1 Clostridium perfringens beta-toxin causes dermonecrosis and oedema in the dorsal skin of animals. In the present study, we investigated the mechanisms of oedema induced by the toxin. 2 The toxin induced plasma extravasation in the dorsal skin of Balb/c mice. 3 The extravasation was signi®cantly inhibited by diphenhydramine, a histamine 1 receptor antagonist. However, the toxin did not cause the release of histamine from mouse mastocytoma cells. ]-SP and spantide, inhibited the toxin-induced leakage in a dose-dependent manner. Furthermore, the non-peptide tachykinin NK 1 receptor antagonist, SR140333, markedly inhibited the toxin-induced leakage. 5 The leakage induced by the toxin was markedly reduced in capsaicin-pretreated mouse skin but the leakage was not aected by systemic pretreatment with a calcitonin gene-related peptide receptor antagonist ]-bradykinin), but was not aected by the selective L-type Ca 2+ channel blocker, verapamil, the P-type Ca 2+ channel blocker, o-agatoxin IVA, tetrodotoxin (TTX), the TTX-resistant Na + channel blocker, carbamazepine, or the sensory nerve conduction blocker, lignocaine. 7 These results suggest that plasma extravasation induced by beta-toxin in mouse skin is mediated via a mechanism involving tachykinin NK 1 receptors.
Clostridium perfringens ␣-toxin, an important agent of gas gangrene with inflammatory myopathies, possesses lethal, hemolytic, and necrotic activities. Here, we show that ␣-toxininduced lethality in mice was inhibited by i.v. preadministration of erythromycin (ERM
The beta-toxin gene isolated from Clostridium perfringens type B was expressed as a glutathione S-transferase (GST) fusion gene in Escherichia coli. The purified GST-beta-toxin fusion protein from the E. coli transformant cells was not lethal. The N-terminal amino acid sequence of the recombinant beta-toxin (r toxin) isolated by thrombin cleavage of the fusion protein was G-S-N-D-I-G-K-T-T-T. Biological activities and molecular mass of r toxin were indistinguishable from those of native beta-toxin (n toxin) purified from C. perfringens type C. Replacement of Cys-265 with alanine or serine by site-directed mutagenesis resulted in little loss of the activity. Treatment of C265A with N-ethylmaleimide (NEM), which inactivated lethal activity of r toxin and n toxin, led to no loss of the activity. The substitution of tyrosine or histidine for Cys-265 significantly diminished lethal activity. In addition, treatment of C265H with ethoxyformic anhydride which specifically modifies histidyl residue resulted in significant decrease in lethal activity, but that of r toxin with the agent did not. These results showed that replacement of the cysteine residue at position 265 with amino acids with large size of side chain or introduction of functional groups in the position resulted in loss of lethal activity of the toxin. Replacement of Tyr-266, Leu-268 or Trp-275 resulted in complete loss of lethal activity. Simultaneous administration of r toxin and W275A led to a decrease in lethal activity of beta-toxin. These observations suggest that the site essential for the activity is close to the cysteine residue.
Background and purpose: Clostridium perfringens beta-toxin, an important agent of necrotic enteritis, causes plasma extravasation due to the release of a tachykinin NK 1 receptor agonist in mouse skin. In this study, we investigated the role of cytokines in beta-toxin-induced plasma extravasation. Experimental approach: Male Balb/c, C3H/HeN and C3H/HeJ mice were anaesthetized with pentobarbitone and beta-toxin was injected i.d. into shaved dorsal skin. SR140333, capsaicin, chlorpromazine and pentoxifylline were given as pretreatment when required before the injection of the toxin. Cytokines in the dorsal skin were measured by ELISA. Key results: Injection (i.d.) of beta-toxin induced a dose-dependent increase in dermal TNF-a and interleukin (IL)-1b levels with a concomitant increase in plasma extravasation, but not the release of IL-6. SR140333 and capsaicin significantly inhibited the toxin-induced release of TNF-a and IL-1b. The plasma extravasation and the release of TNF-a induced by beta-toxin were significantly inhibited by chlorpromazine and pentoxifylline which inhibit the release of TNF-a. The toxin-induced plasma extravasation in mouse skin was attenuated by pretreatment with a monoclonal antibody against TNF-a, but not anti-IL-1b. Furthermore, the toxin caused an increase in plasma extravasation in both C3H/HeN (TLR4-intact) and C3H/HeJ (TLR4-deficient) mice. In C3H/HeN mice, the toxin-induced leakage was not inhibited by pretreatment with anti-TLR4/MD-2 antibody. Conclusions and implications: These observations show that beta-toxin-induced plasma extravasation in mouse skin is related to the release of TNF-a via the mechanism involving tachykinin NK 1 receptors, but not via TLR4.
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