Localization of heat shock proteins in clinical Actinobacillus actinomycetemcomitans strains and their effects on epithelial cell proliferation

Actinobacillus actinomycetemcomitans is an important pathogen in periodontitis. In the present study we localized the GroEL- and DnaK-like heat shock proteins (Hsp) in subcellular fractions of 12 A. actinomycetemcomitans strains of various clinical origin and compared their effects on periodontal epithelial cell proliferation and viability. In all strains, GroEL-like protein was found in the membrane, cytoplasm, and periplasm, whereas DnaK-like protein was present in the cytoplasm and periplasm. No correlation was observed between the Hsp expression and the serotype or origin of A. actinomycetemcomitans strains. The bacterial membrane fractions that expressed the GroEL-like protein moderately or strongly induced epithelial cell proliferation more strongly than strains that expressed the protein weakly. The results suggest that GroEL-like Hsp may play a role in the virulence of A. actinomycetemcomitans by increasing epithelial proliferation.     

Construction and characterization of a live, attenuated apxIICA inactivation mutant of Actinobacillus pleuropneumoniae lacking a drug resistance marker

The apxIIC gene of Actinobacillus pleuropneumoniae serotype 7 was inactivated by homologous recombination using a sucrose counter-selectable marker system, resulting in a mutant strain that had no antibiotic resistance marker and expressed an inactivated ApxII toxin. The safety and immunogenicity of the mutant were evaluated in mice. The mutant strain caused no adverse effects in mice at doses up to 2 x 10(9) CFU via the intraperitoneal route while the parental strain induced total mortality at a dose of 2 x 10(7) CFU. Mice vaccinated intraperitoneally with the mutant strain had 100% and 70% protection against homologous (serotype 7) or heterologous (serotype 1, 3) challenge with A. pleuropneumoniae, respectively. The A. pleuropneumoniae mutant strain HB04C- and the counterselection method used in the study show promise in developing effective live vaccines for porcine pleuropneumonia and for other infections diseases of the respiratory system.     

发酵法生产丁二酸的化学合成培养基的筛选

以产琥珀酸放线杆菌Actinobacillus succinogenes NJ113 为出发菌株,针对该菌株筛选出含有关键生长因子的化学合成培养基,其关键因子为谷氨酸（Glu）、蛋氨酸（Met）和生物素（VH）和烟酸（VPP）。结合原发酵培养基中的磷酸缓冲盐成分,最终得到的化学合成培养基配方（g/L）： CH3COONa 1.36,NaCl 1.0,MgCl2 0.2,CaCl2 0.2,Na2HPO4 0.31,NaH2PO4 1.6, KH2PO4 3,NH4HCO3 1.57,Glu 0.87,Met 0.11,VH 0.010,VPP 0.025。在3 L发酵罐上进行验证实验,50 g/L初始葡萄糖发酵70 h,丁二酸的质量浓度为45.2 g/L,丁二酸收率达到90.4%。与之前的半合成培养基发酵制备丁二酸相比,丁二酸的收率提高了25.2%,副产物也有很大幅度的减少。     

A complete industrial system for economical succinic acid production by Actinobacillus succinogenes

An industrial fermentation system using lignocellulosic hydrolysate, waste yeast hydrolysate, and mixed alkali to achieve high-yield, economical succinic acid production by Actinobacillus succinogenes was developed. Lignocellulosic hydrolysate and waste yeast hydrolysate were used efficiently as carbon sources and nitrogen source instead of the expensive glucose and yeast extract. Moreover, as a novel method for regulating pH mixed alkalis (Mg(OH)₂ and NaOH) were first used to replace the expensive MgCO₃ for succinic acid production. Using the three aforementioned substitutions, the total fermentation cost decreased by 55.9%, and 56.4 g/L succinic acid with yield of 0.73 g/g was obtained, which are almost the same production level as fermentation with glucose, yeast extract and MgCO₃. Therefore, the cheap carbon and nitrogen sources, as well as the mixed alkaline neutralize could be efficiently used instead of expensive composition for industrial succinic acid production.     

Prospects for a bio-based succinate industry

Bio-based succinate is receiving increasing attention as a potential intermediary feedstock for replacing a large petrochemical-based bulk chemical market. The prospective economical and environmental benefits of a bio-based succinate industry have motivated research and development of succinate-producing organisms. Bio-based succinate is still faced with the challenge of becoming cost competitive against petrochemical-based alternatives. High succinate concentrations must be produced at high rates, with little or no by-products to most efficiently use substrates and to simplify purification procedures. Herein are described the current prospects for a bio-based succinate industry, with emphasis on specific bacteria that show the greatest promise for industrial succinate production. The succinate-producing characteristics and the metabolic pathway used by each bacterial species are described, and the advantages and disadvantages of each bacterial system are discussed.     

Expression of the Apx toxins ofActinobacillus pleuropneumoniae inSaccharomyces cerevisiae and its induction of immune response in mice

Actinobacillus pleuropneumoniae is an important pig pathogen, which is responsible for swine pleuropneumonia, a highly contagious respiratory infection. To develop subunit vaccines forA. pleuropneumoniae infection, the Apx toxin genes,apxI andapxII, which are thought to be important for protective immunity, were expressed inSaccharomyces cerevisiae, and the induction of immune responses in mice was examined. TheapxI andapxII genes were placed under the control of a yeast hybridADH2-GPD promoter (AG), consisting of alcohol dehydrogenase II (ADH2) and theGPD promoter. Western blot analysis confirmed that both toxins were successfully expressed in the yeast. The ApxIA and ApxIIA-specific IgG antibody response assays showed dose dependent increases in the antigen-specific IgG antibody titers. The challenge test revealed that ninety percent of the mice immunized with ApxIIA or a mixture of ApxIA and ApxIIA, and sixty percent of mice immunized with ApxIA survived, while none of those in the control groups survived longer than 36 h. These results suggest that vaccination of the yeast expressing the ApxI and ApxII antigens is effective for the induction of protective immune responses againstA. pleuropneumoniae infections in mice.     

胸膜肺炎放线杆菌萘啶酸抗性菌株的选育和信号标签突变株的构建

胸膜肺炎放线杆菌(APP)是重要的猪呼吸道病原菌,给世界养猪业造成严重的经济损失.信号标签突变(STM)技术是在宿主动物体内鉴定病原菌毒力因子的高通量方法.通过体外传代选育出APP血清1型和3型萘啶酸抗性菌株,再以萘啶酸抗性菌株为受体菌,以携带mini-Tn10的标签质粒(pLOF/TAG1-48)的E.coli CC118 λ pir或S17-1λpir为供体菌,在或不在E.coli DH5α(pRK2073)的辅助下,进行三亲本或两亲本接合,通过抗性筛选、PCR和Southern杂交鉴定转座突变株.结果表明:体外萘啶酸加压传代很容易选育出萘啶酸抗性APP菌株,该抗性的产生与DNA促旋酶A亚基基因gyrA的突变有关.在APP与E. coli接合实验中,两亲本接合比三亲本接合操作更简单,效率也较高;APP不同菌株在接合和转座效率上存在很大差异,血清1型菌株高于血清3型菌株,3型标准菌株高于地方分离株JL03-R.本研究为APP STM突变体库的构建与毒力基因的鉴定奠定了基础.     

复合PCR鉴定胸膜肺炎放线杆菌方法的建立及初步应用

根据胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,App)apxIVA毒素基因序列和16SrRNA序列分别设计了一对特异性引物P1P4和一对通用引物S7S10,建立了检测App全部15个血清型的复合PCR方法。对App的15个血清型国际参考株和国内的11个App菌株进行检测,都能得到363bp和692bp的两个扩增片段。而放线杆菌等13株参考菌株只能得到692bp的扩增片段。该方法能将15个血清型的App菌株鉴定到种。检测的灵敏度达9pgDNA1300CFU。用建立的方法检测临床分离的302株可疑菌株,阳性4株,与其它鉴定方法相符。结果表明复合PCR可用于App菌株的鉴定。     

胸膜肺炎放线杆菌三聚体自转运黏附素茎部功能区表达及其与猪肺组织结合蛋白的筛选分析

【目的】胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)可引发猪的传染性胸膜肺炎,给世界养猪业造成了巨大损失。黏附是APP在致病过程中的第一步,新型黏附分子——三聚体自转运黏附素(Trimeric autotransporter adhesin,TAA)是该菌感染肺组织的重要毒力因子,茎部区2 464-2 574氨基酸(BD3)序列区域在细菌的黏附中发挥重要作用,但其如何结合肺脏组织尚属未知。本文表达TAA的茎部功能区,筛选其在肺组织中的结合蛋白。【方法】原核表达及纯化TAA功能区BD3蛋白,与猪肺组织共孵育,通过免疫共沉淀技术捕获BD3的结合蛋白并质谱鉴定。构建猪c DNA文库获取该蛋白核酸序列,进行生物信息学分析。【结果】经质谱分析获得与BD3结合的猪肺组织蛋白的肽段,数据库搜寻比对发现角蛋白1为TAA BD3区与猪肺组织结合的蛋白;构建c DNA文库筛选并测序后获知其基因序列。生物信息学分析显示该序列与猪源及人源角蛋白核酸序列相似性低,猪源细胞角蛋白1作为一个单独的进化分支,与其他角蛋白差异较大;该蛋白在8-100 aa处有一个跨膜区;主要二级结构元件为α螺旋和β折叠;该蛋白在82-362 aa处存在一个G蛋白α亚单位,在515-552 aa处存在一个TSP1区域(凝血酶敏感蛋白1型重复区域)。【结论】与TAA茎部BD3结合的猪肺组织角蛋白1的发现,为TAA黏附肺组织细胞的研究奠定基础,有助于揭示APP专嗜肺组织的致病机制。