Red blood cell lysis induced by the venom of the brown recluse spider: the role of sphingomyelinase D.

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A₂-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.     

How much do we know about hemolytic capability of pathogenic <Emphasis Type="Italic">Candida</Emphasis> species?

Hemolytic factor production by pathogenic Candida species is considered an important attribute in promoting survival within the mammal host through the ability to assimilate iron from the hemoglobin-heme group. Hemolytic capability has been evaluated for Candida species based on hemolysis zones on plate assay, analysis of hemolytic activity in liquid culture medium, and hemolysis from cell-free culture broth. The production of hemolytic factor is variable among Candida species, where C. parapsilosis is the less hemolytic species. In general, no intraspecies differences in beta-hemolytic activities are found among isolates belonging to C. albicans, C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis. The production of hemolytic factor by Candida species is affected by several factors such as glucose supplementation in the culture medium, blood source, presence of erythrocytes and hemoglobin, and presence of electrolytes. On the basis of existing achievements, more researches are still needed in order to extend our knowledge about the biochemical nature of hemolytic molecules produced by distinct Candida species, the mechanism of hemolysis, and the molecular basis of the hemolytic factor expression.     

COMPARATIVE KINETICS OF HEMOLYSIS INDUCED BY BACTERIAL AND OTHER HEMOLYSINS

A study has been made of the kinetics of lysis induced by various hemolytic agents. The course of bemolysis was followed by mixing lysin with washed human erythrocytes, removing samples from the mixture, and estimating colorimetrically the hemoglobin in the supernatant fluid of the centrifuged samples. Most of the curves (but not all of them, e.g. tyrocidine) obtained by plotting degree of hemolysis against time, were S-shaped. The initiation of lysis by streptolysin S'' was delayed, and in this property, streptolysin S'' was similar to Cl. septicum hemolysin. None of the other lysins studied exhibited a long latent period preceding lysis. The maximum rate of hemoglobin liberation was found, in the range of lysin concentrations studied, to be a linear function of concentration when theta toxin of Cl. welchii, pneumolysin, tetanolysin, or streptolysin S'' was the lytic agent. With comparable concentrations of saponin, sodium taurocholate, cetyl pyridinium chloride, tyrocidine, duponol C, lecithin-atrox venom mixture, or streptolysin O, the relation between rate and concentration was non-linear. The critical thermal increment associated with hemolysis was determined for systems containing pneumolysin, theta toxin, streptolysin S'', streptolysin O, tetanolysin, and saponin. The findings concerning the effect of concentration and temperature on the rate of hemolysis provide a basis for classifying hemolytic agents (Tables I and II). Hemolysis induced by Cl. septicum hemolysin was found to be preceded by two phases: a phase of alteration of the erythrocytes and a phase involving swelling. Antihemolytic serum inhibited the first but not the second phase while sucrose inhibited the second but not the first phase.     

Blood-Feeding Induces Reversible Functional Changes in Flight Muscle Mitochondria of Aedes aegypti Mosquito

Background

Hematophagy poses a challenge to blood-feeding organisms since products of blood digestion can exert cellular deleterious effects. Mitochondria perform multiple roles in cell biology acting as the site of aerobic energy-transducing pathways, and also an important source of reactive oxygen species (ROS), modulating redox metabolism. Therefore, regulation of mitochondrial function should be relevant for hematophagous arthropods. Here, we investigated the effects of blood-feeding on flight muscle (FM) mitochondria from the mosquito Aedes aegypti, a vector of dengue and yellow fever.

Methodology/Principal Findings

Blood-feeding caused a reversible reduction in mitochondrial oxygen consumption, an event that was parallel to blood digestion. These changes were most intense at 24 h after blood meal (ABM), the peak of blood digestion, when oxygen consumption was inhibited by 68%. Cytochromes c and a+a ₃ levels and cytochrome c oxidase activity of the electron transport chain were all reduced at 24 h ABM. Ultrastructural and molecular analyses of FM revealed that mitochondria fuse upon blood meal, a condition related to reduced ROS generation. Consistently, BF induced a reversible decrease in mitochondrial H₂O₂ formation during blood digestion, reaching their lowest values at 24 h ABM where a reduction of 51% was observed.

Conclusion

Blood-feeding triggers functional and structural changes in hematophagous insect mitochondria, which may represent an important adaptation to blood feeding.     

Characteristics of the interaction of a treponemal hemolysin with rabbit erythrocytes

The mechanism of action on rabbit red cells of Treponema hyodysenteriae hemolysin was studied using volume analysis and release of hemoglobin. While fixation of the hemolysin on the erythrocytes is temperature independent, it appears that hemolysis is temperature dependent. The kinetics of hemolysis proceed according to a sigmoid curve characterized by a prelytic lag. The duration of the prelytic lag varies inversely with the quantity of hemolysin but the rate and the maximum value of hemolysis are directly proportional to the quantity of hemolysin. The effect of sucrose and trypan blue on the hemolysin and the red cells suggest that erythrocyte lysis is likely to be induced by the hemolysin in a way different from that known for other hemolytic agents.     

Hemolytic activity of Serratia marcescens

A cell-bound hemolytic activity was found in several strains of Serratia marcescens. One Serratia cell per ten erythrocytes was sufficient to cause complete lysis of human erythrocytes within 2 h in the liquid assay. The hemolytic activity resided in the membrane fraction and could be inactivated by incubating cells with proteases. The hemolytic activity was greatly enhanced in actively metabolizing Serratia cells and was partially controlled by the iron supply. Hemolysis was accompanied by degradation of erythrocyte membrane proteins (band 3 and 6, glycophorin) and was independent of the blood group. The exoprotease secreted by S. marcescens in large amounts was not involved in hemolysis. Comparison with various hemolytic strains of Escherichia coli showed that hemolysis of erythrocytes was more pronounced with S. marcescens than with E. coli. In contrast to hemolysis by E. coli, lysis of erythrocytes by S. marcescens was not enhanced by Ca²⁺ ions.Dedicated to Professor Dr. Gerhart Drews on the occasion of his 60th birthday     

Introduction of Culex Toxicity into Bacillus thuringiensis Cry4Ba by Protein Engineering

Bacillus thuringiensis mosquitocidal toxin Cry4Ba has no significant natural activity against Culex quinquefasciatus or Culex pipiens (50% lethal concentrations [LC₅₀], >80,000 and >20,000 ng/ml, respectively). We introduced amino acid substitutions in three putative loops of domain II of Cry4Ba. The mutant proteins were tested on four different species of mosquitoes, Aedes aegypti, Anopheles quadrimaculatus, C. quinquefasciatus, and C. pipiens. Putative loop 1 and 2 exchanges eliminated activity towards A. aegypti and A. quadrimaculatus. Mutations in a putative loop 3 resulted in a final increase in toxicity of >700-fold and >285-fold against C. quinquefasciatus (LC₅₀ 114 ng/ml) and C. pipiens (LC₅₀ 37 ng/ml), respectively. The enhanced protein (mutein) has very little negative effect on the activity against Anopheles or Aedes. These results suggest that the introduction of short variable sequences of the loop regions from one toxin into another might provide a general rational design approach to enhancing B. thuringiensis Cry toxins.     

Screening and sequence analysis of the hemolysin gene of Fusobacterium necrophorum

Fusobacterium necrophorum is the main pathogen that causes numerous necrobacilloses. Hemolysin is one of the major virulence factors involved in fusobacterial infections. In order to investigate the genetic basis of hemolytic activity and the regulation mechanism of the hemolysin expression, a genomic library was constructed from F. necrophorum DNA by ligating DNA fragments generated by partial HindIII digestion with pUC18 vector. The screening of the genomic library with polymerase chain reaction, DNA sequencing and sequence assembly led to a 7.45 kb sequence which includes the putative hly gene and upstream sequence. Clustered putative genes encoding short chain acyl-CoA dehydrogenase (Scad) and electron transfer flavoprotein (Etf) α and β subunits locate upstream of hly. A 535 bp non-coding sequence, possibly with some cis-regulatory elements involved in the regulation of the hemolysin expression in F. necrophorum, locates between etf-β and hly. The nucleotide sequence of the hly gene indicates it encodes hemolysin. It is the first characterized hemolysin coding gene in F. necrophorum.     

Increased Bacterial Hemolytic Activity is Conferred by Expression of TlyA Methyltransferase but not by its 2′-O-methylation of the Ribosome

Bacterial 2′-O-methyltransferase TlyA methylates either both nucleotide C1409 of 16S rRNA and C1920 of 23S rRNA or only the C1920. Both ribosomal methylations increase bacterial susceptibility to ribosome-targeting antibiotics capreomycin and viomycin. However, TlyA has been suggested to also function as a hemolysin. Here, heterologous expression of TlyA from six diverse bacteria (including Mycobacterium tuberculosis and M. smegmatis) was found to increase hemolytic ability in the Escherichia coli host. Characterizing E. coli strains expressing mycobacterial TlyA with mutated rRNA recognition domain and impaired rRNA methylations showed that the abolished C1409 methylation altogether with significantly reduced C1920 methylation did not affect E. coli hemolytic activity. Thus, the increased bacterial hemolytic function is not likely a consequence of TlyA-mediated methylations of the ribosome. Purified water-soluble TlyA showed a weak concentration-dependent hemolysis in vitro. Therefore, the TlyA isoform alone is not a potent hemolysin. The results suggested that the bacterial hemolytic function might relate to the over-expression of TlyA and its interaction to other non-ribosomal target that is associated with the hemolytic ability.     

In vivo hemolytic activity of group B streptococcus is dependent on erythrocyte-bacteria contact and independent of a carrier molecule

Experiments were performed to determine the interaction between the hemolysin of group B streptococcus (GBS) and sheep erythrocytes. Growing GBS were shown to possess a potent hemolysin at a very early stage of the growth cycle. After separation of the cells from the growth medium, all the hemolytic activity remained with the bacterial cells, and no activity could be detected in the growth medium. When fetal calf serum was added to the media, some soluble activity was detected. This activity, however was completely removed by ultracentrifugation, the hemolytic activity being found solely in the pellet. After the hemolysin had formed, no new protein synthesis was needed to cause hemolysis because the addition of chloramphenicol to cells caused no difference in their hemolytic potential. For proof that no short-lived, soluble factors are produced by the bacteria, bacteria and sheep erythrocytes were incubated in contiguous media, separated by a 0.22-m membrane. No hemolytic activity was detected on the erythrocyte side of the membrane, although high amounts of hemolysin could be extracted from the bacteria. Only when a detergent was added to the growth medium was hemolysis detected from the erythrocytes, showing that extracted hemolysin could indeed pass through the membrane. These results suggest that the hemolysin is attached to the surface of the cell and that contact is needed between the bacteria and erythrocyte to cause lysis. Where soluble activity was detected, it was connected to bacterial fragments.     

Binding of active and inactive hemolytic factor of sea anemone nematocyst venom to red blood cells

Sea anemone nematocyst venom, in the presence of Ca²⁺, induced the lysis of red blood cells after an induction period. In the absence of Ca²⁺, however, no lysis occurred, but the hemolytic factor was shown to bind to the cells. This binding was shown to be requisite for the Ca²⁺ dependent lysis to ensue. After freeze thawing, the venom proteins responsible for lysis lost their hemolytic activity, yet still bound to the cells. The freezethawed inactivated venom competitively blocked hemolysis by active venom.     

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.     

Erythrocyte membrane structural features that are critical for the lytic reaction of Spirographis spallanzani coelomic fluid hemolysin

1. Hemolytic activity of Spirographis spallanzani coelomic fluid depends on factor(s) strongly influenced by calcium but not by sulfhydril or disulfide reagents.2. The lytic reaction was suppressed by low zinc ion concentrations but it was not influenced by the presence of proteinase inhibitors.3. These data indicate that S. spallanzani hemolysin is a non-enzymatic, calcium-dependent, zincinhibitable factor that occurs naturally in the coelomic fluid.4. In the absence of calcium, enzymatic desialization converted sheep erythrocytes into susceptible targets, suggesting the involvement of erythrocyte surface sialic acid.5. However, the inhibitory effect of the sugar on anti-rabbit lysis was partially removed by addition of calcium.6. Attempts to characterize membrane components that are critical for hemolysis were performed by inhibition experiments.7. We found that saccharides, glycoproteins, mucosubstances as well as rabbit erythrocyte soluble tryptic fragments were ineffective in inhibiting hemolysis.8. Sonicated dispersion of phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanol, sphingomyelin and cholesterol did not influence the hemolytic reaction.9. Rabbit erythrocyte extracted from membrane lipids (chloroform phase) did not modify the lytic activity against rabbit red blood cells.10. Conversely, the methanol phase consistently reduced the lytic capacity of the fluid.11. The heat-stable, trypsin-resistant inhibitory factor was most probably a small molecule, since dialysis removed the inhibitory effect.     

Erythrocyte Lysis and Xenopus laevis Oocyte Rupture by Recombinant Plasmodium falciparum Hemolysin III

Malaria kills more than 1 million people per year worldwide, with severe malaria anemia accounting for the majority of the deaths. Malaria anemia is multifactorial in etiology, including infected erythrocyte destruction and decrease in erythrocyte production, as well as destruction or clearance of noninfected erythrocytes. We identified a panspecies Plasmodium hemolysin type III related to bacterial hemolysins. The identification of a hemolysin III homologue in Plasmodium suggests a potential role in host erythrocyte lysis. Here, we report the first characterization of Plasmodium falciparum hemolysin III, showing that the soluble recombinant P. falciparum hemolysin III is a pore-forming protein capable of lysing human erythrocytes in a dose-, time-, and temperature-dependent fashion. The recombinant P. falciparum hemolysin III-induced hemolysis was partially inhibited by glibenclamide, a known channel antagonist. Studies with polyethylene glycol molecules of different molecular weights indicated a pore size of approximately 3.2 nm. Heterologous expression of recombinant P. falciparum hemolysin III in Xenopus oocytes demonstrated early hypotonic lysis similar to that of the pore-forming aquaporin control. Live fluorescence microscopy localized transfected recombinant green fluorescent protein (GFP)-tagged P. falciparum hemolysin III to the essential digestive vacuole of the P. falciparum parasite. These transfected trophozoites also possessed a swollen digestive vacuole phenotype. Native Plasmodium hemolysin III in the digestive vacuole may contribute to lysis of the parasitophorous vacuole membrane derived from the host erythrocyte. After merozoite egress from infected erythrocytes, remnant P. falciparum hemolysin III released from digestive vacuoles could potentially contribute to lysis of uninfected erythrocytes to contribute to severe life-threatening anemia.     

A hemolytic peptide from the mycophilic fungus Sepedonium chrysospermum (Bull.) Fr

The hemolytic activity of an extract of the mycoparasite Sepedonium chrysospermum (teleomorph Hypomyces chrysospermus) was detected and characterized. Extraction of the fungal biomass by methanol yielded a fraction in which the hemolytic activity against human red blood cells corresponded to a peptide with a molecular mass of 7,653.72 Da and an isoelectric point of approximately 5.8. The peptide was temperature resistant, and the hemolysis was only partially inhibited, even after a 30-min pre-incubation at 100°C. Its hemolytic activity was unaffected by treatment with proteolytic enzymes such as trypsin. Among the divalent cations assayed, Hg²⁺ was the strongest inhibitor of hemolysis. The reducing agent, dithiothreitol, and the membrane lipid, cholesterol, demonstrated concentration-dependent inhibitory activities. Finally, hemolytic activity triggered by the peptide was analyzed by scanning electron microscopy, and a pore-forming activity was detected.     

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal¹. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis^1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.     

Purification and characterization of hemolysin from periodontopathogenic bacterium Eikenella corrodens strain 1073

Eikenella corrodens 1073 was found to show hemolytic activity when grown on sheep blood agar. A high and dose-dependent hemolytic activity was detected in the cell envelope fraction, which was further purified by ion-exchange and gel-filtration chromatography. Consequently, a 65-kDa protein with hemolytic activity was obtained, suggesting that this protein might be a hemolysin. Its N-terminal amino acid sequence was nearly identical to that of X-prolyl aminopeptidase from E. corrodens ATCC 23834. To confirm that X-prolyl aminopeptidase functions as a hemolytic factor, we expressed the hlyA gene, encoding X-prolyl aminopeptidase, in Escherichia coli. After induction with isopropyl β-D-1-thiogalactopyranoside, a protein of about 65 kDa was purified on a Ni column, and its hemolytic activity was confirmed. Meanwhile, a strain with a disrupted hlyA gene, which was constructed by homologous recombination, did not show any hemolytic activity. These results suggested that X-prolyl aminopeptidase might function as a hemolysin in E. corrodens.     

Mutational analysis of TlyA from Brachyspira hampsonii reveals two key residues conserved in pathogenic bacteria responsible for oligomerization and hemolytic activity

BackgroundTlyA proteins are expressed in a variety of pathogenic bacteria and possess dual hemolytic and ribosomal RNA methyltransferase functions. While the mechanism of TlyA mediated rRNA methylation is well understood, relatively little is known about the mechanism of TlyA induced hemolysis.MethodsTlyA protein from the pig pathogen Brachyspira hampsonii was heterologously expressed and purified from an E. coli host. Hemolytic activity and rRNA methylation were assessed in vitro. Site-directed mutagenesis was used to mutate amino acids believed to be involved in TlyA mediated hemolysis.ResultsPurified TlyA-His protein exhibited both hemolytic and rRNA methyltransferase activities in vitro, with partial inhibition of hemolysis observed under reducing conditions. Mutation of cysteine 80 to alanine impaired hemolytic activity. A C27A/C93A mutant was capable of dimerizing under non-reducing conditions, indicating that a C80-C80 disulfide bond is involved in TlyA oligomerization. A mutation conserved in several avirulent Brachyspira species (S9K) completely abolished hemolytic activity of TlyA. This loss of activity was attributed to impaired oligomerization in the S9K mutant, as assessed by ITC and size-exclusion chromatography experiments.ConclusionsOligomeric assembly and hemolytic activity of TlyA from Brachyspira hampsonii is dependent on the formation of an intermolecular C80-C80 disulfide bond and noncovalent interactions involving serine 9. The conservation of these amino acids in TlyA proteins from pathogenic bacteria suggests a correlation between tlyA gene mutations and bacterial virulence.General significanceOur results further elucidate the mechanisms underlying TlyA mediated hemolysis and provide evidence of a conserved mechanism of oligomerization for TlyA family proteins.