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Smart hydrogels with versatile properties, including a tunable gelation time, nonswelling attributes, and biocompatibility, are in great need in the biomedical field. To meet this urgent demand, we explored novel biomaterials with the desired properties from sessile marine organisms. To this end, a novel protein, Sbp9, derived from scallop byssus was extensively investigated, which features typical epidermal growth factor-like (EGFL) multiple repetitive motifs. Our current work demonstrated that the key fragment of Sbp9 (calcium-binding domain (CBD) and 4 EGFL repeats (CE4)) was able to form a smart hydrogel driven by noncovalent interactions and facilitated by disulfide bonds. More importantly, this smart hydrogel demonstrates several desirable and beneficial features, which could offset the drawbacks of typical protein-based hydrogels, including (1) a redox-responsive gelation time (from <1 to 60 min); (2) tunable mechanical properties, nonswelling abilities, and an appropriate microstructure; and (3) good biocompatibility and degradability. Furthermore, proof-of-concept demonstrations showed that the newly discovered hydrogel could be used for anticancer drug delivery and cell encapsulation. Taken together, a smart hydrogel inspired by marine sessile organisms with desirable properties was generated and characterized and demonstrated to have extensive applicability potential in biomedical applications, including tissue engineering and drug release.

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., (Copyright © 2019. Published by Elsevier B.V.)

Around the world, Clostridium perfringens type A is known to be a common foodborne pathogen. Therefore, the control and treatment of food poisoning caused by this pathogen are important. This study investigated, in vitro, the effects of Bacillus coagulans and its culture extracts on alpha toxin gene expression, growth inhibition, cytotoxicity, and apoptosis induced by C. perfringens spore, germinated spore and its enterotoxin. Flow cytometry was used to evaluate the apoptosis rate, and MTT test was used to evaluate cytotoxicity. Minimum inhibitory concentration was also used to measure the percentage of inhibition in the broth medium. Finally, RT-qPCR was used to evaluate alpha toxin gene expression. The results showed that the B. coagulans culture extract was able to inhibit the growth of the germinated spore of C. perfringens. Moreover, treating the extract with pepsin can reduce growth in the broth medium. MTT and flow cytometry showed that both B. coagulans and its extract can significantly reduce the cytotoxicity and apoptosis rate induced by C. perfringens type A. In addition, it was shown that the co-culture of B. coagulans and C. perfringens decreases alpha toxin gene expression. The findings of this study indicate that B. coagulans, with growth inhibition and reduced expression of alpha toxin in C. perfringens, can reduce the cytotoxicity and apoptosis rate induced on HT-29 cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Clostridium perfringens type A causes gas gangrene characterized by myonecrosis and development of an effective therapy for treating affected patients is of clinical importance. It was recently reported that the expression of granulocyte colony-stimulating factor (G-CSF) is greatly up-regulated by C. perfringens infection. However, the role of G-CSF in C. perfringens -mediated myonecrosis is still unclear. Here, we assessed the destructive changes in C. perfringens -infected skeletal muscles and tested whether inhibition of G-CSF receptor (G-CSFR) signaling or administration of recombinant G-CSF affects the tissue injury. Severe edema, contraction of muscle fiber diameter, and increased plasma creatine kinase activity were observed in mice intramuscularly injected with C. perfringens type A, and the destructive changes were α-toxin-dependent, indicating that infection induces the destruction of skeletal muscle in an α-toxin-dependent manner. G-CSF plays important roles in the protection of tissue against damage and in the regeneration of injured tissue. However, administration of a neutralizing antibody against G-CSFR had no profound impact on the destructive changes to skeletal muscle. Moreover, administration of recombinant human G-CSF, filgrastim, imparted no inhibitory effect against the destructive changes caused by C. perfringens . Together, these results indicate that G-CSF is not beneficial for treating C. perfringens α-toxin-mediated myonecrosis, but highlight the importance of revealing the mechanism by which C. perfringens negates the protective effects of G-CSF in skeletal muscle.

During bacterial infection, granulocyte colony-stimulating factor (G-CSF) is produced and accelerates neutrophil production from their progenitors. This process, termed granulopoiesis, strengthens host defense, but Clostridium perfringens α-toxin impairs granulopoiesis via an unknown mechanism. Here, we tested whether G-CSF accounts for the α-toxin-mediated impairment of granulopoiesis. We find that α-toxin dramatically accelerates G-CSF production from endothelial cells in response to Toll-like receptor 2 (TLR2) agonists through activation of the c-Jun N-terminal kinase (JNK) signaling pathway. Meanwhile, α-toxin inhibits G-CSF-mediated cell proliferation of Ly-6G + neutrophils by inducing degradation of G-CSF receptor (G-CSFR). During sepsis, administration of α-toxin promotes lethality and tissue injury accompanied by accelerated production of inflammatory cytokines in a TLR4-dependent manner. Together, our results illustrate that α-toxin disturbs G-CSF-mediated granulopoiesis by reducing the expression of G-CSFR on neutrophils while augmenting septic shock due to excess inflammatory cytokine release, which provides a new mechanism to explain how pathogenic bacteria modulate the host immune system., Competing Interests: The authors declare no competing interests.

Whereas granulopoiesis during Gram-negative bacterial infection is accelerated through activation of toll-like receptor 4 (TLR4), it has not been elucidated whether Gram-positive bacterial infection can stimulate granulopoiesis. Using the well-known TLR2 agonist peptidoglycan (PGN), it was shown that neutrophils in bone marrow and spleen and plasma granulocyte colony-stimulating factor were increased in mice that had received intraperitoneal administration of PGN. Incorporation of bromodeoxyuridine into bone marrow neutrophils increased in mice administered PGN, demonstrating that PGN promotes granulopoiesis. These results illustrate that bacterial recognition by TLR2 facilitates granulopoiesis during Gram-positive bacterial infection. Thus, granulopoiesis is accelerated to suppress bacterial infection, but some bacteria can still cause severe infections. Clostridium perfringens is a Gram-positive, anaerobic pathogenic bacterium and causes life-threatening gas gangrene in humans. Of the many toxins produced by C. perfringens, α-toxin is known to be a major virulence factor during infection. Recently, it has been revealed that C. perfringens α-toxin impairs the innate immune system by inhibiting neutrophil differentiation, which is crucial for the pathogenesis of C. perfringens. Moreover, the toxin also attenuates erythropoiesis, which would cause severe anemia in clinical settings. The findings provide new insight to understand how hosts strengthen innate immunity to fight pathogenic bacteria and how they evade the hosts' immune systems.

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.

Alpha-toxin produced by Clostridium perfringens is an important virulence factor, causing food poisoning and gas gangrene in humans. As such, it is considered a potential bioterrorism threat. To date, there is still no human effective therapeutic drug against alpha-toxin. In this study, a human single chain antibody against alpha-toxin was produced from synthetic (Tomlinson I + J) naive phage display libraries, and its preventive and therapeutic efficacy in mice was examined. To prove the neutralizing potential of the scFv, alpha-toxin was preincubated with scFv and subsequently tested for its lecithinase and hemolytic activity, as well as its lethal effect in mice following intravenous administration. The equilibrium association constant between scFv and CPA was 2.02 × 10 10 (1/M), as analyzed by SPR. The scFv could inhibit lecithinase and hemolytic activity, and provided effective protection against alpha-toxin when mice were challenged 1-h post scFv injection. In addition, the survival rate reached 80% for mice treated with scFv within 30 min of being challenged with a 2 × LD50 dose of alpha-toxin. These results confirmed that we successfully prepared a human scFv against C. perfringens type A alpha-toxin, which can be used in the prevention and treatment of alpha-toxin-related illness., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

Background: Bovine necrohemorrhagic enteritis is caused by Clostridium perfringens type A. Due to the rapid progress and fatal outcome of the disease, vaccination would be of high value. In this study, C. perfringens toxins, either as native toxins or after formaldehyde inactivation, were evaluated as possible vaccine antigens. We determined whether antisera raised in calves against these toxins were able to protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis., Results: Alpha toxin and perfringolysin O were identified as the most immunogenic proteins in the vaccine preparations. All vaccines evoked a high antibody response against the causative toxins, alpha toxin and perfringolysin O, as detected by ELISA. All antibodies were able to inhibit the activity of alpha toxin and perfringolysin O in vitro. However, the antibodies raised against the native toxins were more inhibitory to the C. perfringens-induced cytotoxicity (as tested on bovine endothelial cells) and only these antibodies protected against C. perfringens challenge in the intestinal loop model., Conclusion: Although immunization of calves with both native and formaldehyde inactivated toxins resulted in high antibody titers against alpha toxin and perfringolysin O, only antibodies raised against native toxins protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis.

The anorectic neuropeptide nesfatin-1 has recently been characterized as a potential mood regulator, but the accurate effect of nesfatin-1 on anxiety and learning and memory behavior and the possible mechanisms remains unknown. In the present study, to test the hypothesis that nesfatin-1 might affect the anxiety-like and learning and memory behaviors in rats via ERK/CREB/BDNF pathway, nesfatin-1 was administered intraperitoneally to rats with the doses (10, 20, 40μg/kg), and the behavioral performance was tested using the open field task, the Morris water maze (MWM), and the Y maze. Moreover, the protein expression of brain-derived neurotrophic factor (BDNF), total and phosphorylated-ERK in the hippocampus and the prefrontal cortex (PFC) were evaluated. The results showed that chronic administration of nesfatin-1 could decrease the moving distance, the duration in the center, and the frequencies of rearing and grooming in the open field task, decrease the moving distance, frequency, and the preference index of new arm in the Y maze, although there was no significant difference of the performance in the MWM task among groups. Furthermore, 3 weeks' consecutive administration of nesfatin-1 resulted in the decrease of protein expression of BDNF and phosphorylated-ERK in the hippocampus and the PFC. These results provided evidence that exogenous nesfatin-1 could decrease exploration and induce anxiety-like behavior in rats, the mechanism of which might be related to the reduced protein expression of BDNF and phosphorylated-ERK in the hippocampus and the PFC., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Clostridium perfringens alpha-toxin is a key mediator of gas gangrene, which is a life-threatening infection that manifests as fever, pain, edema, myonecrosis, and gas production. Alpha-toxin possesses phospholipase C and sphingomyelinase activities. The toxin is composed of an N-terminal domain (1-250 aa, N-domain), which is the catalytic site, and a C-terminal domain (251-370 aa, C-domain), which is the membrane-binding site. Immunization of mice with the C-domain of alpha-toxin prevents the gas gangrene caused by C. perfringens, whereas immunization with the N-domain has no effect. The central loop domain (55-93 aa), especially H….SW(84)Y(85)….G, plays an important role in the interaction with ganglioside GM1a. The toxin binds to lipid rafts in the presence of a GM1a/TrkA complex, and metabolites from phosphatidylcholine to diacylglycerol through the enzymatic activity of alpha-toxin itself. These membrane dynamics leads to the activation of endogenous PLCγ-1 via TrkA. In addition, treatment with alpha-toxin leads to the formation of diacylglycerol at membrane rafts in ganglioside-deficient DonQ cells; this in turn triggers endocytosis and cell death. This article summarizes the current the membrane-binding mechanism of alpha-toxin in detail.

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.

Calcium-binding proteins are ubiquitous modulators of cellular activity and function. Cells possess numerous calcium-binding proteins that regulate calcium concentration in the cytosol by buffering excess free calcium ion. Disturbances in intracellular calcium homeostasis are at the heart of many age-related conditions making these proteins targets for therapeutic intervention. A calcium-binding protein, apoaequorin, has shown potential utility in a broad spectrum of applications for human health and well-being. Large-scale recombinant production of the protein has been successful; enabling further research and development and commercialization efforts. Previous work reported a 90-day subchronic toxicity test that demonstrated this protein has no toxicity by oral exposure in Sprague-Dawley rodents. The current study assesses the allergenic potential of the purified protein using bioinformatic analysis and simulated gastric digestion. The results from the bioinformatics searches with the apoaequorin sequence show the protein is not a known allergen and not likely to cross-react with known allergens. Apoaequorin is easily digested by pepsin, a characteristic commonly exhibited by many non-allergenic dietary proteins. From these data, there is no added concern of safety due to unusual stability of the protein by ingestion., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, plays a key role in the pathogenesis of gas gangrene. CpPLC may lead to cell lysis at concentrations that cause extensive degradation of plasma membrane phospholipids. However, at sublytic concentrations it induces cytotoxicity without inducing evident membrane damage. The results of this work demonstrate that CpPLC becomes internalized in cells by a dynamin-dependent mechanism and in a time progressive process: first, CpPLC colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. Lysosomal damage in the target cells is evident 9 h after CpPLC exposure. Our previous work demonstrated that CpPLCinduces ERK1/2 activation, which is involved in its cytotoxic effect. In this work we found that cholesterol sequestration, dynamin inhibition, as well as inhibition of actin polymerization, prevent CpPLC internalization and ERK1/2 activation, involving endocytosis in the signalling events required for CpPLC cytotoxic effect at sublytic concentrations. These results provide new insights about the mode of action of this bacterial phospholipase C, previously considered to act only locally on cell membrane., (© 2013 John Wiley & Sons Ltd.)

Clostridium perfringens alpha toxin/phospholipase C (CP-PLC) is one of the most potent bacterial toxins known to cause soft tissue infections like gas gangrene in humans and animals. It is the first bacterial toxin demonstrated to be an enzyme with phospholipase, sphingomyelinase and lecithinase activities. The toxin is comprised of an enzymatic N-domain and a binding C-domain interconnected by a flexible linker. The N-domain alone is non-toxic to mammalian cells, but incubation with C-domain restores the toxicity, the mechanism of which is still not elucidated. The objectives of the current study were to investigate the formation of a stable N and C-domain complex, to determine possible interactions between the two domains in silico and to characterize the in vitro and in vivo correlates of the interaction. To establish the existence of a stable N and C-domain hybrid, in vitro pull down assay and dot-Far Western blotting assays were employed, where it was clearly revealed that the two domains bound to each other to form an intermediate. Using bioinformatics tools like MetaPPISP, PatchDock and FireDock, we predicted that the two domains may interact with each other through electrostatic interactions between at least six pairs of amino acids. This N and C-domains interacted with each other in 1:1 ratio and the hybrid lysed mouse erythrocytes in a slower kinetics when compared with wild type native Cp-PLC. BALB/c mice when challenged with N and C-domain hybrid demonstrated severe myonecrosis at the site of injection while no death was observed. Our results provide further insight into better understanding the mechanism for the toxicity of Cp-PLC N and C-domain mixture.

Bovine necrohemorrhagic enteritis is a major cause of mortality in veal calves. Clostridium perfringens is considered as the causative agent, but there has been controversy on the toxins responsible for the disease. Recently, it has been demonstrated that a variety of C. perfringens type A strains can induce necrohemorrhagic lesions in a calf intestinal loop assay. These results put forward alpha toxin and perfringolysin as potential causative toxins, since both are produced by all C. perfringens type A strains. The importance of perfringolysin in the pathogenesis of bovine necrohemorrhagic enteritis has not been studied before. Therefore, the objective of the current study was to evaluate the role of perfringolysin in the development of necrohemorrhagic enteritis lesions in calves and its synergism with alpha toxin. A perfringolysin-deficient mutant, an alpha toxin-deficient mutant and a perfringolysin alpha toxin double mutant were less able to induce necrosis in a calf intestinal loop assay as compared to the wild-type strain. Only complementation with both toxins could restore the activity to that of the wild-type. In addition, perfringolysin and alpha toxin had a synergistic cytotoxic effect on bovine endothelial cells. This endothelial cell damage potentially explains why capillary hemorrhages are an initial step in the development of bovine necrohemorrhagic enteritis. Taken together, our results show that perfringolysin acts synergistically with alpha toxin in the development of necrohemorrhagic enteritis in a calf intestinal loop model and we hypothesize that both toxins act by targeting the endothelial cells.

Dystrophic neurites surrounding β-amyloid (Aβ) plaques precede neuronal death in Alzheimer disease. These neuritic alterations may be one of the initial stages for synaptic loss and dysfunction. However, intracellular pathways that cause local disruption of neuronal processes by Aβ remain to be fully elucidated. The identification of Aβ-induced genes that mediate neuritic pathology would provide considerable insight into the mechanisms of Alzheimer's disease. Previously, we reported that selective up-regulation of calsyntenin-3 (Cst-3) by Aβ and accumulation of neurotoxic Cst-3 in dystrophic neurites surrounding Aβ plaques may lead to local disruption of these neurites. Like amyloid precursor protein, Cst-3 undergoes two-step proteolytic processing: the primary cleavage with α-secretase generates an N-terminal ectodomain and a C-terminal fragment (CTF). The CTF is subsequently cleaved into p3 peptide and an intracellular domain via γ-secretase. It would be interesting to know whether accumulated Cst-3 in dystrophic neurites surrounding Aβ plaques is the full-length version or a CTF. Herein, we show that the CTF but not full-length Cst-3 accumulated in dystrophic neurites surrounding Aβ plaques in Tg2576 mouse and Alzheimer disease brains. In vitro experiments with Cst-3 fragments have revealed that only the CTF resulted in acceleration of neuronal death. These results indicate that accumulation of the neurotoxic CTF in neurites surrounding Aβ plaques may lead to local disruption of neuronal processes and development of dystrophic neurites., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

Clostridium perfringens, the most broadly distributed pathogen in nature, produces a prototype phospholipase C, also called α-toxin, which plays a key role in the pathogenesis of gas gangrene. α-Toxin causes plasma membrane disruption at high concentrations, but the role of intracellular mediators in its toxicity at low concentrations is unknown. This work demonstrates that α-toxin causes oxidative stress and activates the MEK/ERK pathway in cultured cells and furthermore provides compelling evidence that O(2)(-.), hydrogen peroxide, and the OH(.) radical are involved in its cytotoxic and myotoxic effects. The data show that antioxidants and MEK1 inhibitors reduce the cytotoxic and myotoxic effects of α-toxin and demonstrate that edaravone, a clinically used hydroxyl radical trap, reduces the myonecrosis and the mortality caused by an experimental infection with C. perfringens in a murine model of gas gangrene. This knowledge provides new insights for the development of novel therapies to reduce tissue damage during clostridial myonecrosis.

Clostridium perfringens alpha and epsilon toxins produce enterotoxaemia in sheep and goats. However, the information regarding the pathophysiology of alpha and epsilon toxins in the bovine intestine is still scanty. In this study, intestinal loops were performed in the ileum and colon of three one-week-old Holstein and two four-week-old crossbreed calves. Laparotomy was performed in all calves under anaesthesia and four loops -three cm long- were performed in the small and large intestines. For both intestines, loops were inoculated with alpha or epsilon toxins. Tissue samples from all loops were obtained and processed for routine histology and for transmission electron microscopy. Congestion was observed in toxin treated loops. Fluid accumulation in the gut lumen was prominent in all treated loops, but in epsilon treated ones the mucous was also haemorrhagic. The histology revealed large amount of exfoliated epithelial cells in the lumen of alpha toxin treated loops and severe haemorrhage was observed in the lamina propria of epsilon toxin treated colonic loops. Despite some necrotic exfoliated enterocytes, no ultraestructural changes were observed in alpha toxin treated loops, though with epsilon toxin the loops exhibited dilation of the intercellular space in the mucosa of both, small and large intestines. These observations indicate that both, alpha and epsilon toxins can alter the intestinal barrier, in calves and are pathogenic for this species., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Poultry necrotic enteritis (NE) is caused by specific strains of Clostridium perfringens, most of which are type A. The role of alpha toxin (CPA) in NE has been called into question by the finding that an engineered cpa mutant retains full virulence in vivo[9]. This is in contrast to the finding that immunization with CPA toxoids protects against NE. We confirmed the earlier findings, in that 14-day-old Cornish × Rock broiler chicks challenged with a cpa mutant developed lesions compatible with NE in >90% of birds inoculated with the mutant. However, CPA was detected in amounts ranging from 10 to >100 ng per g of gut contents and mucosa in birds inoculated with the cpa mutant, the wildtype strain from which the mutant was constructed, and our positive control strain. There was a direct relationship between lesion severity and amount of CPA detected (R = 0.89-0.99). These findings suggest that the role of CPA in pathogenesis of NE requires further investigation., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The α-toxin produced by Clostridium perfringens is one of the best-studied examples of a toxic phospholipase C. In this study, a nontoxic mutant protein from C. perfringens strain NCTC8237 in which Thr74 is substituted by isoleucine (T74I) has been characterized and is compared with the toxic wild-type protein. Thr74 is part of an exposed loop at the proposed membrane-interfacing surface of the toxin. The mutant protein had markedly reduced cytotoxic and myotoxic activities. However, this substitution did not significantly affect the catalytic activity towards water-soluble substrate or the overall three-dimensional structure of the protein. The data support the proposed role of the 70-90 loop in the recognition of membrane phospholipids. These findings also provide key evidence in support of the hypothesis that the hydrolysis of both phosphatidylcholine and sphingomyelin are required for the cytolytic and toxic activity of phospholipases.

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.

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.

Clostridium perfringens alpha-toxin induces the hemolysis of sheep erythrocytes by activating the metabolism of sphingomyelin (SM) via a GTP binding protein in membranes. alpha-Toxin stimulated the formation of 15-N-nervonoyl sphingosine (C24:1-ceramide), which was identified by positive ion fast atom bombardment-MS and 1H-NMR spectroscopy. C24:1-ceramide stimulated the toxin-induced hemolysis of saponin-pretreated sheep erythrocytes and increased the production of sphingosine 1-phosphate (S1P) in the cells, but N-lignoceroyl sphingosine did not. These events elicited by the toxin in the presence of C24:1-ceramide were significantly attenuated by treatment with dihydrosphingosine, a sphingosine kinase inhibitor. TLC showed that the level of C24:1-ceramide was highest among the ceramides with an unsaturated bond in the fatty acyl chain in the detergent-resistant membranes (DRMs). The toxin specifically bound to DRMs rich in cholesterol, resulting in the hydrolysis of N-nervonoic sphingomyelin (C24:1-SM) in DRMs. Treatment of the cells with pertussis toxin (PT) inhibited the alpha-toxin-induced formation of C24:1-ceramide from C24:1-SM in DRMs and hemolysis, indicating that endogenous sphingomyelinase, which hydrolyzes C24:1-SM to C24:1-ceramide, is controlled by PT-sensitive GTP binding protein in membranes. These results show that the toxin-induced metabolism of C24:1-SM to S1P in DRMs plays an important role in the toxin-induced hemolysis of sheep erythrocytes.

Epsilon toxin (ETX) is the most important virulence factor of Clostridium perfringens type D. Two other important toxins, alpha toxin (CPA) and perfringolysin-O (PFO), are encoded and potentially produced by most C. perfringens type D isolates. The biological effects of these toxins are dissimilar although they are all lethal. Since the possible interaction of these toxins during infection is unknown, the effects of CPA and PFO on the lethal activity of ETX were studied in a mouse model. Mice were injected intravenously or intragastrically with CPA or PFO with or without ETX. Sublethal doses of CPA or PFO did not affect the lethality of ETX when either was injected together with the latter intravenously. However, sublethal or lethal doses of CPA or PFO resulted in reduction of the survival time of mice injected simultaneously with ETX when compared with the intravenous effect of ETX injected alone. When PFO was inoculated intragastrically with ETX, a reduction of the survival time was observed. CPA did not alter the survival time when inoculated intragastrically with ETX. The results of the present study suggest that both CPA and PFO have the potential to enhance the ETX lethal effects during enterotoxemia in natural hosts such as sheep and goats.

Clostridium perfringens alpha-toxin degrades phosphatidylcholine (PC) in the bilayer of liposomes and destroys the membrane. The effect of the type and position of unsaturation in the fatty acyl chain of PC (18:0/18:1 PC) synthesized on the toxin-induced leakage of carboxyfluorescein (CF) from PC liposomes was examined. Differential scanning calorimetry showed that the phase transition temperature (T(m)) was minimal when the triple bond was positioned at C (9) in the sn-2 acyl chain. The toxin-induced CF leakage decreased with the migration of the bond from C (9) to either end of the acyl chain in PC. The PC containing the cis-double bond had a similar T(m) to that with the triple bond, but a lower value than the PC containing the trans-double bond. Furthermore, the toxin-induced leakage from liposomes composed of PC containing the cis-double bond resembled that with PC having the triple bond and was greater than that from liposomes with PC having the trans-double bond. The binding of a H148G mutant to PC liposomes showed a reciprocal relationship in terms of the T(m) value of PC containing the triple bond. These results indicate that the toxin-induced membrane damage is closely related to membrane fluidity in liposomes.

In vitro toxin production is an important tool not only for diagnostic purposes but also for the study of pathogenesis of Clostridium perfringens infections. The present study was carried out to compare the level of toxin production by several strains of C. perfringens type A, isolated from the intestine of animals, when cultured in 3 different conventional culture media. Six strains of C. perfringens type A isolated from the small intestine of healthy sheep were cultured in commercial cooked meat medium (CMM), brain heart infusion (BHI), and tryptone glucose yeast (TGY). Intravenous lethality in mice and phospholipase C (PLC) activity were measured in filtered culture supernatants. Lethality of culture supernatants was highest for all isolates when grown in BHI, followed by CMM. No supernatants from any isolates grown in TGY produced lethality in mice. Phospholipase C activity was highest when the isolates were grown in BHI and CMM and significantly lower when grown in TGY.

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(-).

Aim: To study the cloning of alpha-beta fusion gene from Clostridium perfringens and the immunogenicity of alpha-beta fusion expression., Methods: Cloning was accomplished after PCR amplification from strains NCTC64609 and C58-1 of the protective antigen genes of alpha-toxin and beta-toxin. The fragment of the gene was cloned using plasmid pZCPAB. This fragment coded for the gene with the stable expression of alpha-beta fusion gene binding. In order to verify the exact location of the alpha-beta fusion gene, domain plasmids were constructed. The two genes were fused into expression vector pBV221. The expressed alpha-beta fusion protein was identified by ELISA, SDS-PAGE, Western blotting and neutralization assay., Results: The protective alpha-toxin gene (cpa906) and the beta-toxin gene (cpb930) were obtained. The recombinant plasmid pZCPAB carrying alpha-beta fusion gene was constructed and transformed into BL21(DE3). The recombinant strain BL21(DE3)(pZCPAB) was obtained. After the recombinant strain BL21(DE3)(pZCPAB) was induced by 42 degC, its expressed product was about 22.14% of total cellular protein at SDS-PAGE and thin-layer gel scanning analysis. Neutralization assay indicated that the antibody induced by immunization with alpha-beta fusion protein could neutralize the toxicity of alpha-toxin and beta-toxin., Conclusion: The obtained alpha-toxin and beta-toxin genes are correct. The recombinant strain BL21(DE3)(pZCPAB) could produce alpha-beta fusion protein. This protein can be used for immunization and is immunogenic. The antibody induced by immunization with alpha-beta fusion protein could neutralize the toxicity of alpha-toxin and beta-toxin.

As shown previously, a recombinant alphatoxin variant (rAT121A/91) constructed from the naturally occurring Clostridium perfringens mutant strain 121A/91, was devoid of enzymatic (PLC), hemolytic and lethal activity (18). In the present study, the recombinant variant was altered by an oligonucleotide-directed reversion of an arginine in position 212 for a histidine residue, corresponding to the sequence of the wild-type alphatoxin. The new variant rAT121A/91R212H proved to be negative in enzymatic, hemolytic and lethal activity as well. RAT121A/91 as well as rAT121A/91R212H was used for i.p. immunization of balb/c mice. The immune response was studied in ELISA as well as in the mouse neutralization test. Furthermore, immunized mice were challenged by i.p. application of active C. perfringens alphatoxin. In all immunized groups, mice developed high anti-alphatoxin titers (up to 1:128000). Antisera of both groups were able to reduce the hemolytic effect of native alphatoxin with predominance of anti-rAT121A/91R212H sera. During neutralization experiments, mice receiving a mixture of anti-rAT121A/91R212H and wild-type toxin were protected completely, whereas an anti-rAT121A/91/toxin mixture prolonged time until death but failed in protection. I.p immunization with rAT121A/91R212H yielded a significant protection rate (76%) when mice were challenged intraperitoneal with wild-type toxin. Our cumulative data indicates that the reversion of arginine in position 212 to histidine for rAT121A/91R212H was necessary to induce production of protective antibodies against wild-type alphatoxin of C. perfringens.

Objective: To evaluate the morphologic and physiologic changes induced by Clostridium perfringens type A (alpha toxin in the ileum and colon of sheep., Sample Population: 16 ligated intestinal loops in 4 Merino lambs and 18 explants of ileum and colon from slaughtered lambs., Procedure: alpha Toxin-induced fluid accumulation was evaluated in ligated ileal and colonic loops of sheep. Tissues were evaluated morphologically by use of gross and histologic examination. Effects of toxin on in vitro intestinal net water transport were tested in modified Ussing chambers., Results: Ovine ileal and colonic loops incubated with C perfringens type A alpha toxin retained more fluid than control loops. Histologically, in the ileum of lambs inoculated with 300 LD50 of alpha toxin/mL, there was a mild to moderate multifocal infiltration of neutrophils in the lamina propria and submucosa. The colonic loops of lambs inoculated with 30 or 300 LD50 of alpha toxin/mL had excessive mucus in the lumen, a moderate amount of neutrophils mixed with mucus in the intestinal lumen, and moderate multifocal infiltration of the lamina propria and submucosa with neutrophils; the blood vessels of these layers were engorged with neutrophils. In vitro measurements of water transport also revealed inhibition of net epithelial water absorption in ileum and colon incubated with alpha toxin on the mucosal side., Conclusions and Clinical Relevance: These results indicate that alpha toxin induces alterations in sheep intestine. Clostridium perfringens type A organisms that produce alpha toxin could be responsible for diseases of intestinal origin in some ruminants.

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.

Clostridium perfringens alpha-toxin is able to lyse various erythrocytes. Exposure of horse erythrocytes to alpha-toxin simultaneously induced hot-cold hemolysis and stimulated production of diacylglycerol and phosphorylcholine. When A23187-treated erythrocytes were treated with the toxin, these events were dependent on the concentration of extracellular Ca2+ . Incubation with the toxin of BAPTA-AM-treated horse erythrocytes caused no hemolysis or production of phosphorylcholine, but that of the BAPTA-treated erythrocytes did. When Quin 2-AM-treated erythrocytes were incubated with the toxin in the presence of 45Ca2+, the cells accumulated 45Ca2+ in a dose- and a time-dependent manner. These results suggest that the toxin-induced hemolysis and hydrolysis of phosphatidylcholine are closely related to the presence of Ca2+ in the cells. Flunarizine, a T-type Ca2+ channel blocker, and tetrandrine, an L- and T-type Ca2+ channel blocker, inhibited the toxin-induced hemolysis and Ca2+ uptake. However, L-type Ca2+ channel blockers, nifedipine, verpamil and diltiazem, an N-type blocker, omega-conotoxin SVIB, P-type blockers, omega-agatoxin TK and omega-agatoxin IVA, and a Q-type blocker, omega-conotoxin MVII C, had no such inhibitory effect. The observation suggests that Ca2+ taken up through T-type Ca2+ channels activated by the toxin plays an important role in hemolysis induced by the toxin.

Several angiogenesis inhibitors are fragments of larger proteins that are themselves not active as angiogenesis inhibitors. Vasostatin, the N-terminal domain of calreticulin inclusive of amino acids 1-180, is an angiogenesis inhibitor that exerts antitumor effects in vivo. In the present study, we examined whether the full-length calreticulin molecule shares the antiangiogenic and antitumor activities of vasostatin. Similar to vasostatin, calreticulin selectively inhibited endothelial cell proliferation in vitro, but not cells of other lineages, and suppressed angiogenesis in vivo. When inoculated into athymic mice, calreticulin inhibited Burkitt tumor growth comparably with vasostatin. Calreticulin lacking the N-terminal 1-120 amino acids inhibited endothelial cell proliferation in vitro and Burkitt tumor growth in vivo comparably with vasostatin. An internal calreticulin fragment encompassing amino acids 120-180 also inhibited endothelial cell proliferation in vitro and angiogenesis in vivo comparably with calreticulin and vasostatin. These results suggest that the antiangiogenic activities of vasostatin reside in a domain that is accessible from the full-length calreticulin molecule and localize to calreticulin N-terminal amino acids 120-180. Thus, calreticulin and calreticulin fragments are inhibitors of angiogenesis that directly target endothelial cells, inhibit angiogenesis, and suppress tumor growth. This information may be critical in designing targeted inhibitors of pathological angiogenesis that underlies cancer and other diseases.

Purpose: Recoverin, a calcium-binding protein that selectively localizes to the retina and pineal gland, has been identified as the target for the putative pathogenic autoimmune process of cancer-associated retinopathy (CAR). The present study was aimed at testing the capacity of recoverin to induce experimental autoimmune uveoretinitis and pinealitis in Lewis rats., Methods: Lewis rats were immunized against recombinant myristoylated recoverin by a single footpad injection of the protein, at various doses, emulsified in complete Freund's adjuvant. Development of uveoretinitis was monitored by clinical and histologic examinations, whereas pinealitis was detected by histologic examination., Results: Immunization with recoverin induced severe panuveitic changes that closely resemble those induced by S-antigen (arrestin). The effect was dose-dependent, with 10 micrograms/rat the lowest immunopathogenic dose. Rats immunized with recoverin also developed pineal inflammation., Conclusion: Recoverin is highly immunopathogenic in Lewis rats. Although the ocular changes induced in rats differ from those seen in CAR, the data recorded here are in line with the concept that recoverin can initiate pathogenic autoimmune processes in the eye.

To study the interaction between calcium and the medullary hypoxic lesions found in isolated perfused rat kidneys, the acute effects of high extracellular calcium upon renal function and morphology were evaluated in kidneys perfused with cell-free medium at a total calcium concentration of 8 to 9 mg/dl (controls), 13 to 14 and 19 to 20 mg/dl (high Ca++). High Ca++ increased hypoxic damage to medullary thick ascending limbs from 58.2 +/- 4.0% of tubules in controls to 80.2 +/- 4.0% (P less than 0.005) in the deepest area of the outer medulla. Morphological changes in the cortex were minimal. The increase in damage to medullary thick limbs induced by high Ca++ was prevented by the calcium channel blocker verapamil. Addition of the calcium ionophore A23187 to controls reproduced the effects of high Ca++ with an increase in the proportion of damaged thick limbs to 92.1 +/- 4.1% (P less than 0.001 vs. controls). Addition of equimolar amounts of magnesium chloride did not reproduce the effect of high calcium perfusions. When transport activity was reduced with ouabain, high calcium perfusions were no longer associated with structural damage. In kidneys perfused with a medium enriched with amino acids, the proportion of tubules with severe, irreversible damage increased from 12 +/- 3 to 43 +/- 10% (P less than 0.01) after high calcium perfusion, and to 75 +/- 12% (P less than 0.001) after perfusion with the calcium ionophore. High extracellular and intracellular calcium appear to act in concert with hypoxia to increase the susceptibility of the renal medulla to injury by mechanisms potentially operative in hypercalcemic and ischemic nephropathy.(ABSTRACT TRUNCATED AT 250 WORDS)