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Photorhabdus luminescens W-14 insecticidal activity consists of at least two similar but distinct proteins -: Purification and characterization of toxin A and toxin B

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第14期274卷 9836-9842页

作者： Guo, LN Fatig, RO Orr, GL Schafer, BW Strickland, JA Sukhapinda, K Woodsworth, AT Petell, JK Dow AgroSci LLC Input Agr Gene Discovery Biotechnol Indianapolis IN 46268 USA

Both the bacterium Photorhabdus luminescens alone and its symbiotic Photorhabdus-nematode complex are known to be highly pathogenic to insects, The nature of the insecticidal activity of Photorhabdus bacteria was investigated for its potential application as an insect control agent. It was found that in the fermentation broth of P. luminescens strain W-14, at least two proteins, toxin A and toxin B, independently contributed to the oral insecticidal activity against Southern corn rootworm, Purified toxin A and toxin B exhibited single bands on native polyacrylamide gel electrophoresis and two peptides of 208 and 63 kDa on SDS-polyacrylamide gel electrophoresis. The native molecular weight of both the toxin A and toxin B was determined to be approximately 860 kDa, suggesting that they are tetrameric, NH2-terminal amino acid sequencing and Western analysis using monospecific antibodies to each toxin demonstrated that the two toxins were distinct but homologous, The oral potency (LD,,) of toxin A and toxin B against Southern corn rootworm larvae was determined to be similar to that observed with highly potent Bt toxins against lepidopteran pests, In addition, it was found that the two peptides present in toxin B could be processed in vitro from a 281-kDa protoxin by endogenous P. luminescens proteases. Proteolytic processing was shown to enhance insecticidal activity.

关键词：氨基酸序列细菌毒素类/化学细菌毒素类/分离和提纯细菌毒素类/药理学印迹法蛋白质电泳聚丙烯酰氨凝胶肠杆菌科/化学发酵杀虫药幼虫/药物作用分子序列数据分子量害虫防治生物学植物光谱法质量基质辅助激光解吸电离动物

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A novel cytotoxin from Clostridium difficile serogroup F is a functional hybrid between two other large clostridial cytotoxins

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第16期274卷 11046-11052页

作者： Chaves-Olarte, E Löw, P Freer, E Norlin, T Weidmann, M von Eichel-Streiber, C Thelestam, M Karolinska Inst Ctr Microbiol & Tumor Biol S-17177 Stockholm Sweden Univ Costa Rica Unidad Microscopia Elect San Jose Costa Rica Karolinska Inst Nobel Inst Neurophysiol Dept Neurosci S-17177 Stockholm Sweden Johannes Gutenberg Univ Mainz Inst Med Mikrobiol & Hyg Verfugungagebaude Forsch & Entwicklung D-55101 Mainz Germany

The large clostridial cytotoxins (LCTs) constitute a group of high molecular weight clostridial cytotoxins that inactivate cellular small GTP-binding proteins. We demonstrate that a novel LCT (TcdB-1470) from Clostridium difficile strain 1470 is a functional hybrid between "reference" TcdB-10463 and Clostridium sordellii TcsL-1522. It bound to the same specific receptor as TcdB-10463 but glucosylated the same GTP-binding proteins as TcsL-1522. Ah three toxins had equal enzymatic potencies but were equally cytotoxic only when micro injected. When applied extracellularly TcdB-1470 and TcdB-10463 were considerably more potent cytotoxins than TcsL-1522. The small GTP-binding protein R-Ras was identified as a target for TcdB-1470 and also for Test-1522 but not for TcdB-10463. R-Ras is known to control integrin-extracellular matrix interactions from inside the cell. Its glucosylation may be a major determinant for the cell rounding and detachment induced by the two R-Ras-attacking toxins. In contrast, fibroblasts treated with TcdB-10463 were arborized and remained attached, with phosphotyrosine containing structures located at the cell-to-cell contacts and beta(3)-integrin remaining at the tips of cellular protrusions. These components were absent from cells treated with the R-Ras-inactivating toxins. The novel hybrid toxin will broaden the utility of the LCTs for clarifying the functions of several small GTPases, now including also R-Ras.

关键词： 3T3细胞细菌毒素类/代谢细菌毒素类/药理学碱基序列细胞黏附/药物作用难辨梭菌/代谢细胞毒素类/代谢细胞毒素类/药理学 DNA引物糖基化显微镜检查电子扫描重组融合蛋白质类/代谢动物小鼠

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第11期275卷 7764-7770页

作者： Doussau, F Gasman, S Humeau, Y Vitiello, F Popoff, M Boquet, P Bader, MF Poulain, B CNRS Lab Neurobiol Cellulaire UPR 9009 F-67084 Strasbourg France INSERM U338 F-67084 Strasbourg France Inst Pasteur F-75724 Paris 15 France INSERM U452 F-06107 Nice 2 France

Rho, Rac, and Cdc42 monomeric GTPases are well known regulators of the actin cytoskeleton and phosphoinositide metabolism and have been implicated in hormone secretion in endocrine cells. Here, we examine their possible implication in Ca2+-dependent exocytosis of neurotransmitters. Using subcellular fractionation procedures, we found that RhoA,RhoB, Rac1, and Cdc42 are present in rat brain synaptosomes;however, only Rad was associates with highly purified synaptic vesicles. To determine the synaptic function of these GTPases, toxins that impair Rho-related proteins were microinjected into Aplysia neurons. We used lethal toxin from Clostridium sordellii, which inactivates Rac;toxin B from Clostridium difficile, which inactivates Rho, Rac, and Cdc42;and C3 exoenzyme from Clostridium bo;botulinum and cytotoxic necrotizing factor 1 from Escherichia coli, which mainly affect Rho. Analysis of the toxin effects on evoked acetylcholine release revealed that a member of the Rho family, most likely Rad, was implicated in the control of neurotransmitter release. Strikingly, blockage of acetylcholine release by lethal toxin and toxin B could be completely removed in <1 s by high frequency stimulation of nerve terminals. Further characterization of the inhibitory action produced by lethal toxin suggests that Rac1 protein regulates a late step in Ca2+-dependent neuroexocytosis.

关键词： ADP核糖转移酶类/药理学乙酰胆碱/代谢海螺细菌毒素类/药理学肉毒毒素类脑/代谢钙/代谢细胞分级分离梭菌属细胞毒素类/药理学电刺激大肠杆菌蛋白质类胞吐作用/药物作用突触小泡/代谢 rac1GTP结合蛋白质/代谢 ρGTP结合蛋白质类/代谢动物大鼠

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Regulation of dendritic spine morphology by the Rho family of small GTPases: Antagonistic roles of Rac and Rho

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CEREBRAL CORTEX 2000年第10期10卷 927-938页

作者： Tashiro, A Minden, A Yuste, R Columbia Univ Dept Biol Sci New York NY 10027 USA

Dendritic spines mediate most excitatory transmission in the mammalian CNS and have been traditionally considered stable structures. Following the suggestion that spines may 'twitch', it has been recently shown that spines are capable of rapid morphological rearrangements. Because of the role of the small GTPases from the Rho family in controlling neuronal morphogenesis, we investigated the effects of several members of this biochemical signaling pathway in the maintenance of the morphology of extant dendritic spines by combining biolistic transfection of pyramidal neurons in cultured cortical and hippocampal slices with two-photon microscopy. We find a variety of effects on the density and morphology of dendritic spines by expressing either constitutively active or dominant negative forms of several small GTPases of the Rho family, by blocking the entire pathway with Clostridium difficile toxin B or by blocking Rho with C3 transferase. We propose a model where Rac promotes spine formation, while Rho prevents it. We conclude that the small GTPases provide antagonistic control mechanisms of spine maintenance in pyramidal neurons.

关键词：细菌蛋白质类细菌毒素类/药理学培养技术树突/药物作用树突/生理学树突/超微结构绿色荧光蛋白质类指示剂和试剂/药理学发光蛋白质类/遗传学发光蛋白质类/药理学小鼠近交C57BL 信号传导/生理学转染 cdc42GTP结合蛋白质/药理学 racGTP结合蛋白质类/药理学 racGTP结合蛋白质类/生理学 ρGTP结合蛋白质类/药理学 ρGTP结合蛋白质类/生理学动物小鼠

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Cytosolic delivery of granzyme B by bacterial toxins: Evidence that endosomal disruption, in addition to transmembrane pore formation, is an important function of perforin

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MOLECULAR AND CELLULAR BIOLOGY 1999年第12期19卷 8604-8615页

作者： Browne, KA Blink, E Sutton, VR Froelich, CJ Jans, DA Trapani, JA Austin Res Inst Heidelberg Vic 3084 Australia Australian Natl Univ John Curtin Sch Med Res Nucl Signalling Lab Canberra ACT 2600 Australia ENH Res Inst Cell Death Program Evanston IL 60201 USA

Granule-mediated cell killing by cytotoxic lymphocytes requires the combined actions of a membranolytic protein, perforin, and granule-associated granzymes, but the mechanism by which they jointly kill cells is poorly understood. me have tested a series of membrane-disruptive agents including bacterial pore-farming toxins and hemolytic complement for their ability to replace perforin in facilitating granzyme B-mediated cell death. As with perforin, low concentrations of streptolysin O and pneumolysin (causing <10% Cr-51 release) permitted granzyme B-dependent apoptosis of Jurkat and Yac-l cells, but staphylococcal alpha-toxin and complement were ineffective, regardless of concentration. The ensuing nuclear apoptotic damage was caspase dependent and included cleavage of poly(ADP-ribose) polymerase, suggesting a mode of action similar to that of perforin. The plasma membrane lesions formed at low dose by perforin, pneumolysin, and streptolysin did not permit diffusion of fluorescein-labeled proteins as small as 8 kDa into the cell, indicating that large membrane defects are not necessary for granzymes (32 to 65 kDa) to enter the cytosol and induce apoptosis. The endosomolytic toxin, listeriolysin O, also effected granzyme B-mediated cell death at concentrations which produced no appreciable tell membrane damage. Cells pretreated with inhibitors of endosomal trafficking such as brefeldin A took up granzyme B normally but demonstrated seriously impaired nuclear targeting of granzyme B when perforin was also added, indicating that an important role of perforin is to disrupt vesicular protein trafficking. Surprisingly, cells exposed to granzyme B,vith perforin concentrations that produced nearly maximal Cr-51 release (1,600 U/ml) also underwent apoptosis despite excluding a 8-kDa fluorescein-labeled protein marker. only at concentrations of >4,000 U/mI were perforin pores demonstrably large enough to account for transmembrane diffusion of granzyme B. We conclude that pore

关键词：细胞凋亡细菌蛋白质类细菌毒素类/代谢细菌毒素类/药理学细胞核胞质溶胶内颗粒/代谢颗粒酶类热休克蛋白质类/代谢热休克蛋白质类/药理学溶血素蛋白质类/代谢溶血素蛋白质类/药理学 Jurkat细胞膜糖蛋白类/生理学穿孔素成孔毒素蛋白质类丝氨酸内肽酶类/代谢链球菌溶血素类/代谢链球菌溶血素类/药理学肿瘤细胞培养的动物人类小鼠

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Photorhabdus toxins:: novel biological insecticides

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CURRENT OPINION IN MICROBIOLOGY 1999年第3期2卷 284-288页

作者： ffrench-Constant, R Bowen, D Univ Bath Dept Biol & Biochem Bath BA2 7AY Avon England Russell Labs 237 Dept Entomol Madison WI 53706 USA

Following concerns over the potential for insect resistance to insecticidal Bacillus thuringiensis toxins expressed in transgenic plants, there has been recent interest in novel biological insecticides. Over the past year there has been considerable progress in the cloning of several alternative toxin genes from the bacteria Photorhabdus luminescens and Xenorhabdus nematophilus. These genes encode large insecticidal toxin complexes with little homology to other known toxins.

关键词：氨基酸序列细菌毒素类/遗传学细菌毒素类/药理学克隆分子肠杆菌科/化学肠杆菌科/遗传学杀虫药/药理学昆虫/药物作用分子序列数据序列同源性氨基酸动物

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Bacillus thuringiensis Cry1Ac protoxin is a potent systemic and mucosal adjuvant

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SCANDINAVIAN JOURNAL OF IMMUNOLOGY 1999年第6期49卷 578-584页

作者： Vázquez, RI Moreno-Fierros, L Neri-Bazán, L De La Riva, GA López-Revilla, R Inst Politecn Nacl CINVESTAV Dept Cell Biol Mexico City 07000 DF Mexico Ctr Genet Engn & Biotechnol Havana Cuba Univ Nacl Autonoma Mexico ENEP Iztacala Unidad Morfol & Funct Tlalnepantla Mexico

Recently we demonstrated that recombinant Cry1Ac protoxin from Bacillus thuringiensis is a potent systemic and mucosal immunogen. In this study we compared the adjuvant effects of Cry1Ac and cholera toxin (CT) for the hepatitis B surface antigen (HBsAg) and bovine serum albumin (BSA). The antibody responses of intestinal secretions and serum were determined by ELISA in Balb/c mice immunized through the intragastric (IG) or intraperitoneal (IP) routes. When HBsAg was administered via TG, the anti-HBsAg intestinal response was not enhanced by either Cry1Ac or CT, whereas via IP Cry1Ac increased the anti-HBsAg intestinal immunoglobulin (IE)G response and CT increased the intestinal IgA and IgM responses. Serum anti-BSA antibodies increased when BSA was co-administered with CT or Cry1Ac by both routes. Cholera toxin and Cry1Ac co-administered via IP increased the IgG anti-BSA response in fluid of the large intestine and CT also increased the IgA and IgM responses slightly. When co-administered via IF, CT and Cry1Ac did not affect the IgG anti-BSA response of the small intestine significantly. We conclude that Cry1Ac is a mucosal and systemic adjuvant as potent as CT which enhances mostly serum and intestinal Ige antibody responses, especially at the large intestine, and its effects depend on the route and antigen used. These features make Cry1Ac of potential use as carrier and/or adjuvant in mucosal and parenteral vaccines.

关键词：佐剂免疫苏云金芽孢杆菌细菌蛋白质类/免疫学细菌蛋白质类/药理学细菌毒素类/免疫学细菌毒素类/药理学内毒素类/免疫学内毒素类/药理学溶血素蛋白质类乙型肝炎表面抗原/免疫学免疫/药物作用免疫/免疫学免疫黏膜/药物作用免疫黏膜/免疫学小鼠近交BALBC 血清白蛋白牛/免疫学动物牛小鼠

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Hormone-stimulated calcium release is inhibited by cytoskeleton-disrupting toxins in AR4-2J cells

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CELL CALCIUM 2000年第2期28卷 73-82页

作者： Bozem, M Kuhlmann, S Blum, R Feick, P Schulz, I Univ Saarland Dept Physiol D-66421 Homburg Germany

We have studied the role of the actin cytoskeleton in bombesin-induced inositol 1,4,5-trisphosphate (IP3)-production and Ca2+ release in the pancreatic acinar tumour cell line AR4-2J. Intracellular and extracellular free Ca2+ concentrations were measured in cell suspensions, using Fura-2. Disruption of the actin cytoskeleton by pretreatment of the cells with latrunculin B (10 mu M), cytochalasin D (10 mu M) or toxin B from Clostridium difficile (20 ng/ml) for 5-29 h led to inhibition of both, bombesin-stimulated IP3-production and Ca2+ release. The toxins had no effect on binding of bombesin to its receptor, on Ca2+ uptake into intracellular stores and on resting Ca2+ levels. Ca2+ mobilization from intracellular stores, induced by thapsigargin (100 nM) or IP3 (1 mu M) was not impaired by latrunculin B. In latrunculin B-pretreated cells inhibition of both, bombesin-stimulated IP3 - production and Ca2+ release was partly suspended in the presence of aluminum fluoride, an activator of G-proteins. Aluminum fluoride had no effect on basal IP3 and Ca2+ levels of control and toxin-pretreated cells. We conclude that disruption of the actin cytoskeleton impairs coupling of the bombesin receptor to its G-protein, resulting in inhibition of phospholipase C-activity with subsequent decreases in IP3- production and Ca2+ release. (C) 2000 Harcourt Publishers Ltd.

关键词：铝化合物/药理学细菌蛋白质类细菌毒素类/药理学二环化合物杂环/药理学铃蟾肽/代谢铃蟾肽/药理学钙/代谢细胞膜通透性/药物作用细胞松弛素D/药理学细胞骨架/药物作用胞质溶胶/药物作用胞质溶胶/代谢氟化物/药理学 GTP结合蛋白质类/药物作用 GTP结合蛋白质类/代谢激素类/药理学肌醇1 4 5-三磷酸/代谢胰腺/细胞学胰腺/药物作用胰腺/代谢胰腺肿瘤噻唑类/药理学噻唑烷类肿瘤细胞培养的动物大鼠

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Bacillus thuringiensis δ-endotoxin proteins show a correlation in toxicity and short circuit current inhibition against Helicoverpa zea

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CURRENT MICROBIOLOGY 2000年第3期41卷 214-219页

作者： Karim, S Gould, F Dean, DH Ohio State Univ Dept Biochem Columbus OH 43210 USA N Carolina State Univ Dept Entomol Raleigh NC 27695 USA

Pesticidal activity of Bacillus thuringiensis delta-endotoxins, Cry1Aa, Cry1Ab, Cry1Ac, and Cry2A, was determined by using the force-feeding bioassay method to 4(th) instar larvae of Helicoverpa tea. H. tea was susceptible to Bt toxins in the order Cry1Ac > Cry1Ab > Cry1Aa > Cry2A with 63.60, 89.04, 159.65, and 375.78 ng/larvae respectively. The abilities of selected Bacillus thuringiensis toxins to inhibit short circuit current (I-SC) in midgut epithelia of H. ten were also investigated by voltage clamp assay. The voltage-clamp studies were conducted on isolated midguts, measuring the inhibition of short circuit current (I-SC by activated toxin. A Cry1Aa toxin dilution of 33.3 and 500 ng/ml resulted in inhibition of I-SC of -2.29 mu A/min (lag time 15 min) and -4.48 mu A/min (lag time, 2 min) respectively. The Cry1Ab dilution of 25 ng/ml inhibited I-SC to -1.39 mu A/min, a lag time of 14 min, and 333.3 ng/ml dilution resulted in decay of I-SC -2.49 mu A/min, lag time 1 min respectively. The Cry1Ac lower dilution 16.7 ng/ml inhibited I-SC to -1.39 mu A/min, lag time 4 min, and a high dilution 333.3 ng/ml decay I-SC to -2.44 mu A/min, lag time 1 min. The inhibition of I-SC (-1.10 mu A/min, lag time 25) at lower dilution (33.3 ng/ml) and high dilution (500 ng/ml), decay (-2.38 mu A/min, lag time 5 min), showed a correlation between toxin concentration and inhibitory response with Cry2A toxin. The lag time decreased with increasing concentration of toxin applied, which is additional evidence of dose response besides direct correlation of toxicity assays and I-SC.

关键词：芽孢杆菌苏云金/化学细菌蛋白质类/生物合成细菌蛋白质类/遗传学细菌蛋白质类/药理学细菌毒素类/药理学消化系统/药物作用剂量效应关系药物导电性内毒素类/生物合成内毒素类/遗传学内毒素类/药理学溶血素蛋白质类杀虫药/药理学幼虫/药物作用蛾/药物作用蛾/生理学时间因素动物

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Actin cytoskeleton depolymerization with Clostridium spiroforme toxin enhances the secretory activity of rat melanotrophs

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JOURNAL OF PHYSIOLOGY-LONDON 1999年第2期521卷 389-395页

作者： Chowdhury, HH Popoff, MR Zorec, R Sch Med Inst Pathophysiol Lab Neuroendocrinol Mol Cell Physiol Ljubljana 1001 Slovenia Inst Pasteur F-75724 Paris 15 France

1. We measured membrane capacitance (C-m) in cultured rat melanotrophs pretreated with Clostridium spiroforme toxin (CST), which specifically depolymerises cortical filamentous actin (F-actin). Phalloidin staining confirmed that CST treatment depolymerised the F-actin. 2. In control cells, cytosol dialysis with 1 mu M Ca-i(2+) increased C-m by 23 +/- 4% (n = 11) relative to the resting C-m 400 s after the start of patch rupture. In CST-treated cells the increase in C-m was 32 +/- 5% (n = 15), not significantly different from controls. The rate of C-m increase was affected transiently by CST treatment, peaking at 1 min after patch rupture. The maximal rate of C-m increase was 4.27 +/- 0.85 fF s(-1) (n = 12;measured 200 s after the start of patch rupture) in controls and 8.0 +/- 1.35 fF s(-1) (n = 23;measured 75 s after the start of patch rupture) in CST-treated cells (P < 0.01). 3. In control cells cytosol dialysis with 0 mu M Ca-i(2+) decreased C-m by 9 +/- 3% (n = 7), in CST-treated cells C-m increased by 11 +/- 3% (n = 7) relative to resting C-m 400 s after the start of cytosol dialysis. The rate of change in C-m remained constant (controls: -1 to -2 fF s(-1);CST treatment: 1-2 fF s(-1)). 4. Transient and sustained effects of CST treatment on changes in C-m at high or low [Ca2+](i), respectively, suggest a distinct role of cytoskeleton in Ca2+-dependent and Ca2+-independent changes in C-m. Transient enhancement of the rate of C-m by CST is consistent with a barrier role of cytoskeleton in regulated exocytosis. The sustained effect of CST on Ca2+-independent changes in C-m suggests cytoskeletal involvement, in endocytosis.

关键词： ADP核糖转移酶类肌动蛋白类/药物作用肌动蛋白类/代谢细菌毒素类/药理学钙/药理学细胞骨架/药物作用细胞骨架/代谢导电性胞吐作用/药物作用胞吐作用/生理学黑素细胞/药物作用黑素细胞/代谢黑素细胞/分泌显微镜检查荧光膜片钳术鬼笔环肽聚合物/代谢大鼠 Wistar 动物男(雄)性大鼠

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