In silico design and in vitro analysis of a recombinant trivalent fusion protein candidate vaccine targeting virulence factor of Clostridium perfringens.

Necrotic enteritis (NE) is a multifactorial disease in broiler that is caused by colonization of Clostridium perfringens in their gastrointestinal tract. Recently several immunogenic proteins from virulent C. perfringens have been considered as vaccines to provide protection against NE. In this study, a novel trivalent fusion protein including immunogenic epitopes of three virulence factors of, NetB, alpha toxin and a metallopeptidase protein (NAM) was designed using in silico studies. Circular dichroism spectra was applied for determination of secondary structure and folding properties of the purified recombinant NAM (rNAM) expressed in E. coli. The antigenicity of rNAM was confirmed by induction of immune response in rabbit and neutralization experiments of the toxins in cell culture studies. To this end, anti-rNAM antisera neutralized the crude toxins produced by a wild type virulent C. perfringens strain using chicken hepatocellular carcinoma (LMH) cell lines. The cells were exposed to a mixture of anti-rNAM antisera and 2 × LD50 doses of the toxins. The result showed 94% viability of the cells against the crude toxins, in the presence of anti-rNAM antisera. Our study suggests that combination of metallopeptidase protein along with alpha toxin and NetB toxins is a potent immunogen which is able to neutralize the toxicity of crude extracellular toxins. The recombinant chimeric NAM could be a suitable and effective subunit vaccine candidate to prevent NE disease caused by C. perfringens.

Clostridium perfringens α-toxin impairs erythropoiesis by inhibition of erythroid differentiation.

Clostridium perfringens α-toxin induces hemolysis of erythrocytes from various species, but it has not been elucidated whether the toxin affects erythropoiesis. In this study, we treated bone marrow cells (BMCs) from mice with purified α-toxin and found that TER119+ erythroblasts were greatly decreased by the treatment. A variant α-toxin defective in enzymatic activities, phospholipase C and sphingomyelinase, had no effect on the population of erythroblasts, demonstrating that the decrease in erythroblasts was dependent of its enzymatic activities. α-Toxin reduced the CD71+TER119+ and CD71-TER119+ cell populations but not the CD71+TER119- cell population. In addition, α-toxin decreased the number of colony-forming unit erythroid colonies but not burst-forming unit erythroid colonies, indicating that α-toxin preferentially reduced mature erythroid cells compared with immature cells. α-Toxin slightly increased annexinV+ cells in TER119+ cells. Additionally, simultaneous treatment of BMCs with α-toxin and erythropoietin greatly attenuated the reduction of TER119+ erythroblasts by α-toxin. Furthermore, hemin-induced differentiation of human K562 erythroleukemia cells was impaired by α-toxin, whereas the treatment exhibited no apparent cytotoxicity. These results suggested that α-toxin mainly inhibited erythroid differentiation. Together, our results provide new insights into the biological activities of α-toxin, which might be important to understand the pathogenesis of C. perfringens infection.

Lipidomic profile of GM95 cell death induced by Clostridium perfringens alpha-toxin.

Clostridium perfringens alpha-toxin (ATX) is considered as a prototype of cytotoxic bacterial phospholipases C, and is the major virulence factor in C. perfringens-induced gas gangrene. It is known that, depending on the dose, ATX causes membrane disruption and cytolysis or only limited hydrolysis of its substrates. In the latter case, toxin activity leads to the unregulated generation of bioactive lipids that can ultimately induce cell death. We have characterized apoptosis and necrosis in highly ATX-sensitive, ganglioside-deficient cells exposed to different concentrations of ATX and we have studied the lipidomic profile of cells treated with ATX as compared to native cells to detect the main changes in the lipidomic profile and the possible involvement of lipid signals in cell death. ATX causes both apoptosis and necrosis, depending on dose and time. ATX activates cell death, stimulating the release of cytochrome C from mitochondria and the consequent activation of caspases-3. Moreover GM95 cells treated with ATX showed important lipidomic alterations, among them we detected a general decrease in several phospholipid species and important changes in lipids involved in programmed cell death e.g. ceramide. The data suggest two different mechanisms of cell death caused by ATX, one leading to (mainly saturated) glycerophospholipid hydrolysis related to an increase in diacylglycerols and associated to membrane damage and necrosis, and a second mechanism involving chiefly sphingomyelin hydrolysis and generation of proapoptotic lipidic mediators such as ceramide, N-acylethanolamine and saturated non-esterified fatty acids.

Clostridium perfringens α-Toxin Impairs Innate Immunity via Inhibition of Neutrophil Differentiation.

Although granulopoiesis is accelerated to suppress bacteria during infection, some bacteria can still cause life-threatening infections, but the mechanism behind this remains unclear. In this study, we found that mature neutrophils in bone marrow cells (BMCs) were decreased in C. perfringens-infected mice and also after injection of virulence factor α-toxin. C. perfringens infection interfered with the replenishment of mature neutrophils in the peripheral circulation and the accumulation of neutrophils at C. perfringens-infected sites in an α-toxin-dependent manner. Measurements of bacterial colony-forming units in C. perfringens-infected muscle revealed that α-toxin inhibited a reduction in the load of C. perfringens. In vitro treatment of isolated BMCs with α-toxin (phospholipase C) revealed that α-toxin directly decreased mature neutrophils. α-Toxin did not influence the viability of isolated mature neutrophils, while simultaneous treatment of BMCs with granulocyte colony-stimulating factor attenuated the reduction of mature neutrophils by α-toxin. Together, our results illustrate that impairment of the innate immune system by the inhibition of neutrophil differentiation is crucial for the pathogenesis of C. perfringens to promote disease to a life-threatening infection, which provides new insight to understand how pathogenic bacteria evade the host immune system.

Inflammatory responses to a Clostridium perfringens type A strain and α-toxin in primary intestinal epithelial cells of chicken embryos.

The causative pathogen of necrotic enteritis is the Gram-positive bacterium Clostridium perfringens. Its main cell wall component, peptidoglycan (PGN), can be recognized by Toll-like receptor 2 and nucleotide-binding oligomerization domain (NOD). Consequently, the immune response is initiated via activation of nuclear factor kappa B (NF-κB) signalling pathway. An in vitro study was conducted to investigate chicken intestinal inflammatory responses to C. perfringens type A and one of its virulence factors, α-toxin. In primary intestinal epithelial cells, C. perfringens as well as commercially available PGN and α-toxin challenge upregulated mRNA expression of interleukin (IL)-6, IL-8 and inducible nitric oxide synthase (iNOS) with a dosage-dependent manner at 3 h post infection (p.i.; P ≤ 0.001). Time-course effects of three stimulators at high concentration were further examined. C. perfringens infection elevated IL-6, IL-8 and iNOS levels from 1 h to 9 h p.i., while PGN treatment increased IL-6 and IL-8 expression at 1 h and 3 h p.i. (P < 0.05). Bacterial and PGN treatments induced NOD1 expression at 6 h p.i. and only bacterial infection boosted NF-κB p65 expression at 6 h and 9 h p.i. (P < 0.05). α-Toxin treatment upregulated IL-6 and IL-8 expression throughout infection, as well as iNOS, TNF-α and NF-κB p65 expression at later hours p.i. (P < 0.05). In conclusion, both C. perfringens and α-toxin challenge induced intense cytokine expression associated with NF-κB activation in chicken intestinal epithelial cells. The receptors for the recognition of PGN component of C. perfringens need further investigation.

Cross-complementation of Clostridium perfringens PLC and Clostridium septicum alpha-toxin mutants reveals PLC is sufficient to mediate gas gangrene.

Clostridium perfringens and Clostridium septicum are the most common causes of clostridial myonecrosis or gas gangrene. Although they mediate a similar disease pathology, they elaborate functionally very different alpha-toxins. We used a reciprocal complementation approach to assess the contribution of the primary toxin of each species to disease and found that C. perfringens alpha-toxin (PLC) was able to mediate the gross pathology of myonecrosis even in a C. septicum background, although it could not induce vascular leukostasis. Conversely, while C. septicum alpha-toxin restored some virulence to a C. perfringens plc mutant, it was less active than in its native background.

In vitro effects of alpha toxin from Clostridium perfringens on the electrophysiological parameters of jejunal tissues from laying hens preincubated with inulin and N-acetyl-L-cysteine.

The present report demonstrates the effect of alpha toxin from Clostridium perfringens on electrophysiological indexes of jejunal mucosa from laying hens pretreated with inulin and N-acetyl-l-cysteine (ACC), a mucolytic agent. In a first set of experiments, the effect of alpha toxin with or without pretreatment with ACC on the electrophysiological parameters was determined when jejunal tissues from laying hens were mounted in Ussing chambers. The short-circuit current remained unchanged when alpha toxin was added mucosally in the tissues whether pretreated with ACC or not. The change in the transmural tissue conductance (DeltaGt) was higher (P = 0.18) after 90 min exposure of toxin independent of pretreament with ACC. The effect of alpha toxin on DeltaGt became significant (P < or = 0.05) after 120 min of incubation. In the second set of experiments, the effect of alpha toxin on the jejunal tissues preincubated with inulin (0.1%) was investigated. The effect of toxin was also time dependent, and DeltaGt became significantly higher (P < or = 0.05) after 120 min of incubation independent of preinubation with inulin. Inulin did not influence the DeltaGt during the experimental period when compared with control tissues. In conclusion, alpha toxin from C. perfringens can impair the intestinal mucosal barrier. The effect is obviously not dependent on the presence of a mucolytic agent nor can it be affected by direct addition of inulin under in vitro conditions. Whether there is an effect of inulin after long-term supplementation in feeding trials or it is due to fermentation bacterial metabolites remains an open question.

Signal transduction mechanism involved in Clostridium perfringens alpha-toxin-induced superoxide anion generation in rabbit neutrophils.

Clostridium perfringens alpha-toxin induces the generation of superoxide anion (O2(-)) via production of 1,2-diacylglycerol (DG) in rabbit neutrophils. The mechanism of the generation, however, remains poorly understood. Here we report a novel mechanism for the toxin-induced production of O2(-) in rabbit neutrophils. Treatment of the cells with the toxin resulted in tyrosine phosphorylation of a protein of about 140 kDa. The protein reacted with anti-TrkA (nerve growth factor high-affinity receptor) antibody and bound nerve growth factor. Anti-TrkA antibody inhibited the production of O2(-) and binding of the toxin to the protein. The toxin induced phosphorylation of 3-phosphoinositide-dependent protein kinase 1 (PDK1). K252a, an inhibitor of TrkA receptor, and LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), reduced the toxin-induced production of O2(-) and phosphorylation of PDK1, but not the formation of DG. These inhibitors inhibited the toxin-induced phosphorylation of protein kinase C theta (PKCtheta). U73122, a phospholipase C (PLC) inhibitor, and pertussis toxin inhibited the toxin-induced generation of O2(-) and formation of DG, but not the phosphorylation of PDK1. These observations show that the toxin independently induces production of DG through activation of endogenous PLC and phosphorylation of PDK1 via the TrkA receptor signaling pathway and that these events synergistically activate PKCtheta in stimulating an increase in O2(-). In addition, we show the participation of mitogen-activated protein kinase-associated signaling events via activation of PKCtheta in the toxin-induced generation of O2(-).

Multiple virulence factors are required for Staphylococcus aureus-induced apoptosis in endothelial cells.

Staphylococcus aureus infections can result in sepsis and septic shock associated with vascular damage and multiple organ failure. Apoptosis appears to play a key role during sepsis, and the ability of S. aureus to induce apoptosis in endothelial cells might contribute to metastatic infection. In contrast to leukocytes, in human umbilical vein endothelial cells and two endothelial cell lines neither purified alpha-toxin nor staphylococcal supernatants were sufficient to induce apoptosis. Apoptosis induction instead required staphylococcal invasion as well as signals from metabolically active intracellular staphylococci. Only strongly haemolytic and invasive staphylococci, but not non-invasive strains induced apoptosis that was caspase-dependent but Fas-independent. However, only a subgroup of clinical isolates with an invasive and haemolytic phenotype induced apoptosis. Expression of alpha-toxin in a non-haemolytic strain partially restored apoptosis induction, suggesting a role of alpha-toxin as a trigger of apoptosis. Furthermore, infection of endothelial cells with isogenic mutants of various regulator genes revealed that apoptosis induction was dependent on the global regulator agr and the alternative sigma factor sigB, but not influenced by sarA. Together, our results indicate that the ability of S. aureus to induce apoptosis in endothelial cells is determined by multiple virulence factors.

Effects of Clostridium perfringens alpha-toxin (PLC) and perfringolysin O (PFO) on cytotoxicity to macrophages, on escape from the phagosomes of macrophages, and on persistence of C. perfringens in host tissues.

Clostridium perfringens is the most common cause of clostridial myonecrosis (gas gangrene). Polymorphonuclear cells (PMNs) appear to play only a minor role in preventing the onset of myonecrosis in a mouse animal model of the disease (unpublished results). However, the importance of macrophages in the host defense against C. perfringens infections is still unknown. Two membrane-active toxins produced by the anaerobic C. perfringens, alpha-toxin (PLC) and perfringolysin O (PFO), are thought to be important in the pathogenesis of gas gangrene and the lack of phagocytic cells at the site of infection. Therefore, C. perfringens mutants lacking PFO and PLC were examined for their relative cytotoxic effects on macrophages, their ability to escape the phagosome of macrophages, and their persistence in mouse tissues. C. perfringens survival in the presence of mouse peritoneal macrophages was dependent on both PFO and PLC. PFO was shown to be the primary mediator of C. perfringens-dependent cytotoxicity to macrophages. Escape of C. perfringens cells from phagosomes of macrophage-like J774-33 cells and mouse peritoneal macrophages was mediated by either PFO or PLC, although PFO seemed to play a more important role in escape from the phagosome in peritoneal macrophages. At lethal doses (10(9)) of bacteria only PLC was necessary for the onset of myonecrosis, while at sublethal doses (10(6)) both PFO and PLC were necessary for survival of C. perfringens in mouse muscle tissue. These results suggest PFO-mediated cytotoxicity toward macrophages and the ability to escape macrophage phagosomes may be important factors in the ability of C. perfringens to survive in host tissues when bacterial numbers are low relative to those of phagocytic cells, e.g., early in an infection.

Sensitivity of polarized epithelial cells to the pore-forming toxin aerolysin.

Aerolysin is one of the major virulence factors produced by Aeromonas hydrophila, a human pathogen that produces deep wound infection and gastroenteritis. The toxin interacts with target mammalian cells by binding to the glycan core of glycosylphosphatidyl inositol (GPI)-anchored proteins and subsequently forms a pore in the plasma membrane. Since epithelial cells of the intestine are the primary targets of aerolysin, we investigated its effect on three types of polarized epithelial cells: Caco-2 cells, derived from human intestine; MDCK cells, a well-characterized cell line in terms of protein targeting; and FRT cells, an unusual cell line in that it targets its GPI-anchored proteins to the basolateral plasma membrane in contrast to other epithelial cells, which target them almost exclusively to the apical surface. Surprisingly, we found that all three cell types were sensitive to the toxin from both the apical and the basolateral sides. Apical sensitivity was always higher, even for FRT cells. In contrast, FRT cells were more sensitive from the basolateral than from the apical side to the related toxin Clostridium septicum alpha-toxin, which also binds to GPI-anchored proteins but lacks the lectin binding domain found in aerolysin. These observations are consistent with the notion that a shuttling mechanism involving low-affinity interactions with surface sugars allows aerolysin to gradually move toward the membrane surface, where it can finally encounter the glycan cores of GPI-anchored proteins.

Role of Clostridium perfringens phospholipase C in the pathogenesis of gas gangrene.

Gas gangrene is an acute and devastating infection most frequently caused by Clostridium perfringens and characterized by severe myonecrosis, intravascular leukocyte accumulation, and significant thrombosis. Several lines of evidence indicate that C. perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in this disease. This toxin is a Zn2+ metalloenzyme with lecithinase and sphingomyelinase activities. Its three dimensional structure shows two domains, an N-terminal domain which contains the active site, and a C-terminal domain required for the Ca2+dependent interaction with membranes. Cp-PLC displays several biological activities: it increases capillary permeability, induces platelet aggregation, hemolysis, myonecrosis, decreases cardiac contractility, and is lethal. Experiments with genetically engineered Cp-PLC variants have revealed that the sphingomyelinase activity and the C-terminal domain are required for toxicity. The myotoxicity of Cp-PLC is largely dependent on its membrane damaging effect. In addition, it has been suggested that the alterations in the blood flow induced by this toxin also contribute to muscle damage. In gas gangrene, Cp-PLC dysregulates transduction pathways in endothelial cells, platelets and neutrophils leading to the uncontrolled production of several intercellular mediators and adhesion molecules. Thus, Cp-PLC alters the traffic of neutrophils to the infected tissue and promotes thrombotic events, enhancing the conditions for anaerobic growth.

Synergistic effects of alpha-toxin and perfringolysin O in Clostridium perfringens-mediated gas gangrene.

To examine the synergistic effects of alpha-toxin and perfringolysin O in clostridial myonecrosis, homologous recombination was used to construct an alpha-toxin deficient derivative of a perfringolysin O mutant of Clostridium perfringens. The subsequent strain was complemented with separate plasmids that carried the alpha-toxin structural gene (plc), the perfringolysin O gene (pfoA), or both toxin genes, and the resultant isogenic strains were examined in a mouse myonecrosis model. Synergistic effects were clearly observed in these experiments. Infection with the control strain, which did not produce either toxin, resulted in very minimal gross pathological changes, whereas the isogenic strain that was reconstituted for both toxins produced a pathology that was clearly more severe than when alpha-toxin alone was reconstituted. These changes were most apparent in the rapid spread of the disease, the gross pathology of the footpad and in the rate at which the mice had to be euthanatized for ethical reasons. Elimination of both alpha-toxin and perfringolysin O production removed most of the histopathological features typical of clostridial myonecrosis. These effects were restored when the mutant was complemented with the alpha-toxin structural gene, but reconstituting only perfringolysin O activity produced vastly different results, with regions of coagulative necrosis, apparently enhanced by vascular disruption, being observed. Reconstitution of both alpha-toxin and perfringolysin O activity produced histopathology most similar to that observed with the alpha-toxin reconstituted strain. The spreading of myonecrosis was very rapid in these tissues, and coagulative necrosis appeared to be restricted to the lumen of the blood vessels. The results of these virulence experiments clearly support the hypothesis that alpha-toxin and perfringolysin O have a synergistic effect in the pathology of gas gangrene.

The pathogenesis of clostridial myonecrosis.

These pieces of evidence can be assimilated into a molecular and cellular model of pathogenesis which is initiated by direct toxin effects upon venous capillary endothelial cell function, leading to expression of pro-inflammatory mediators and adhesion molecules, and initiation of platelet aggregation. Toxin-induced hyperadhesion of leukocytes (see above section) with enhanced respiratory burst activity (due to toxins directly or to toxin-induced IL-8 or PAF synthesis by host cells) and toxin-induced chemotaxis deficits could result in neutrophil-mediated vascular injury. Direct toxin-induced cytopathic effects on EC may also contribute to vascular abnormalities associated with gas gangrene. Over prolonged incubation periods, PLC at sublytic concentrations causes EC to undergo profound shape changes similar to those described following prolonged TNF or interferon gamma exposure. In vivo, conversion of EC to this fibroblastoid morphology could contribute to the localized vascular leakage and massive swelling observed clinically with this infection. Similarly, the direct cytotoxicity of PFO could disrupt endothelial integrity and contribute to progressive edema both locally and systemically. Thus, via the mechanisms outlined above, both PLC and PFO may cause local, regional and systemic vascular dysfunction. For instance, local absorption of exotoxins within the capillary beds could affect the physiological function of the endothelium lining the postcapillary venules, resulting in impairment of phagocyte delivery at the site of infection. Toxin-induced endothelial dysfunction and microvascular injury could also cause loss of albumin, electrolytes, and water into the interstitial space resulting in marked localized edema. These events, combined with intravascular platelet aggregation and leukostasis, would increase venous pressures and favor further loss of fluid and protein in the distal capillary bed. Ultimately, a reduced arteriolar flow would impair oxygen delivery thereby attenuating phagocyte oxidative killing and facilitating anaerobic glycolysis of muscle tissue. The resultant drop in tissue pH, together with reduced oxygen tension, might further decrease the redox potential of viable tissues to a point suitable for growth of this anaerobic bacillus. As infection progresses and additional toxin is absorbed, larger venous channels would become affected, causing regional vascular compromise, increased compartment pressures and rapid anoxic necrosis of large muscle groups. When toxins reach arterial circulation, systemic shock and multiorgan failure rapidly ensue, and death is common.

Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangrene.

Clostridium perfringens phospholipase C (PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. The toxic activities of genetically engineered alpha-toxin variants harboring single amino-acid substitutions in three loops of its C-terminal domain were studied. The substitutions were made in aspartic acid residues which bind calcium, and tyrosine residues of the putative membrane-interacting region. The variants D269N and D336N had less than 20% of the hemolytic activity and displayed a cytotoxic potency 103-fold lower than that of the wild-type toxin. The variants in which Tyr275, Tyr307, and Tyr331 were substituted by Asn, Phe, or Leu had 11-73% of the hemolytic activity and exhibited a cytotoxic potency 102- to 105-fold lower than that of the wild-type toxin. The results demonstrated that the sphingomyelinase activity and the C-terminal domain are required for myotoxicity in vivo and that the variants D269N, D336N, Y275N, Y307F, and Y331L had less than 12% of the myotoxic activity displayed by the wild-type toxin. This work therefore identifies residues critical for the toxic activities of C. perfringens PLC and provides new insights toward understanding the mechanism of action of this toxin at a molecular level.

[3H]-noradrenaline secretion from rat cortex synaptosomes perforated with Staphylococcus aureus alpha-toxin.

Rat cortex synaptosomes have been successfully perforated with high concentrations (> or = 400 U/ml) of Staphylococcus aureus alpha-toxin. The free Ca2+-concentration dependence of [3H]-noradrenaline release was similar to that observed for PC 12 and chromaffin cells. Release from the alpha-toxin perforated synaptosomes was not significantly inhibited by omega-conotoxin GVIA. Initially, Ca2+-dependent release was independent of MgATP (for 0.5 min), but became increasingly dependent on MgATP with time. Lactate dehydrogenase efflux from alpha-toxin-perforated synaptosomes was not different than efflux from control synaptosomes, and an antibody to N-ethylmaleimide-sensitive fusion protein did not enter the synaptosomes. [3H]-noradrenaline release was temperature and alpha-toxin-concentration dependent. Ca2-dependent release was more resistant to rundown from alpha-toxin- than from streptolysin-O-perforated synaptosomes. This preparation of perforated synaptosomes should be useful for studies of regulated exocytosis from nerve endings.

Neutralization of hemolytic and mouse lethal activities of C. perfringens alpha-toxin need simultaneous blockade of two epitopes by monoclonal antibodies.

Three murine monoclonal antibodies (MAbs 3B4, 1E8, 1F9) were produced by fusion of X63-Ag8.653 myeloma cells and splenocytes of mice immunized with glutaraldehyde-inactivated alpha-toxin of Clostridium perfringens. All MAbs belonged to the immunoglobulin G (IgG) class and possessed a kappa light chain. All the MAbs were specific for alpha-toxin of C. perfringens as demonstrated by immunoblotting experiments performed with culture supernatants of C. perfringens, C. bifermentans, C. sordellii, and Bacillus cereus. Competition analysis in an ELISA revealed that the MAbs recognized different epitopes on the alpha-toxin molecule. In an immunoblot assay based on a recombinant protein expressed in Escherichia coli, the binding site of MAb 1E8 but not those of MAbs 3B4 and 1 F9 were mapped to the COOH-terminal fragment of alpha-toxin (aa 248-370). To prove the neutralizing potential of the MAbs, alpha-toxin was preincubated with MAbs and subsequently tested for its lecithinase activity in an egg yolk diffusion turbidity (EYDT) assay, its hemolytic activity in a hemolysis test, and its lethal effect on mice after intraperitoneally administration. When the MAbs were tested individually, neutralization was only seen in the EYDT assay, where MAb 3B4 completely abolished the lecithinase activity of alpha toxin. However, when MAbs 3B4 and 1 E8 were used in combination, they acted synergistically and inhibited the lysis of rabbit erythrocytes in vitro. The same mixture of MAbs was also able to completely neutralize the lethal effect of three LD50 of alpha-toxin on Balb/c mice. Our results suggest that the alpha-toxin molecule contains several domains which are differently involved in the various activities of the toxin. We conclude that the hemolytic domain(s) of alpha-toxin is (are) identical with or very closely located to the domain(s) that cause the mouse lethal effect. The lecithinase activity may be involved in the mechanisms of hemolysis and mouse lethality but appears not to be the only determinant.

cGMP-kinase mediates cGMP- and cAMP-induced Ca2+ desensitization of skinned rat artery.

(Rp)-8-Bromo-guanosine 3',5'-cyclic monophosphorothioate (Rp-8-Br-cGMPS) inhibited competitively both isozymes of type I alpha and I beta cGMP-dependent protein kinase (cGMP-kinase) purified from porcine aorta with apparent Ki values (microM) of 3.7 for I alpha and 1.8 for I beta. The compound also inhibited bovine heart type II cAMP-dependent protein kinase (cAMP-kinase), but with a Ki of 25 microM. Thus, it is a selective inhibitor of cGMP-kinase. In alpha-toxin-skinned smooth muscle preparations from rat mesenteric artery, 8-Br-cGMP (10(-7) M) and 8-Br-cAMP (10(-6) M) produced a rightward shift of the concentration-contraction curves for Ca2+, denoting a decrease in Ca2+ sensitivity of the contractile elements. The shift by 8-Br-cAMP as well as by 8-Br-cGMP was completely reversed by Rp-8-Br-cGMPS, while a selective inhibitor of activation of cAMP-kinase, (Rp)-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPS), was without effects on the shift produced by these two compounds. These findings indicate the pivotal role that the activation of cGMP-kinase plays in the production of a decrease in Ca2+ sensitivity of contractile elements.