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C-terminal amino acid residues are required for the folding and cholesterol binding property of perfringolysin O, a pore-forming cytolysin

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第26期274卷 18536-18542页

作者： Shimada, Y Nakamura, M Naito, Y Nomura, K Ohno-Iwashita, Y Tokyo Metropolitan Inst Gerontol Dept Prot Biochem Itabashi Ku Tokyo 1730015 Japan Inst Phys & Chem Res Div Biomol Characterizat Wako Saitama 5310198 Japan

Perfringolysin O (theta-toxin) is a pore-forming cytolysin whose activity is triggered by binding to cholesterol in the plasma membrane. The cholesterol binding activity is predominantly localized in the beta-sheet-rich C-terminal half. In order to determine the roles of the C-terminal amino acids in theta-toxin conformation and activity, mutants were constructed by truncation of the C terminus. While the mutant with a two-amino acid C-terminal truncation retains full activity and has similar structural features to native theta-toxin, truncation of three amino acids causes a 40% decrease in hemolytic activity due to the reduction in cholesterol binding activity with a slight change in its higher order structure. Furthermore, both mutants were found to be poor at in vitro refolding after denaturation in 6 M guanidine hydrochloride, resulting in a dramatic reduction in cholesterol binding and hemolytic activities. These activity losses were accompanied by a slight decrease in beta-sheet content. A mutant toxin with a five-amino acid truncation expressed in Escherichia coli is recovered as a further truncated form lacking the C-terminal 21 amino residues. The product retains neither cholesterol binding nor hemolytic activities and shows a highly disordered structure as detected by alterations in the circular dichroism and tryptophan fluorescence spectra. These results show that the C-terminal region of theta-toxin has two distinct roles;the last 21 amino acids are involved to maintain an ordered overall structure, and in addition, the last two amino acids at the C-terminal end are needed for protein folding in vitro, in order to produce the necessary conformation for optimal cholesterol binding and hemolytic activities.

关键词：氨基酸序列氨基酸类/生理学细菌毒素类/化学细菌毒素类/遗传学细菌毒素类/代谢细胞膜/代谢胆固醇/代谢酶联免疫吸附测定溶血素蛋白质类/化学溶血素蛋白质类/遗传学溶血素蛋白质类/代谢溶血模型分子分子序列数据诱变定点蛋白质折叠绵羊构效关系动物

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Prevalence and characteristics of verotoxin-producing Escherichia coli (VTEC) in stool samples from asymptomatic human carriers working in the meat processing industry in Switzerland

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第2期88卷 335-341页

作者： Stephan, R Ragettli, S Untermann, F Univ Zurich Fac Vet Med Inst Food Safety & Hyg CH-8057 Zurich Switzerland

A total of 5590 stool samples from healthy employees in the meat industry were screened by PCR for verotoxin-producing Escherichia coli (VTEC). The PCR product of VT-encoding genes was detected in 3.5% of the samples. Phenotypic and genotypic traits of 47 VTEC strains isolated from asymptomatic carriers were characterized. A variety of serotypes was found;one strain belonged to the serotype O157:H7. The majority of the isolates proved to be VT2-positive. Fifty-seven percent of the verotoxin-producing strains harboured the genes for one or several additional virulence associated factors, including intimin (eae, 8.5%), the 60 MDa plasmid (42.5%), enterohaemolysin (EHEC-hlyA, 38.3%), the heat-stable enterotoxin (astA, 6.4%), a serin protease (espP, 6.4%), colicin production (col D157, 12.8%) and a secretion system II (etpD, 10.6%). None of the strains was positive for a specific enzyme with catalase-peroxidase activity (katP).

关键词：细菌毒素类/生物合成细菌毒素类/遗传学细菌分型技术带病原状态/微生物学电泳凝胶脉冲场大肠杆菌/分类大肠杆菌/分离和提纯大肠杆菌/代谢大肠杆菌/致病力大肠杆菌感染/流行病学大肠杆菌感染/微生物学大肠杆菌O157/分类大肠杆菌O157/分离和提纯大肠杆菌O157/代谢大肠杆菌O157/致病力粪便/微生物学食品处理和加工肉类加工业聚合酶链反应患病率血清分型志贺毒素1 瑞士/流行病学毒力/遗传学人类

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Shiga-toxin-producing Escherichia coli in faeces of healthy dairy cows, sheep and goats:: prevalence and virulence properties

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LETTERS IN APPLIED MICROBIOLOGY 2000年第3期31卷 203-208页

作者： Zschöck, M Hamann, HP Kloppert, B Wolter, W Staatliches Med Lebensmittel & Veterinaruntersuch Giessen Germany

The prevalence of Shiga-toxin-producing Escherichia coli (STEC) in healthy dairy ruminants was investigated between 1996 and 1998 by a multiplex polymerase chain reaction (mPCR) technique. A total of 13 552 E. coli colonies from 726 cows, 28 sheep and 93 goats out of 112 randomly selected dairy farms in Hessia, Germany were analysed. STEC strains were recovered from 131 (18.0%) cows, nine (32.1%) sheep and 70 (75.3%) goats. Further characterization of the STEC isolates showed that 89 (0.66% of the investigated colonies) of animal field strains carried stx1 gene, 64 (0.47%) stx2 gene and 57 (0.42%) stx1 and stx2 gene. Sixty (93.8%) out of 64 stx2 field strains were harboured by cows. In contrast, 74 (83.1%) out of 89 stx1 dairy animal field strains were from ovine or caprine origin. Only 17 (8.1%) stx-positive isolates (13 from cattle, three from sheep and only one from goat) were positive for eneA gene. Eight (9.0%) of the stx1, five (7.8%) of the stx2 and four (7.0%) of the stx1/stx2 gene-positive field strains carried the eneA gene. The prevalence of EHEC-haemolysin (EHEC-hlyA) gene sequence was 88.8% (79 isolates) of the stx1 and 68.8% (44 isolates) of the stx2 isolates. Out of 57 stx1- and stx2-positive field-strains, 34 (59.6%) carried the EHEC-hlyA gene. E. coli O serovars O:157 and O:111 were not found. Only one isolate was positive with O26 antiserum.

关键词：黏着素细菌细菌外膜蛋白质类/遗传学细菌毒素类/遗传学载体蛋白质类牛/微生物学乳制品业大肠杆菌/分离和提纯大肠杆菌/代谢大肠杆菌/致病力大肠杆菌蛋白质类粪便/微生物学山羊/微生物学核酸扩增技术聚合酶链反应/方法患病率反刍动物/微生物学绵羊/微生物学志贺毒素1/生物合成志贺毒素1/遗传学志贺毒素2/生物合成志贺毒素2/遗传学毒力/遗传学动物人类

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Importance of the carboxyl terminus in the folding and function of α-hemolysin of Staphylococcus aureus

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第14期274卷 9193-9199页

作者： Sangha, N Kaur, S Sharma, V Krishnasastry, MV Natl Ctr Cell Sci Pune 411007 Maharashtra India

The physical state of two model mutants of alpha-hemolysin (alpha HL), alpha HL(1-289), a carboxyl-terminal deletion mutant (CDM), and alpha HL(1-331), a carboxyl-terminal extension mutant (CEM), were examined in detail to identify the role of the carboxyl terminus in the folding and function of native alpha HL, Denatured alpha HL can be refolded efficiently with nearly total recovery of its activity upon restoration of nondenaturing conditions. Various biophysical and biochemical studies on the three proteins have revealed the importance of an intact carboxyl terminus in the folding of alpha HL. The CDM exhibits a marked increase in susceptibility to proteases as compared with alpha HL, alpha HL and CEM exhibit similar fluorescence emission maxima, and that of the CDM is red-shifted by 9 nm, which indicates a greater solvent exposure of the tryptophan residues of the CDM, In addition, the CDM binds 8-anilino-1-naphthalene sulfonic acid (ANS) and increases its fluorescence intensity significantly unlike alpha HL and GEM, which show marginal binding. The circular dichroism studies point that the CDM possesses significant secondary structure, but its tertiary structure is greatly diminished as compared with alpha HL, These data show that the CDM has several of the features that characterize a molten globule state. Experiments with freshly translated mutants, using coupled in vitro transcription and translation, have further supported our observations that deletion at the carboxyl terminus leads to major structural perturbations in the water-soluble form of alpha HL. The studies demonstrate a critical role of the carboxyl terminus of alpha HL in attaining the native folded state.

关键词：细菌毒素类/化学细菌毒素类/遗传学克隆分子电泳聚丙烯酰氨凝胶外毒素类/化学外毒素类/遗传学外毒素类/生理学溶血素蛋白质类/化学溶血素蛋白质类/遗传学氢离子浓度诱变定点神经毒素类/化学神经毒素类/遗传学蛋白质折叠光谱法荧光分光光度法紫外线金黄色葡萄球菌构效关系

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PCR detection, characterization, and distribution of virulence genes in Aeromonas spp.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1999年第12期65卷 5293-5302页

作者： Bin Kingombe, CI Huys, G Tonolla, M Albert, MJ Swings, J Peduzzi, R Jemmi, T Swiss Fed Vet Off Microbiol Sect CH-3003 Liebefeld Bern Switzerland State Univ Ghent Microbiol Lab B-9000 Ghent Belgium Ist Cantonale Batteriosierol Lugano Switzerland Int Ctr Diarrhoeal Dis Res Dhaka 2 Bangladesh

We found 73.1 to 96.9% similarity by aligning the cytolytic enterotoxin gene of Aeromonas hydrophila SSU (AHCYTOEN;GenBank accession no. M84709) against aerolysin genes of Aeromonas spp,, suggesting the possibility of selecting common primers. Identities of 90 to 100% were found among the eight selected primers from those genes, Amplicons obtained from Aeromonas sp, reference strains by using specific primers for each gene or a cocktail of primers were 232 bp long. Of hybridization group 4/5A/5B (HG4/5A/5B), HG9, and HG12 or non-Aeromonas reference strains, none were positive. PCR-restriction fragment Length polymorphism (PCR-RFLP) with HpaII yielded three types of patterns. PCR-RFLP 1 contained two fragments (66 and 166 bp) found in HG6, HG7, HCS, HG10, and HG11. PCR-RFLP 2 contained three fragments (18, 66, and 148 bp) found in HG1, HG2, HG3, and HG11. PCR-RFLP 3, with four fragments (7, 20, 66, and 139 bp), was observed only in HG13, PCR-amplicon sequence analysis (PCR-ASA) revealed three main types. PCR-ASA 1 had 76 to 78% homology with AHCYTOEN and included strains in HG6, HG7, HG8, HG10, and HG11. PCR-ASA 2, with 82% homology, was found only in HG13. PCR-ASA 3, with 91 to 99% homology, contained the strains in HG1, HG2, HG3, and HG11. This method indicated that 37 (61%) of the 61 reference strains were positive with the primer cocktail master mixture, and 34 (58%) of 59 environmental isolates, 93 (66%) of 141 food isolates, and 100 (67%) of 150 clinical isolates from around the world carried a virulence factor when primers AHCF1 and AHCR1 were used. In conclusion, this PCR-based method is rapid, sensitive, and specific for the detection of virulence factors of Aeromonas spp. It overcomes the handicap of time-consuming biochemical and other DNA-based methods.

关键词：气单胞菌属/遗传学气单胞菌属/分离和提纯气单胞菌属/致病力细菌毒素类/遗传学 DNA引物脱氧核糖核酸酶HpaⅡ 欧洲溶血素蛋白质类/遗传学肉/微生物学分子序列数据聚合酶链反应/方法多态性限制性片段长度成孔毒素蛋白质类家禽海味/微生物学猪蔬菜/微生物学毒力/遗传学动物牛

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Transduction of enteric Escherichia coli isolates with a derivative of Shiga toxin 2-encoding bacteriophage φ3538 isolated from Escherichia coli O157:H7

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1999年第9期65卷 3855-3861页

作者： Schmidt, H Bielaszewska, M Karch, H Univ Wurzburg Inst Hyg & Mikrobiol D-97080 Wurzburg Germany Charles Univ Prague Fac Med 2 Inst Med Microbiol Prague 15006 Czech Republic

We investigated the ability of a detoxified derivative of a Shiga toxin 2 (Stx2)-encoding bacteriophage to infect and lysogenize enteric Escherichia coli strains and to develop infectious progeny from such lysogenized strains. The stx(2) gene of the patient E. coli O157:H7 isolate 3538/95 was replaced by the chloramphenicol acetyltransferase (cat) gene from plasmid pACYC184, Phage phi 3538(Delta stx(2)::cat) was isolated after induction of E. coli O157:H7 strain 3538/95 with mitomycin. A variety of strains of enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Stx-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and E. coli from the physiological stool microflora were infected with phi 3538 (Delta stx(2)::cat), and plaque formation and lysogenic conversion of wild-type E. coli strains were investigated. With the exception of one EIEC strain, none of the E. coli strains supported the formation of plaques when used as indicators for phi 3538(Delta stx(2)::cat). However, 2 of 11 EPEC, 11 of 25 STEC, 2 of 7 EAEC, 1 of 3 EIEC, and 1 of 6 E. coli isolates from the stool microflora of healthy individuals integrated the phage in their chromosomes and expressed resistance to chloramphenicol. Following induction with mitomycin, these lysogenic strains released infectious particles of phi 3538(Delta sfx(2)::cat) that formed plaques on a lawn of E. coli laboratory strain C600, The results of our study demonstrate that phi 3538(Delta stx(2)::cat) was able to infect and lysogenize particular enteric strains of pathogenic and nonpathogenic E. coli and that the lysogens produced infectious phage progeny. Stx-encoding bacteriophages are able to spread stx genes among enteric E. coli strains.

关键词：细菌毒素类/遗传学氯霉素O-乙酰转移酶/遗传学氯霉素抗药性/遗传学大肠杆菌噬菌体/遗传学大肠杆菌/药物作用大肠杆菌/遗传学大肠杆菌感染/微生物学大肠杆菌O157/病毒学溶源性微生物敏感性试验聚合酶链反应/方法重组遗传志贺毒素类转导遗传人类

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Detection of PCR products in solution using surface plasmon resonance

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ANALYTICAL CHEMISTRY 1999年第4期71卷 796-800页

作者： Kai, E Sawata, S Ikebukuro, K Iida, T Honda, T Karube, I Univ Tokyo Adv Sci & Technol Res Ctr Meguro Ku Tokyo 1538904 Japan Natl Space Dev Agcy Japan Tsukuba Ibaraki 3050047 Japan Osaka Univ Microbial Dis Res Inst Dept Bacterial Infect Suita Osaka 5650871 Japan

Polymerase chain reaction (PCR) products were detected using a now injection-type sensor based on surface plasmon resonance. Asymmetric PCR was used to amplify the target DNA sequence, and two products with different length were produced. The novelty of our DNA detection system was that our target DNA was double stranded but the probe binding site, located in the 3'-terminus, was single stranded. This avoids the formation of intra- and intermolecular complexes. This novel design permitted us not only to detect PCR product but also to develop a rapid detection system for the detection of the verotoxin 2 gene of Escherichia coli O157:H7.

关键词：细菌毒素类/分析细菌毒素类/遗传学碱基序列 DNA/分析 DNA/化学大肠杆菌/遗传学分子序列数据聚合酶链反应/方法志贺毒素2 溶液表面等离激元/方法温度

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Activation process of dipteran-specific insecticidal protein produced by Bacillus thuringiensis subsp israelensis

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1999年第8期65卷 3464-3469页

作者： Yamagiwa, M Esaki, M Otake, K Inagaki, M Komano, T Amachi, T Sakai, H Kyoto Univ Grad Sch Agr Div Appl Life Sci Kyoto 6068502 Japan Kinki Univ Dept Genet Engn Wakayama 6496493 Japan

Dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 130 kDa in Bacillus thuringiensis subsp. israelensis. Here we performed the in vitro processing of Cry4A and showed that the 130-kDa protoxin of Cry4A was processed into the two protease-resistant fragments of 20 and 45 kDa through the intramolecular cleavage of a 60-kDa intermediate. The processing into these two fragments was also observed in vivo. To investigate functional properties of the two fragments, GST (glutathione S-transferase) fusion proteins of the 60-kDa intermediate and the 20- and 45-kDa fragments mere constructed. Neither the GST-20-kDa fusion protein (GST-20) nor the GST-45-kDa fusion protein (GST-45) was actively toxic against mosquito larvae of Culex pipiens, whereas the GST-60-kDa intermediate fusion protein (GST-60) exhibited significant toxicity. However, when the two fusion proteins GST-20 and GST-45 coexisted, significant toxicity was observed. The coprecipitation experiment demonstrated that the two fragments associated with each other. Therefore, it is strongly suggested that the two fragments formed an active complex of apparently 60 kDa. A mutant of the 60-kDa protein which was apparently resistant to the intramolecular cleavage with the midgut extract of C. pipiens larvae had toxicity slightly lower than that of GST-60.

关键词：氨基酸序列芽孢杆菌苏云金/遗传学芽孢杆菌苏云金/代谢细菌蛋白质类/遗传学细菌蛋白质类/代谢细菌蛋白质类/毒性细菌毒素类/遗传学细菌毒素类/代谢细菌毒素类/毒性碱基序列库蚊属/药物作用 DNA探针/遗传学内毒素类/遗传学内毒素类/代谢内毒素类/毒性溶血素蛋白质类杀虫药/代谢杀虫药/毒性分子序列数据诱变定点质粒/遗传学蛋白质加工转译后重组融合蛋白质类/遗传学重组融合蛋白质类/代谢重组融合蛋白质类/毒性动物

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Rapid SLT gene detection on polyethylene-coacrylic acid film without molecular labels or surface-fouling agents

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ANALYTICAL BIOCHEMISTRY 2000年第2期282卷 218-226页

作者： Zhang, P Fawcett, NC Evans, JA Hurt, T Harvey, KG Craven, RC Univ So Mississippi Dept Chem & Biochem Hattiesburg MS 39406 USA

The commercially available copolymer of 10 mol% acrylic acid and polyethylene is easily formed into a nonfluorescing, non-polynucleotide-adsorbing film. The film has surface carboxylate functions whose concentration can be increased by heating to 80 degrees C in 30% NaOH. The carboxylate groups will react at pH approximate to 7 with commercially available, oligo-DNA, 2-8 ng/mu l, that has been synthesized with a C-12-alkylamino tail on the 5'-end. The reaction is mediated with water-soluble carbodiimide reagent and is assumed to result in a primary amide bond between the polymer film and the modified oligo-DNA. The tethered oligo-DNA retains its hybridization activity, and its surface concentration is sufficient to permit qualitative, labelless detection of hybridized target by fluorescence after brief staining with ethidium bromide. The film is used to detect Shiga-like toxin gene II: (SLT-II) from Escherichia coli O157:H7 after asymmetric, capillary, PCR amplification, and a 4-h hybridization. Captured target may be removed from the film using distilled water, after which the film can be used again without noticeable loss of activity. The method provides relatively rapid detection of PCR amplimers without having to use molecular labels, or surface-fouling agents. (C) 2000 Academic Press.

关键词：丙烯酸盐类/化学细菌毒素类/遗传学大肠杆菌/遗传学胡米胺乙基二甲基氨基丙碳化二亚胺/化学荧光基因细菌核酸杂交/方法聚乙烯/化学聚合酶链反应聚合物/化学氢氧化钠模板遗传

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Detection of Escherichia coli O157:H7 in heifers' faecal samples using an automated immunoconcentration system

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LETTERS IN APPLIED MICROBIOLOGY 2000年第3期30卷 217-222页

作者： Vernozy-Rozand, C Feng, P Montet, MP Ray-Gueniot, S Villard, L Bavai, C Meyrand, A Mazuy, C Atrache, V Ecole Natl Vet Lyon Unite Microbiol Alimentaire & Microbiol Prevision F-69280 Marcy Letoile France US FDA Washington DC 20204 USA BioMerlieux Marcy Letoile France

Pre-treatment of a 5-h enrichment culture with an automated immunoconcentration (ICE) system greatly improved the isolation of Escherichia call O157:H7 from spiked heifer faecal samples. Enrichment samples plated directly onto sorbitol MacConkey agar (SMAC) and SMAC agar supplemented with cefixime and potassium tellurite (CT-SMAC) showed recovery rates of 8% and 56%, respectively. However, after ICE treatment, E. coli O157:H7 was recovered from 92% of the samples on SMAC and 100% on CT-SMAC. Immunoconcentration analysis of heifers' faecal samples collected from a slaughterhouse in France, during March to June 1998, showed that 1% (three of 300) was positive for E. coli O157:H7. Phenotypic and genotypic analysis showed that all three isolates carried both the O157 and H7 antigens, did not ferment sorbitol or had beta-glucuronidase activity and carried trait virulence factors for E. coli O157:H7 (uidA allele, eaeA and pO157 plasmid). However, only one strain was toxigenic and this strain produced a single toxin, namely verotoxin 2.

关键词：黏着素细菌等位基因动物新生细菌外膜蛋白质类/遗传学细菌毒素类/分析细菌毒素类/遗传学载体蛋白质类牛疾病/微生物学培养基大肠杆菌感染/兽医学大肠杆菌O157/遗传学大肠杆菌O157/分离和提纯大肠杆菌蛋白质类粪便/微生物学葡糖醛酸糖苷酶/代谢免疫学技术免疫磁化分离质粒聚合酶链反应志贺毒素2 山梨醇/代谢动物牛

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