which supports both growth and hemolysin production by Haemophilus pleuropneumoniae. Although the growth rate in stationary cultures was substantially slower in CDM than in trypticase soy broth plus 0.6% yeast extract (TSBYE) and slightly slower than in heart infusion broth (HIB), extracellular hemolysin activity in CDM was slightly higher than in HIB and 16-fold greater than in TSBYE. Maximum hemolytic activity was produced in CDM in early to mid log phase of growth. Hemolytic activity in sterile, cell-free culture supernatant fluids persisted for over 10 days at 4 degrees C and 3-5 days at 37 degrees C, but was completely destroyed at 56 degrees C after 30 min. Total hemolysin inactivation was also achieved in the presence of trypsin or pronase (10 units/mL), but no decrease in hemolytic activity was noted in the presence of DNase or RNase. Iron had little effect on the hemolytic activity in the early stages of growth. However, in the later stages of growth, iron had a pronounced effect with hemolytic activity decreasing as the iron concentration increased from 1 to 500 microM. None of these iron concentrations had any effect on the hemolytic activity when added directly to prepared cell-free culture supernatant fluids. The extracellular hemolysin produced by H. pleuropneumoniae in CDM appears to be a heat-labile protein the activity of which is influenced by iron at certain phases of growth.     

Mechanism of free radical-induced hemolysis of human erythrocytes: comparison of calculated rate constants for hemolysis with experimental rate constants.

We previously developed a simple competitive reaction model between lipid peroxidation and protein oxidation in erythrocyte membranes that accounts for radical-induced hemolysis of human erythrocytes. In this study, we compared the rate constants calculated from the hemolysis curves of erythrocytes in the presence of radical initiators with those obtained from experiments using erythrocyte ghosts treated with radicals. 2,2'-Azobis(amidinopropane) dihydrochloride and 2,2'-azobis(2,4-dimethylvaleronitrile) were used as radical initiators. Plots of the logarithm of concentration of the radical initiator against the logarithm of the rate constant gave straight lines. The slope of the lines for the calculated lipid peroxidation was nearly equal with the experimental value. Similar results were obtained for oxidation of membrane proteins, except for band 3 oxidation. The values for the rate constants calculated from hemolysis curves seem to be accurate. The slope of the lines for the calculated rate constants for proteins was larger than the experimental value for band 3 oxidation, because band 3 oxidation is accompanied by aggregation or redistribution of band 3 proteins to form hemolytic holes. These results indicate that the competitive reaction model may be useful for analyzing radical-induced hemolysis.     

Studies on the role of goat heart galectin-1 as an erythrocyte membrane perturbing agent

Galectins are β-galactoside binding lectins with a potential hemolytic role on erythrocyte membrane integrity and permeability. In the present study, goat heart galectin-1 (GHG-1) was purified and investigated for its hemolytic actions on erythrocyte membrane. When exposed to various saccharides, lactose and sucrose provided maximum protection against hemolysis, while glucose and galactose provided lesser protection against hemolysis. GHG-1 agglutinated erythrocytes were found to be significantly hemolyzed in comparison with unagglutinated erythrocytes. A concentration dependent rise in the hemolysis of trypsinized rabbit erythrocytes was observed in the presence of GHG-1. Similarly, a temperature dependent gradual increase in percent hemolysis was observed in GHG-1 agglutinated erythrocytes as compared to negligible hemolysis in unagglutinated cells. The hemolysis of GHG-1 treated erythrocytes showed a sharp rise with the increasing pH up to 7.5 which became constant till pH 9.5. The extent of erythrocyte hemolysis increased with the increase in the incubation period, with maximum hemolysis after 5 h of incubation. The results of this study establish the ability of galectins as a potential hemolytic agent of erythrocyte membrane, which in turn opens an interesting avenue in the field of proteomics and glycobiology.     

Schistosoma mansoni: characterization of hemolytic activity from adult worms

Homogenates of adult Schistosoma mansoni worms contain a hemolytically active component(s). Centrifugation at 10,000 g shows the major activity is present in the pellet fraction. Red blood cell lysis with the schistosome hemolytic agent is optimal at acid pH (5.0) and highly temperature dependent. The hemolytic component is resistant to boiling (5 min) and stable for extended periods of time at 38 C (22 hr). The length of the lag phase prior to hemolysis and the rate of hemolysis are both concentration and temperature dependent. Following hemolysis, red blood cell ghosts remain.     

Presence and characterization of complement-like activity in the amphioxus Branchiostoma belcheri tsingtauense

The humoral fluid of Branchiostoma belcheri tsingtauense was examined for the presence of complement-like activity. The humoral fluid showed hemolytic activity for rabbit erythrocytes and those from species representing mammals, birds, amphibians and fish, but not sensitized sheep erythrocytes. There was no relationship between phylogeny of the target erythrocytes and degree of hemolysis. The hemolytic activity was optimally assayed at 20 degrees C, at pH 7.5, and in the presence of 10 mM Mg2+. The hemolytic activity was Mg2+-dependent and heat-sensitive, and was abrogated by treatment with rabbit anti-human C3 serum, zymosan, methylamine, hydrazine, and phenylmethylenesulfonyl fluoride. In addition, Western blotting and titration by turbidimetric immunoassay (TIA) revealed that amphioxus humoral fluid contained C3 component, and its concentration is about 1.17 mg/ml, which is comparable to C3 concentration in human or dog sera. These suggest that the hemolytic activities displayed by amphioxus humoral fluid appear to represent the vertebrate complement system probably operating via the alternative pathway.     

Kinetics of erythrocyte lysis by snake venom cardiotoxins

Hemolysis of guinea pig erythrocytes by snake venom cardiotoxins was investigated with a semi-automatic method based on light-scattering changes of erythrocyte suspensions at 700 nm which are directly related to hemoglobin release. Small amounts of phospholipase-free cardiotoxin (<100 μg) could be conveniently and rapidly assayed with the high reproducibility in a recording spectrophotometer, and reliable kinetic data were accumulated.Cardiotoxins from two different genera (Hemachatus haemachates and Naja mossambica mossambica) displayed virtually identical hemolytic properties. Hemolysis increased linearly with time, in contrast with a sigmoidal pattern when phospholipase was present as an impurity. Low concentrations of Ca²⁺ (<1 mM) stimulated cardiotoxin action. A limiting plateau rate of hemolysis reached during concentration dependence experiments in which the level of either cardiotoxin or of erythrocytes was varied, suggested that the interaction of cardiotoxin with erythrocyte membranes is a saturation phenomenon only at a high ratio of cardiotoxin: erythrocytes. No hemolysis was observed with an homologous neurotoxin of S-methylated cardiotoxin, providing evidence for specificity. The linear Arrhenius plots obtained for the temperature dependence of cardiotoxin-induced hemolysis strengthened the conclusion that its action involves more than a detergent-like effect on membrane phospholipids.