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The methanol extract of the leaves of Illicium lanceolatum, indigenous to Fujian Province, People’s Republic of China, was found to exhibit antimicrobial activity against the periodontal pathogen Porphyromonas gingivalis, and a bioassay-guided fractionation led to the isolation of two new compounds, 1and 2, along with two known santalane-type sesquiterpenoids, 3and 4. The structures of lanceolactone A (1) and lanceolactone B (2) were elucidated by analyzing their 2D NMR spectroscopic data. Compounds 1and 2were assigned as new tetranorsesquiterpenoids with a spiroacetal ring and tricyclic structure, respectively. Compound 3(α-santal-11-en-10-one) showed potent antimicrobial activity against the oral pathogen P. gingivalis.

ABSTRACTClostridium perfringensbeta-toxin is an important agent of necrotic enteritis and enterotoxemia. Beta-toxin is a pore-forming toxin (PFT) that causes cytotoxicity. Two mitogen-activated protein kinase (MAPK) pathways (p38 and c-Jun N-terminal kinase [JNK]-like) provide cellular defense against various stresses. To investigate the role of the MAPK pathways in the toxic effect of beta-toxin, we examined cytotoxicity in five cell lines. Beta-toxin induced cytotoxicity in cells in the following order: THP-1 = U937 > HL-60 > BALL-1 = MOLT-4. In THP-1 cells, beta-toxin formed oligomers on lipid rafts in membranes and induced the efflux of K+from THP-1 cells in a dose- and time-dependent manner. The phosphorylation of p38 MAPK and JNK occurred in response to an attack by beta-toxin. p38 MAPK (SB203580) and JNK (SP600125) inhibitors enhanced toxin-induced cell death. Incubation in K+-free medium intensified p38 MAPK activation and cell death induced by the toxin, while incubation in K+-high medium prevented those effects. While streptolysin O (SLO) reportedly activates p38 MAPK via reactive oxygen species (ROS), we showed that this pathway did not play a major role in p38 phosphorylation in beta-toxin-treated cells. Therefore, we propose that beta-toxin induces activation of the MAPK pathway to promote host cell survival.

ABSTRACTClostridium perfringensiota-toxin is composed of an enzymatic component (Ia) and a binding component (Ib). Ib binds to a cell surface receptor, undergoes oligomerization in lipid rafts, and binds Ia. The resulting complex is then endocytosed. Here, we show the intracellular trafficking of iota-toxin. After the binding of the Ib monomer with cells at 4°C, oligomers of Ib formed at 37°C and later disappeared. Immunofluorescence staining of Ib revealed that the internalized Ib was transported to early endosomes. Some Ib was returned to the plasma membrane through recycling endosomes, whereas the rest was transported to late endosomes and lysosomes for degradation. Degraded Ib was delivered to the plasma membrane by an increase in the intracellular Ca2+concentration caused by Ib. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of Ib in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted Ib's movement in the cytosol. These results indicated that an internalized Ia and Ib complex was delivered to early endosomes and that subsequent delivery of Ia to the cytoplasm occurs mainly in early endosomes. Ib was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation. Degraded Ib was transported to plasma membranes.

ABSTRACTClostridium perfringensiota-toxin is a binary toxin composed of an enzyme component (Ia) and a binding component (Ib). Each component alone lacks toxic activity, but together they produce cytotoxic effects. We examined the cytotoxicity of iota-toxin Ib in eight cell lines. A431 and A549 cells were susceptible to Ib, but MDCK, Vero, CHO, Caco-2, HT-29, and DLD-1 cells were not. Ib bound and formed oligomers in the membranes of A431 and MDCK cells. However, Ib entered MDCK cells but not A431 cells, suggesting that uptake is essential for cellular survival. Ib also induced cell swelling and the rapid depletion of cellular ATP in A431 and A549 cells but not the insensitive cell lines. In A431 cells, Ib binds and oligomerizes mainly in nonlipid rafts in the membranes. Disruption of lipid rafts by methyl-β-cyclodextrin did not impair ATP depletion or cell death caused by Ib. Ib induced permeabilization by propidium iodide without DNA fragmentation in A431 cells. Ultrastructural studies revealed that A431 cells undergo necrosis after treatment with Ib. Ib caused a disruption of mitochondrial permeability and the release of cytochrome c. Staining with active-form-specific antibodies showed that the proapoptotic Bcl-2-family proteins Bax and Bak were activated and colocalized with mitochondria in Ib-treated A431 cells. We demonstrate that Ib by itself produces cytotoxic activity through necrosis.

Clostridium perfringens TpeL belongs to a family of large clostridial cytotoxins that encompasses Clostridium difficile toxin A (TcdA) and B (TcdB) and Clostridium sordellii lethal toxin (TcsL). We report here the identification of the TpeL-catalyzed modification of small GTPases. A recombinant protein (TpeL1-525) derived from the TpeL N-terminal catalytic domain in the presence of streptolysin O (SLO) induced the rounding of Vero cells and the glycosylation of cellular Rac1. Among several hexoses tested, UDP-N-acetyl-glucosamine (UDP-GlcNAc) and UDP-glucose (UDP-Glc) served as cosubstrates for TpeL1-525-catalyzed modifications. TpeL1-525 catalyzed the incorporation of UDP-Glc into Ha-Ras, Rap1B, and RalA and of UDP-GlcNAc into Rac1, Ha-Ras, Rap1B, and RalA. In Rac1, TpeL and TcdB share the same acceptor amino acid for glycosylation, Thr-35. In Vero cells treated with TpeL1-525 in the presence of SLO, glycosylation leads to a translocation of the majority of Rac1 and Ha-Ras to the membrane. We demonstrate for first time that TpeL uses both UDP-GlcNAc and UDP-Glc as donor cosubstrates and modifies the Rac1 and Ras subfamily by glycosylation to mediate its cytotoxic effects.

ABSTRACTClostridium perfringensTpeL belongs to a family of large clostridial cytotoxins that encompasses Clostridium difficiletoxin A (TcdA) and B (TcdB) and Clostridium sordelliilethal toxin (TcsL). We report here the identification of the TpeL-catalyzed modification of small GTPases. A recombinant protein (TpeL1-525) derived from the TpeL N-terminal catalytic domain in the presence of streptolysin O (SLO) induced the rounding of Vero cells and the glycosylation of cellular Rac1. Among several hexoses tested, UDP-N-acetyl-glucosamine (UDP-GlcNAc) and UDP-glucose (UDP-Glc) served as cosubstrates for TpeL1-525-catalyzed modifications. TpeL1-525 catalyzed the incorporation of UDP-Glc into Ha-Ras, Rap1B, and RalA and of UDP-GlcNAc into Rac1, Ha-Ras, Rap1B, and RalA. In Rac1, TpeL and TcdB share the same acceptor amino acid for glycosylation, Thr-35. In Vero cells treated with TpeL1-525 in the presence of SLO, glycosylation leads to a translocation of the majority of Rac1 and Ha-Ras to the membrane. We demonstrate for first time that TpeL uses both UDP-GlcNAc and UDP-Glc as donor cosubstrates and modifies the Rac1 and Ras subfamily by glycosylation to mediate its cytotoxic effects.

Clostridium perfringensα-toxin, an important agent of gas gangrene with inflammatory myopathies, possesses lethal, hemolytic, and necrotic activities. Here, we show that α-toxin-induced lethality in mice was inhibited by i.v. preadministration of erythromycin (ERM). Administration of ERM resulted in a drastic reduction in the release of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 and systemic hemolysis induced by α-toxin, whereas the administration of kitasamycin did not. Furthermore, the lethality and systemic hemolysis caused by α-toxin were blocked by the preinjection of anti-TNF-α, but not the anti-IL-1β- or anti-IL-6-antibody. In addition, TNF-α-deficient mice were resistant to α-toxin, indicating that TNF-α plays an important role in the lethality. ERM inhibited the toxin-induced release of TNF-α from neutrophils and phosphorylation of toropomyosin-related kinase receptor A (TrkA) and extracellular-regulated kinase (ERK) 1/2. Furthermore, K252a, a TrkA inhibitor, and PD98059 (2′-amino-3′-methoxyflavone), an ERK1/2 inhibitor, inhibited the toxin-induced release of TNF-α from neutrophils. The observation shows that the toxin-induced release of TNF-α is dependent on the activation of ERK/mitogen-activated protein kinase signal transduction via TrkA in neutrophils and that ERM specifically blocks the toxin-induced events through the activation of neutrophils.

Clostridium perfringens alpha-toxin, an important agent of gas gangrene with inflammatory myopathies, possesses lethal, hemolytic, and necrotic activities. Here, we show that alpha-toxin-induced lethality in mice was inhibited by i.v. preadministration of erythromycin (ERM). Administration of ERM resulted in a drastic reduction in the release of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 and systemic hemolysis induced by alpha-toxin, whereas the administration of kitasamycin did not. Furthermore, the lethality and systemic hemolysis caused by alpha-toxin were blocked by the preinjection of anti-TNF-alpha, but not the anti-IL-1beta- or anti-IL-6-antibody. In addition, TNF-alpha-deficient mice were resistant to alpha-toxin, indicating that TNF-alpha plays an important role in the lethality. ERM inhibited the toxin-induced release of TNF-alpha from neutrophils and phosphorylation of toropomyosin-related kinase receptor A (TrkA) and extracellular-regulated kinase (ERK) 1/2. Furthermore, K252a, a TrkA inhibitor, and PD98059 (2'-amino-3'-methoxyflavone), an ERK1/2 inhibitor, inhibited the toxin-induced release of TNF-alpha from neutrophils. The observation shows that the toxin-induced release of TNF-alpha is dependent on the activation of ERK/mitogen-activated protein kinase signal transduction via TrkA in neutrophils and that ERM specifically blocks the toxin-induced events through the activation of neutrophils.

Clostridium perfringensα-toxin induces the hemolysis of sheep erythrocytes by activating the metabolism of sphingomyelin (SM) via a GTP binding protein in membranes. α-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 α-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.

ABSTRACTClostridium perfringensalpha-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 θ (PKCθ). 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 PKCθ in stimulating an increase in O2−. In addition, we show the participation of mitogen-activated protein kinase-associated signaling events via activation of PKCθ in the toxin-induced generation of O2−.

The vulnerable plant Dalbergia tonkinensisPrain, is a rare species, native to Vietnam. Phytochemical investigations and biological evaluations of this species are quite limited. Antimicrobial screening has suggested that, at the low dose of 1.0 mg/mL, the methanol extracts of the leaf, stem bark, and root, as well as chloroform fraction of heartwood can serve as useful sources against seven gram-positive skin microbacteria, Bacillus cereus(ATCC27522), Escherichia coli(JM109), Staphylococcus aureus(ATCC25923), Staphylococcus epidermidis(ATCC14990), Streptococcus pneumonia(ATCC49619), Streptococcus pyogenes(ATCC12344), and Vibrio parahaemolyticus(RIMD2210010), and four oral gram-positive microbacteria Streptococcus mutans(ATCC 25175), Streptococcus mitis(ATCC903), Streptococcus sobrinus(ATCC 33478), and Porphyromonas gingivalis(ATCC33277), with an inhibitory percentage of 60-80% growth for several strains Bacillus cereus, Escherichia coli, and Streptococcus pneumonia. We further assessed antimicrobial activities of the enriched diverse flavonoids from chloroform fraction of its heartwood. Extensive structural activity relationship studies showed structure function closely related to the antimicrobial activity, in which methoxylation at C-2’, and 4’ in isoflavanones, hydroxylation at C-3’ in flavones, substitution at C-5 in isoflavones, and lactone opened ring in neoflavonoids were found to increase the effective inhibitions. In the second antimicrobial assessment, the isolated flavonoid liquiritigenin showed the MIC values of 50, and 100 μg/mL against the microbacterial strains Staphylococcus aureussubsp. aureus(ATCC 11632), and Aspergillus niger(439), respectively, whereas the MIC value of 100 μg/mL was assignable to biochanin A against microbacterium Staphylococcus aureussubsp. aureus.