Assembly mechanism of the oligomeric streptolysin O pore: the early membrane lesion is lined by a free edge of the lipid membrane and is extended gradually during oligomerization

105

A recombinant form of CAMP factor of Streptococcus agalactiae has been expressed as glutathione S-transferase-CAMP fusion protein in Escherichia coli. After thrombin cleavage of the fusion protein, the recombinant CAMP factor exhibited hemolytic activity comparable with that of the native form. Osmotic protection experiments with polyethylene glycols show that CAMP factor forms discrete transmembrane pores with a diameter upward of 1.6 nm on susceptible membranes; electron microscopy reveals circular membrane lesions of heterogeneous size, up to 12-15 nm in diameter. Liposome permeabilization studies show that pore formation is a highly cooperative process, which suggests that it involves the oligomerization of CAMP factor. Chemical cross-linking experiments also support an oligomeric mode of action.The CAMP reaction consists of a distinct zone of hemolysis on blood agar plates produced by Streptococcus agalactiae when grown near the colonies of Staphylococcus aureus (1). It has been used in diagnostic microbiology to identify the S. agalactiae strains ever since its discovery in 1944 by Christie et al. (1). The proteins responsible for the CAMP reaction are sphingomyelinase from S. aureus and CAMP factor, a protein secreted by S. agalactiae that has a molecular weight of 25 kDa and a pI of 8.9 (2). Sphingomyelinase initially hydrolyzes sphingomyelin to ceramide, which renders the erythrocytes susceptible to the lytic activity of CAMP factor. Erythrocytes from different mammalian species support the CAMP reaction to different extents depending on the sphingomyelin content in their cell membranes (3). Sheep red blood cells are the most susceptible, in keeping with their sphingomyelin content as high as 51% (by moles) (4). Human red blood cells and rabbit red blood cells, with 26 and 19% mol of sphingomyelin, respectively (4, 5), were reportedly not sensitive to CAMP factor after sphingomyelinase treatment (3). The latter, however, were rendered susceptible to the CAMP factor after phospholipase C treatment, which converted the glycerophospholipids to diacylglycerol (6), indicating that ceramide is not specifically required for CAMP activity. Previous work with liposomes also suggested that the fraction of cholesterol in the membrane influences the activity of CAMP factor (6, 7). CAMP factor has long been known as a pathogenic determinant that exerted lethal effects when administered to rabbits and mice (8). Apart from its membrane damaging activity, CAMP factor was found to bind to the F c fragments of immunoglobulins, and it was therefore designated as protein B in analogy to protein A of S. aureus (9).The CAMP factor genes of S. agalactiae (10), Streptococcus uberis (11), and Streptococcus pyogenes (12) have been cloned in Escherichia coli, and their sequences were found to be highly homologous with each other (12). Co-hemolytic phenomena have also been reported with various other bacterial genera, but the proteins responsible for those, although sometimes referred to by the name "CAMP factor" as well, are not...

Section: Fig 2 Lysis Of Sheep Erythrocytes By Camp Factorsupporting

confidence: 88%

Characterization of Streptococcus agalactiae CAMP Factor as a Pore-forming Toxin

Lang

Palmer

2003

105

“…Washed human platelets from healthy donors were prepared (24), resuspended in ice-cold Buffer A (50 mM Hepes/KOH, pH 7.2, 78 mM KCl, 4 mM MgCl 2 , 0.2 mM CaCl 2 , 2 mM EGTA, 1 mM dithiothreitol, and the calculated free [Ca 2ϩ ] was ϳ20 nM (25)) containing 4 mg/ml bovine serum albumin and 20 ng/ml prostaglandin E 1 and kept at 4°C. The platelets were incubated with 0.6 g/ml SLO at 4°C for 10 min and washed once to remove unbound SLO (19,20,26,27), The treated platelets were resuspended in ice-cold Buffer A containing 4 mg/ml bovine serum albumin at a density of 5 ϫ 10 8 /ml, quantified with a Coulter Counter, and incubated at 30°C for 5 min to make holes in their plasma membrane (19,20,26,27). The permeabilized platelets were kept on ice for 15-30 min with 2 mg of proteins/ml human platelet cytosol (or rat brain cytosol), an ATP-regenerating system (19,20,26,27), and tested substances.…”

Section: Methodsmentioning

confidence: 99%

“…The platelets were incubated with 0.6 g/ml SLO at 4°C for 10 min and washed once to remove unbound SLO (19,20,26,27), The treated platelets were resuspended in ice-cold Buffer A containing 4 mg/ml bovine serum albumin at a density of 5 ϫ 10 8 /ml, quantified with a Coulter Counter, and incubated at 30°C for 5 min to make holes in their plasma membrane (19,20,26,27). The permeabilized platelets were kept on ice for 15-30 min with 2 mg of proteins/ml human platelet cytosol (or rat brain cytosol), an ATP-regenerating system (19,20,26,27), and tested substances. Because fibrinogen has been added in previously established aggregation assays using washed platelets at 0.4 mg/ml by Kinlough-Rathbone et al (28) and 0.38 mg/ml by Santos et al (29), we also added fibrinogen (Sigma) in our assay at the concentration of 0.4 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

“…We have established an aggregation assay with platelets permeabilized by SLO, which has been shown to form pores of ϳ30 nm in diameter in the membrane (27). In this method, we permeabilized only the plasma membrane judging from observations that the intracellular membrane structures of the platelets appeared to be intact morphologically (30) and that vWF stored in ␣-granules did not leak out (19,20).…”

Section: Establishment Of An Assay Analyzing the Ca 2ϩ -Induced Aggrementioning

confidence: 99%

See 1 more Smart Citation

Direct Demonstration of Involvement of Protein Kinase Cα in the Ca2+-induced Platelet Aggregation

Tabuchi¹,

Yoshioka²,

Higashi³

et al. 2003

Platelets play critical roles in hemostasis and thrombosis through their aggregation following activation of integrin ␣ IIb ␤ 3 . However, the molecular mechanism of the integrin activation inside platelets remains largely unknown. Pharmacological experiments have demonstrated that protein kinase C (PKC) plays an important role in platelet aggregation. Because PKC inhibitors can have multiple substrates and given that non-PKC-phorbol ester-binding signaling molecules have been demonstrated to play important roles, the precise involvement of PKC in cellular functions requires re-evaluation. Here, we have established an assay for analyzing the Ca 2؉ -induced aggregation of permeabilized platelets. The aggregation of platelets was inhibited by the addition of the arginine-glycine-aspartate-serine peptide, an integrin-binding peptide inhibitor of ␣ IIb ␤ 3 , suggesting that the aggregation was mediated by the integrin. The aggregation was also dependent on exogenous ATP and platelet cytosol, indicating the existence of essential cytosolic factors required for the aggregation. To examine the role of PKC in the aggregation assay, we immunodepleted PKC␣ and ␤ from the cytosol. The PKC-depleted cytosol lost the aggregation-supporting activity, which was recovered by the addition of purified PKC␣. Furthermore, the addition of purified PKC␣ in the absence of cytosol did not support the aggregation, whereas the cytosol containing less PKC supported it efficiently, suggesting that additional factors besides PKC would also be required. Thus, we directly demonstrated that PKC␣ is involved in the regulation of Ca 2؉ -induced platelet aggregation.Platelets play critical roles in hemostasis and thrombosis through aggregation following activation of integrin ␣ IIb ␤ 3 (1-3). Although ␣ IIb ␤ 3 of platelets in the resting stage does not bind fibrinogen or von Willebrand factors (vWF), 1 the activation of platelets results in conformation changes, which allow ␣ IIb ␤ 3 to bind these ligands (1-3). When fibrinogen or vWF binds to ␣ IIb ␤ 3 , the ligand-occupied integrin signals downstream to stabilize platelet aggregation through reorganization of the actin cytoskeletal network and the release of bioactive substances stored in the granules (1-3). Thus, the process of platelet activation and aggregation consists of a series of orchestrated responses (1-3). However, the molecular mechanisms that underlie this process remain unclear because it is difficult to use molecular biology and biochemistry in platelets that do not synthesize new proteins. To overcome this difficulty, semi-intact assays using permeabilized platelets have been established for the study of granule secretion (4 -8). Although some semi-intact aggregation assays have now been developed, it has been difficult to demonstrate a cytosol dependence in these experiments (8).Protein kinase C (PKC) family members are important signaling molecules regulating many cellular functions (9, 10). Among the family members, conventional PKCs (cPKC), which include PKC␣, ␤I, ␤II, and ␥...

“…Two models of pore formation by the toxic protein have been proposed: (a) Palmer et al (9) proposed an assembly model for streptolysin O, a member of the family of cholesterol-dependent cytolysins, in which the pore formation is initiated by insertion of a small oligomeric complex of streptolysin O into the membrane and then enlarged in a progressive manner by the addition of soluble toxin monomers; (b) Hotze et al (10) proposed a pre-pore model for perfringolysin O in which the formation of oligomeric pre-pore complex precedes its membrane insertion.…”

mentioning

confidence: 99%

Functional Domains of a Pore-forming Cardiotoxic Protein, Volvatoxin A2

Weng

Lin

Hsu

et al. 2004

Volvatoxin A2 (VVA2), a novel pore-forming cardiotoxic protein was isolated from the mushroom Volvariella volvacea. We identified an N-terminal fragment (NTF) (1-127 residues) of VVA2 as a domain for oligomerization by limited tryptic digestion. On preincubation of NTF with VVA2, NTF was found to inhibit VVA2 hemolytic activity by inducing VVA2 oligomerization in the solution in the same manner as liposomes. By site-directed mutagenesis, the amphipathic ␣-helix B of NTF or VVA2 was shown to be indispensable for its biological functions. Interestingly, at a molar ratio of recombinant NTF (reNTF)/VVA2 as low as 0.01, reNTF was able to inhibit VVA2 hemolytic activity and induce VVA2 oligomerization. This indicates that reNTF can trigger VVA2 oligomerization by a seeding effect. Furthermore, the recombinant C-terminal fragment (128 -199 residues) was found to be a functional domain that mediates the membrane binding of VVA2. We found a fragment localized at the C-terminal half of VVA2 containing ␤6, -7, and -8, which is protected from protease digestion because of its insertion of a membrane. We also identified a putative heparin binding site (HBS) located in the VVA2 C terminus (166 -194 residues), which was conserved among 10 kinds of snake venom cardiotoxins. VVA2 or the reHBS fragment was shown to interact with sulfated glycoaminoglycans by affinity column chromatography. The finding of a higher number of glycoaminoglycans in the membrane of cardiac myocytes suggested that they could be the specific membrane target for VVA2. Taken together, these findings indicate that VVA2 contains two functional domains, NTF and CTF. The NTF domain is responsible for VVA2 oligomerization and the CTF domain for membrane binding and insertion. Our results support a model whereby the formation of VVA2 oligomeric pre-pore complexes precedes their membrane insertion.Volvatoxin A (VVA), 1 first isolated from the edible mushroom Volvariella volvacea (1, 2), is a novel pore-forming cardiotoxic protein that causes cardiac arrest by activation of Ca 2ϩ -dependent ATPase activity and inhibition of Ca 2ϩ accumulation in the microsomal fraction of the sarcoplasmic recticulum of the ventricular muscle (3). VVA is composed of VVA1 and VVA2, of which only VVA2 is endowed with hemolytic and cytotoxic activity (1). Our previous studies showed that VVA2 consists of 199 amino acid residues without cysteine residues, and that it forms pores in response to its oligomer formation in human erythrocyte membranes and liposomes. This toxic protein also permeabilized the cell membrane and allowed the passage of 70-kDa dextran rhodamine B into cultured Xenopus myocytes. Furthermore, conformational changes occurred when VVA2 interacted with liposomes (2).Naturally occurring hemolytic toxic proteins can be classified into three groups based on their mechanisms of lysis of red blood cells: (a) pore forming, (b) enzymatic degradation of membrane lipids, and (c) solubilizing cell membrane by a detergentlike action (4). Several hemolytic toxic proteins isolated...