Simplified procedure for purification of Bordetella bronchiseptica dermonecrotic toxin

Abstract Bordetella bronchiseptica dermonecrotic toxin was purified by a simplified method. The method consisted of SP Toyopearl 650M chromatography and high performance liquid chromatography on a TSK gel G3000SW column. 47.5% of the activity of the crude cell extract was recovered. The purified toxin behaved as a homogeneous protein in sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance liquid chromatography, and agar gel double diffusion tests.     

支气管败血波氏杆菌皮肤坏死毒素的重组表达及其生物学特性

皮肤坏死毒素(DNT)是支气管败血波氏杆菌的主要致病因子之一.通过PCR分段扩增和克隆获得了全长4 356 bp的dnt基因,并利用pET-28a/BL21系统对其进行了融合表达.Western blotting检测结果表明,表达产物具有良好的免疫学活性.使用His-band purification kit纯化试剂盒纯化后,得到纯度为93.2％的融合蛋白His6-DNT.在乳鼠皮肤坏死试验中,表达产物His6-DNT和天然DNT均能导致乳鼠皮肤产生坏死性病变.在乳鼠皮肤坏死阻断试验中,兔抗His6-DNT血清能中和天然DNT使其失去对乳鼠的皮肤坏死毒性.试验结果表明,重组蛋白具有天然DNT的生物学毒性和免疫原性,所产生的抗体具有中和活性.文中所获得的重组DNT蛋白具有良好的生物学活性,为DNT的结构和功能研究奠定基础.     

Effects of Bordetella bronchiseptica dermonecrotizing toxin on bone formation in calvaria of neonatal rats

Abstract The effects of Bordetella bronchiseptica dermonecrotizing toxin on bone formation were investigated using a purified toxin preparation. Single injection of 4.3 ng of the toxin into the subcutaneous tissue overlying the calvariae of neonatal rats necrotized periosteum of parietal bone and degenerated osteoblasts within two days. Nine days after the injection, the lesion of the bone tissue became severe; the bone matrix became thin and fragmented. These observations indicate that dermonecrotizing toxin without other factors produced by the organisms impairs bone formation.     

Pseudomonas aeruginosa rhamnolipids disperse Bordetella bronchiseptica biofilms

We have previously reported that the respiratory pathogen Bordetella bronchiseptica can form biofilms in vitro. In this report, we demonstrate the disruption of B. bronchiseptica biofilms by rhamnolipids secreted from Pseudomonas aeruginosa. This suggests that biosurfactants such as rhamnolipids may be utilized as antimicrobial agents for removing Bordetella biofilms.     

兔支气管败血波氏杆菌单克隆抗体的制备及鉴定

目的建立能稳定分泌抗兔支气管败血波氏杆菌（Bb）的单克隆抗体杂交瘤细胞株,为今后进一步建立该菌的免疫检测技术奠定基础。方法以Bb分离株BLJ05的灭活菌液为免疫原,腹腔免疫BALB/c小鼠,采用常规杂交瘤技术制备Bb单克隆抗体（McAb）,用间接ELISA、Western-blot等方法对McAb特性进行鉴定。结果获得两株能稳定分泌抗Bb单克隆抗体的杂交瘤细胞株,分别命名为A7D5和D6B2,其小鼠腹水抗体效价分别为1∶409600和1∶102400;且不与兔大肠杆菌、多杀性巴氏杆菌、产气荚膜梭菌等兔的常见病原菌反应,特异性强。两株单抗亲和力实验表明A7D5亲和力略高于D6B2。ELISA相加试验表明它们针对相同的抗原表位。结论成功建立了两株能稳定分泌抗兔支气管败血波氏杆菌单克隆抗体的杂交瘤细胞株,效价高、特异性强,为今后建立该菌的免疫检测技术建立奠定了基础。     

重组百日咳杆菌黏附素蛋白免疫小鼠可完全抵抗支气管败 血波氏杆菌的致死性感染

[目的]通过建立的小鼠呼吸道感染模型评价重组百日咳杆菌黏附素蛋白(GST-PRN)对小鼠的免疫保护效力.[方法和结果]在主动免疫保护试验中,GST-PRN免疫组小鼠能产生较高的PRN抗体水平,在使用3xLD50的支气管败血波氏杆菌HH0809株进行呼吸道气雾攻毒后,其保护率为100%(20/20),但载体蛋白GST和PBS对照组小鼠的存活率仅为15%(3/20)和20%(4/20).在被动免疫保护试验中,腹腔免疫GST-PRN兔抗血清能100%(10/10)保护小鼠抵抗10×LD50的HH0809株的腹腔攻击,但GST兔抗血清和PBS免疫组小鼠的存活率均为0(0/10和0/9).[结论]研究结表明重组PRN蛋白具有良好的免疫学活性,可作为亚单位疫苗或疫苗添加成分.     

Bordetella bronchiseptica dermonecrotizing toxin stimulates protein synthesis in an osteoblastic clone, MC3T3-E1 cells

Abstract The effects of Bordetella bronchiseptica dermonecrotizing toxin on protein synthesis in an osteoblastic clone, MC3T3-E1 cells, were investigated. The rate of protein synthesis in the serum-starved cells was increased by the toxin after a latent period of about 4 h, and reached 2.5 times that of the control 24 h after addition of toxin. The toxin raised the level of protein synthesis even in actively proliferating cells. The stimulatory effect of the toxin on protein synthesis occurred earlier than other toxic events so far reported, such as the stimulation of DNA synthesis and the inhibition of osteoblastic differentiation, and was apparently dependent on the toxin concentrations over the range 0.05 ng ml⁻¹ to 6.0 ng ml⁻¹. Therefore, the stimulatory effect of the toxin on protein synthesis could be useful in determining the mode of action of the toxin.     

家兔支气管败血波氏杆菌重组蛋白DNT1的原核表达及其间接ELISA方法的建立

目的表达支气管败血波氏杆菌（Bordetella bronchiseptica,Bb）DNT蛋白,并以此建立检测Bb抗体的间接ELISA方法。方法参照GenBank公布的猪源支气管败血波氏杆菌dnt基因序列（AB020025）针对其N-端设计了一对特异性引物,PCR扩增出相应的核苷酸片段。将PCR扩增产物连接至原核表达载体pET-28a（＋）载体中,以E.coli BL21（DE3）为表达菌株进行诱导表达,以纯化重组蛋白DNT1作为诊断抗原,通过探索最佳抗原包被量和抗体血清稀释倍数,建立检测支气管败血波氏杆菌重组蛋白DNT1抗体的ELISA方法。结果成功克隆了dntN-端的基因序列,并在E.coli BL21（DE3）中获得高效表达,经SDS-PAGE、Western blot分析显示重组蛋白DNT1具有良好的抗原性。应用重组蛋白DNT1为抗原建立了检测Bb血清抗体的间接ELISA诊断方法。试验确定重组蛋白DNT1抗原的包被浓度为6.25μg/mL,最适血清稀释度为1∶100。结论建立的ELISA检测方法,不仅为Bb抗体检测提供了一种比较实用的血清学检测手段,也为进一步开发Bb检测试剂盒奠定了基础。     

Bordetella dermonecrotic toxin binds to target cells via the N-terminal 30 amino acids

Bordetella dermonecrotic toxin (DNT) affects the biological function of host cells by activating intracellular Rho GTPases. The toxin binds to unidentified receptor(s) via 54 N-terminal amino acids, undergoes intramolecular cleavage on the C-terminal side of Arg(44) by furin or furin-like protease, and eventually enters the cytoplasm where the Rho GTPases reside. The binding to the receptor(s) and intramolecular cleavage are essential for DNT to intoxicate cells, and the 54 amino-acid binding domain encompasses the cleavage site, however, it is unclear whether these two events are related. In this study, we could narrow down the cell-binding domain to the N-terminal amino acids 2-30. The region does not contain the furin-recognition site, indicating that the cell binding and the intramolecular cleavage are independent events.     

兔支气管败血波氏杆菌PCR检测方法的建立及应用

目的建立一种简便、快速、敏感、特异的适用于支气管败血波氏杆菌的PCR检测方法。方法根据兔支气管败血波氏杆菌（Bordetella bronchiseptica）的fim2基因序列设计了一对特异性引物,进行PCR扩增、特异性和敏感性试验,并将其应用于临床样品的检测。结果利用该PCR方法扩增出425bp的目的基因片段,该产物序列与GeneBank上公布的基因序列同源性为100％。特异性试验表明,该方法对大肠埃希氏菌、多杀性巴氏杆菌、魏氏梭菌和金黄色葡萄球菌均无交叉性反应;并且最小可检出菌液浓度为3．6CPU。用该PCR方法检测了从江苏、山东等地采集的146份兔鼻拭子,结果检出支气管败血波氏杆菌阳性92例,阳性率为63．01％。结论建立了快速检测支气管败血波氏杆菌的PCR方法。     

Variation in Bordetella bronchiseptica lipopolysaccharide during human infection

We previously reported the case of a human chronic Bordetella bronchiseptica respiratory infection, due to contact with infected rabbits. Lipopolysaccharides of the human isolates, of one rabbit isolate and of isolates from other origins were analyzed with sera from infected mice, rabbit and human. Antigenicity and length of the lipopolysaccharide molecules varied between isolates. We showed a progressive loss of O-chain during infection, associated with an enhanced susceptibility of the isolates to the bactericidal effect of normal serum. This observation suggests the existence of an intracellular niche which selects for strains with distinct lipopolysaccharide types.     

Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from Loxosceles intermedia (brown spider) venom gland

Brown spider (Genus Loxosceles) bites are normally associated with necrotic skin degeneration, gravitational spreading, massive inflammatory response at injured region, platelet aggregation causing thrombocytopenia and renal disturbances. Brown spider venom has a complex composition containing many different toxins, of which a well-studied component is the dermonecrotic toxin. This toxin alone may produce necrotic lesions, inflammatory response and platelet aggregation. Biochemically, dermonecrotic toxin belongs to a family of toxins with 30-35 kDa characterized as sphingomyelinase-D. Here, employing a cDNA library of Loxosceles intermedia venom gland, we cloned and expressed two recombinant isoforms of the dermonecrotic toxin LiRecDT2 (1062 bp cDNA) and LiRecDT3 (1007 bp cDNA) that encode for signal peptides and complete mature proteins. Phylogenetic tree analysis revealed a structural relationship for these toxins compared to other members of family. Recombinant molecules were expressed as N-terminal His-tag fusion proteins in Escherichia coli and were purified to homogeneity from cell lysates by Ni(2+) chelating chromatography, resulting in proteins of 33.8 kDa for LiRecDT2 and 34.0 kDa for LiRecDT3. Additional evidence for related toxins containing sequence/epitopes identity comes from antigenic cross-reactivity using antibodies against crude venom toxins and antibodies raised with a purified dermonecrotic toxin. Recombinant toxins showed differential functionality in rabbits: LiRecDT2 caused a macroscopic lesion with gravitational spreading upon intradermal injection, while LiRecDT3 evoked transient swelling and erythema upon injection site. Light microscopic analysis of skin biopsies revealed edema, a collection of inflammatory cells in and around blood vessels and a proteinaceous network at the dermis. Moreover, differential functionality for recombinant toxins was also demonstrated by a high sphingomyelinase activity for LiRecDT2 and low activity for LiRecDT3 as well as greater in vitro platelet aggregation and blood vessel permeability induced by LiRecDT2 and residual activity for LiRecDT3. Cloning and expression of two recombinant dermonecrotic toxins demonstrate an intraspecific family of homologous toxins that act in synergism for deleterious activities of the venom and open possibilities for biotechnological applications for recombinant toxins as research tools for understanding the inflammatory response, vascular integrity and platelet aggregation modulators.     

Pertactin antigens extracted from Bordetella pertussis and Bordetella bronchiseptica differ in the isoelectric point

Pertactin, which is a membrane-associated antigen of Bordetella pertussis and which is present in many acellular vaccines against whooping cough, has been reported to be similar to the homologous protein in Bordetella bronchiseptica. By running parallel experiments using proteins derived from the two species, we show that the isoelectric point of pertactin from B. pertussis is lower than reported and clearly distinguishable from the homologous protein of B. bronchiseptica. Received: 9 April 1997 / Accepted: 20 May 1997     

支气管败血波氏杆菌外膜蛋白的提取及其免疫效果的检测

本研究以小鼠为实验动物模型,研究支气管败血波氏杆菌的外膜蛋白(OMP)和其中的有效保护抗原成分(OMP68)的免疫原性及免疫保护作用.本研究利用改进的Wooldridge的方法提取了支气管败血波氏杆菌P11和P13的OMP,利用SDS-PAGE比较了2株之间差异,采用Western-blotting进行了分析并确定了P13菌株OMP(P13-OMP)中的有效保护性抗原成分,采用电洗脱方法获的分子量为68 kD的P13-OMP有效保护抗原成分(OMP68),然后制备了油乳剂P13的全菌、P13-OMP和OMP68免疫抗原.抗体动态变化:将清洁级小鼠70只随机分成7组,10只/组,用油乳剂P13的全茵和不同剂量的P13-OMP和OMP68免疫抗原分别免疫,采用间接ELISA检测免疫小鼠的相应抗体水平并分析抗体动态变化;主动免疫保护试验:将清洁级小鼠80只随机分成8组,10只/组,分别采用制备的油乳剂P13-OMP(OMP25 μg/只)、OMP68(25 μg/只)和P13菌体免疫抗原在免疫10 d后,分别用100 LD50的P11和P13腹腔攻毒,然后统计保护率并分析免疫原所提供的免疫保护力.免疫P13-OMP和OMP68油乳剂抗原后,7 d时,抗体水平开始逐渐上升,42 d时,抗体水平分别达到215.3和214.8,然后逐渐下降,70 d时,抗体水平分别达到29.1和210.2.主动免疫时,P11和P13攻毒的P13-OMP免疫组分别均得到9/10和9/10的保护,OMP68免疫组分别均得到9/10和10/10的保护,而P13全菌免疫组分别得到4/10和6/10保护,对照组全部死亡.P13-OMP和OMP68抗原均具有良好的免疫原性和免疫保护作用,为支气管败血波氏杆菌OMP亚单位疫苗的研制奠定了良好的理论基础.     

猪源支气管败血波氏杆菌百日咳杆菌黏附素基因的原核表达及其抗体检测ELISA方法

[目的]以猪源支气管败血波氏杆菌(Bordetella bronchiseptica,Bb)百日咳杆菌黏附素(PRN)基因的原核表达产物为抗原建立检测PRN抗体的间接ELISA方法.[方法和结果]利用谷胱甘肽-S-转移酶(GST)表达系统对PRN基因在大肠杆菌中进行融合表达.SDS-PAGE和Western blot检测证实该基因获得高效表达,产物易于纯化且具有良好的免疫学活性.通过凝血酶酶切GST-PRN并回收,获得不含GST载体蛋白的PRN蛋白片段.以PRN蛋白片段为抗原建立检测天然PRN抗体的间接ELISA方法.该方法对猪巴氏杆菌病等7种常见细菌性疾病阳性血清的检测结果均为阴性,其敏感性比乳胶凝集试验提高4～128倍,能检测到人工感染14 d后的仔猪血清抗体IgG,对临床送检的1,229份猪血清的检测阳性率为32.7%.ELISA方法对阳性猪场的监测结果预示了保育期仔猪的合群导致猪群大量感染支气管败血波氏杆菌.[结论]该方法具有特异性强、敏感性高、重复性好的特点,可用于猪群PRN抗体水平监测和猪波氏菌病流行病学调查.     

百日咳杆菌粘附素对猪源支气管败血波氏杆菌的保护性抗原的筛选

摘要：【目的】本研究通过百日咳杆菌黏附素(PRN)基因的分段克隆表达及其在BALB/c小鼠的主动和被动免疫保护试验筛选PRN中的保护性抗原肽。【方法和结果】利用大肠杆菌进行PRN的完整蛋白、N端和C端多肽及其RI和RII区域多肽（双拷贝）的表达,命名为GST-PRN、GST-PN、GST-PC、GST-2PRI和GST-2PRII。Western blot检测证实5种表达产物均具有良好的反应原性。在主动免疫保护试验中,5种表达产物均能诱导小鼠产生较高的PRN抗体水平;当使用3 LD50的支气管败血波氏杆菌     

A deep rough type structure in Bordetella bronchiseptica lipopolysaccharide modulates host immune responses

The present authors have previously obtained the Bordetella bronchiseptica mutant BbLP39, which contains a deep-rough lipopolysaccharide (LPS) instead the wild type smooth LPS with O antigen. This mutant was found to be altered in the expression of some proteins and in its ability to colonize mouse lungs. Particularly, in BbLP39 the expression of pertactin is decreased. To differentiate the contribution of each bacterial component to the observed phenotype, here mice defective in the LPS sensing receptor TLR4 (TLR4-defective mice) were used. In contrast to wild-type mice, infection of TLR4-defective mice with BbLP39 resulted in lung infection, which persisted for more than 10 days post-challenge. Comparative analysis of the immune responses induced by purified mutant and wild type LPSs showed that the mutant LPS induced significantly higher degrees of expression of TNF-α and IL-10 mRNA than did the wild type. UV matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry analysis revealed that both LPSs had the same penta-acylated lipid A structure. However, the lipid A from BbLP39 contained pyrophosphate instead of phosphate at position 1. This structural difference, in addition to the lack of O-antigen in BbLP39, may explain the functional differences between BbLP39 and wild type strains.     

Iron starvation regulates the type III secretion system in Bordetella bronchiseptica

The type III secretion system (T3SS) plays a key role in the exertion of full virulence by Bordetella bronchiseptica. However, little is known about the environmental stimuli that induce expression of T3SS genes. Here, it is reported that iron starvation is a signal for T3SS gene expression in B. bronchiseptica. It was found that, when B. bronchiseptica is cultured under iron-depleted conditions, secretion of type III secreted proteins is greater than that in bacteria grown under iron-replete conditions. Furthermore, it was confirmed that induction of T3SS-dependent host cell cytotoxicity and hemolytic activity is greatly enhanced by infection with iron-depleted Bordetella. In contrast, production of filamentous hemagglutinin is reduced in iron-depleted Bordetella. Thus, B. bronchiseptica controls the expression of virulence genes in response to iron starvation.     

Expression of urease does not affect the ability of Bordetella bronchiseptica to colonise and persist in the murine respiratory tract

To investigate the role played by urease during the Bordetella bronchiseptica infection process, the ability to colonise and persist in the mouse respiratory tract of a urease-negative B. bronchiseptica BB7865 and a BB7865 derivative constitutively expressing urease was compared with that of the wild-type strain. The results obtained showed that neither constitutive expression nor abolishment of urease activity had any significant effect on the course of B. bronchiseptica infection. Therefore, under our experimental conditions, urease is not essential for B. bronchiseptica to colonise and persist within the murine host.