Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.     

炭疽杆菌保护性抗原基因的克隆与序列测定

采用聚合酶链反应从炭疽芽孢杆菌减毒株ＹＢ１中扩增其保护性抗原（ＰＡ）的编码区基因,将其克隆至ｐＧＥＭ－Ｔ载体中,并分步测定其序列。序列测定表明,该基因长２２０５ｂｐ,编码７３５个氨基酸残基,与献报道的标准菌株Ｓｔｅｒｎｅ株的ＰＡ序列只有４个碱基的差异。     

炭疽杆菌保护性抗原在大肠杆菌中的表达及其纯化

利用基因重组技术获取炭疽杆菌保护性抗原(PA)。将炭疽杆菌保护性抗原编码基因pag与pET载体连接构建重组质粒,转化大肠杆菌DE3株,诱导表达炭疽杆菌保护性抗原,并经亲和层析及凝胶过滤纯化此抗原。实验成功构建了表达炭疽杆菌保护性抗原的重组菌株,纯化后PA纯度达90%,且经检测纯化产物具有天然PA的生物学活性。同时表明从大肠杆菌中纯化PA较以往从炭疽杆菌中获取PA简便易行。     

炭疽芽孢杆菌保护性抗原在大肠杆菌中的表达及纯化

按照炭疽芽孢杆菌保护性抗原（PA）基因成熟肽编码序列设计引物,从炭疽杆菌pOX1质粒中扩增出PA基因片段,将该片段定向插入到原核表达载体pET-28a中,获得了pET-PA原核表达重组质粒,限制性酶切分析和DNA序列测定均证实该克隆插入片段为PA基因的成熟呔编码序列。将该重组质粒转化大肠杆菌BL21（DE3）,经IPTG诱导,重组蛋白在大肠杆菌表达系统中获得了高效表达;Western印迹分析表明表达产物具有良好的免疫学活性。     

A Bacillus thuringiensis strain producing a polyglutamate capsule resembling that of Bacillus anthracis

Bacillus thuringiensis serovar Monterrey strain BGSC 4AJ1 produced a microscopically visible capsule that reacted with a fluorescent antibody specific for the poly-gamma-d-glutamic acid (PGA) capsule of Bacillus anthracis. PGA capsule biosynthesis genes with 75%, 81%, 72%, 65% and 63% similarity, respectively, to those of the B. anthracis capBCADE cluster were present on a plasmid (pAJ1-1). Strain BGSC 4AJ1, together with five strains of Bacillus cereus that hybridized to a PGA cap gene probe, were analyzed phylogenetically using six housekeeping genes of a B. cereus multilocus sequence typing scheme. Bacillus thuringiensis BGSC 4AJ1 shared four identical alleles with B. anthracis and was the second most closely related to this bacterium of the 674 isolates in the multilocus sequence typing database. The other cap+ strains were distributed among various lineages of Clade 1 of the B. cereus group.     

Production and characterization of neutralizing monoclonal antibodies that recognize an epitope in domain 2 of Bacillus anthracis protective antigen

Antibodies against the protective antigen (PA) of Bacillus anthracis play a key role in response to infection by this important pathogen. The aim of this study was to produce and characterize monoclonal antibodies (mAbs) specific for PA and to identify novel neutralizing epitopes. Three murine mAbs with high specificity and nanomolar affinity for B. anthracis recombinant protective antigen (rPA) were produced and characterized. Western immunoblot analysis, coupled with epitope mapping using overlapping synthetic peptides, revealed that these mAbs recognize a linear epitope within domain 2 of rPA. Neutralization assays demonstrate that these mAbs effectively neutralize lethal toxin in vitro.     

The extracellular and cytoplasmic proteomes of the non-virulent Bacillus anthracis strain UM23C1-2

The recently published genome sequence of Bacillus anthracis Ames has facilitated the prediction of proteins associated with the virulence of this bacterium. The aim of this study was to define reference maps for the extracellular and cytoplasmic proteomes of the avirulent B. anthracis strain UM23C1-2 that are useful for physiological studies and the development of improved vaccines. Using 2-DE and subsequent MALDI-TOF-TOF MS, 64 proteins were identified in the extracellular proteome, only 29 of which were predicted to be exported into the culture medium. The latter included chitinases, proteases, nucleotidases, sulfatases, phosphatases and proteins of unknown function. Of the remaining proteins in the culture medium, 18 were predicted to be associated with the cell wall or anchored on the trans side of the cytoplasmic membrane while 17 other proteins lacked identifiable export signals and were predicted to be cytoplasmic proteins. Among the S-layer proteins, Sap and Eag account for 10% of the total extracellular proteome. Many of the proteins are predicted to contribute to the virulence and antigenic signature of B. anthracis. We have also studied the composition of the cytoplasmic proteome, identifying 300 distinct proteins. The most abundant cytoplasmic proteins are primarily those involved in glycolysis, amino acid metabolism, protein translation, protein folding and stress adaptation. The presence of a variety of proteases, peptidases, peptide binding proteins, as well as enzymes required for the metabolism of amino acids, suggests that B. anthracis is adapted to life in a protein-rich environment rather than the soil. We therefore speculate that proteases and peptidases could be useful targets for the development of improved vaccines. In addition, both of these B. anthracis compartment-specific proteomes can be used as reference maps to monitor changes in the production of secreted and cytosolic proteins that occur, for example, during growth in macrophages.     

炭疽杆菌S层的研究进展

S层是广泛存在于古细菌和真细菌细胞壁最外层的结构独特、功能特殊的成分,对它的研究具有重要的意义。炭疽杆菌是引起人畜炭疽病的病原体,对其S层的研究将为深入认识该菌的生理特性及致病机制等提供坚实的基础。就近年来对炭疽杆菌S层的结构、功能特点及应用前景等方面的研究进展做一简要的综述。     

Differential identification of Bacillus anthracis from environmental Bacillus species using microarray analysis

AIMS: To determine whether microarray analysis could be employed for the differential identification of a range of environmental Bacillus sp. from four strains of Bacillus anthracis. METHODS AND RESULTS: Oligonucleotide probes were designed that were specific to virulence factor genes of B. anthracis (pag, lef and cap), the variable number tandem repeat region of the B. anthracis vrrA gene and to the 16S-23S rRNA intergenic transcribed spacer region (ITS) and pleiotropic regulator (plcR) regions of the Bacillus cereus subgroup species. Generic probes were also designed to hybridize with conserved regions of the 16S rRNA genes of Bacillus (as a positive control), Neisseria sp., Pseudomonas sp., Streptococcus sp., Mycobacterium sp. and to all members of the Enterobacteriaceae to allow simultaneous detection of these bacteria. Identification of B. anthracis was found to rely entirely on hybridization of DNA specific to regions of the pag, lef and cap genes. Cross-reaction was observed between B. anthracis and other Bacillus species with all the other Bacillus probes tested. Results obtained using microarray hybridizations were confirmed using conventional microbiological techniques and found to have very high comparability. CONCLUSIONS: Microarray-based assays are an effective method for the identification of B. anthracis from mixed-culture environmental samples without problems of false-positivity that have been observed with conventional PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of environmental Bacillus sp. by conventional PCR is prone to potential for reporting false-positives. This study provides a method for the exclusion of such isolates.     

Unfolding transitions of Bacillus anthracis protective antigen

Protective antigen (PA) is an 83kDa protein which, although essential for toxicity of Bacillus anthracis, is harmless and an effective vaccine component. In vivo it undergoes receptor binding, proteolysis, heptamerisation and membrane insertion. Here we probe the response of PA to denaturants, temperature and pH. We present analyses (including barycentric mean) of the unfolding and refolding behavior of PA and reveal the origin of two critical steps in the denaturant unfolding pathway in which the first step is a calcium and pH dependent rearrangement of domain 1. Thermal unfolding fits a single transition near 50 degrees C. We show for the first time circular dichroism (CD) spectra of the heptameric, furin-cleaved PA63 and the low-pH forms of both PA83 and PA63. Although only PA63 should reach the acidic endosome, both PA83 and PA63 undergo similar acidic transitions and an unusual change from a beta II to a beta I CD spectrum.     

炭疽杆菌芽孢外壁胶原样蛋白(BclA)的多态性分析

炭疽杆菌芽孢外壁胶原样蛋白（BclA）是芽孢外壁发状菌丝的主要结构成分,也是芽孢的主要免疫原。从国内分离的3株炭疽杆菌中克隆出BclA基因并进行了序列分析,结果发现有2株（A16R和40048）的BclA与国外报道菌株长度不同,分别含有388个和322个氨基酸,72个和50个GXX三氨基酸重复序列,5个和3个含21个氨基酸的（GPT）5 GDTGTT重复序列（BclA重复）。另一株40022的BclA与国外报道的53169株完全一敛,含有370个氨基酸,66个GXX重复,5个BclA重复。对我国炭疽杆菌BclA蛋白多态性的分析为进行炭疽杆菌的基因分型以及研究炭疽芽孢的免疫原性和致病机理打下基础。     

2-D reference map of Bacillus anthracis vaccine strain A16R proteins

Bacillus anthracis has always been an important pathogen because it can cause lethal inhalational anthrax, and may be used as a bioweapon or by bioterrorists. In this study, a 2-DE reference map and database of B. anthracis A16R was constructed. In total, 534 spots were processed, and 406 spots representing 299 proteins were identified. Gel-estimated pIs and molecular masses mostly matched well with their theoretical predictions, but some discrepancies also existed. Spot and protein corresponding analysis revealed that post-translational modifications might be common in B. anthracis. Through the MASCOT search, the similarity of B. anthracis, B. cereus and B. thuringiensis was further verified by protein level and a possible annotation error in B. anthracis strain Ames 0581 genome was found. Proteins of energy metabolism, fatty acid and phospholipid metabolism, protein synthesis, and cellular processes represented a large part of the most abundant proteins. At the same time, 27 hypothetical proteins were experimentally proved. There were 28 proteins also identified as spore composition in recently spore-related research, which indicated that they might play some roles in different phases such as growth, sporulation and outgrowth. Maps and information about all identified proteins are available on the Internet at http://www.mpiib-berlin.mpg.de/2D-PAGE and http://www.proteomics.com.cn.     

利用组氨酸合成酶基因作为同源双交换序列构建在乳酸乳球菌中表达炭疽杆菌保护性抗原的载体

目的:为了实现炭疽杆菌保护性抗原(PA)在乳酸乳球菌中的整合性表达,利用组氨酸合成酶基因(HISB)作为同源交换序列构建表达PA的载体。方法:采用PCR等方法将酸启动子P170、红霉素抗性基因、HISB及酶切位点克隆到pMD18-T载体上,命名为pHEC-P170-PA。结果:构建好的双交换载体经酶切电泳鉴定并测序,其序列中含有PA基因。结论:构建了炭疽杆菌保护性抗原基因同源双交换载体。     

Evaluation of two selective media for the isolation of Bacillus anthracis

Aims:  To evaluate two selective media, polymyxin, lysozyme, ethylenediaminetetraacetic acid, thallium acetate (PLET) agar and R&F Anthracis chromogenic agar (ChrA), for the isolation and selection of Bacillus anthracis .
Methods and Results:  Sixteen genotypically diverse B. anthracis strains were sub-cultured onto PLET and ChrA to test the sensitivity (ability of B. anthracis to grow and produce expected colony morphology) of both media. Fourteen of the 16 B. anthracis strains produced the expected morphology on PLET (88% sensitive) while 13/16 produced the expected morphology on the ChrA medium (81% sensitive). Seventeen other Bacillus strains and 18 non Bacillus spp. strains were used to evaluate the media's selectivity (ability to inhibit non- B. anthracis growth). PLET inhibited growth of 14/35 strains (40% selective), including six Bacillus strains, while ChrA inhibited 3/35 (9% selective). In addition, we did not observe any differences between the recovered CFU on PLET or ChrA when plating extractions of spiked soil.
Conclusions:  Polymyxin, lysozyme, ethylenediaminetetraacetic acid, thallium acetate agar was more selective and sensitive than ChrA.
Significance and Impact of the Study:  Although both media are more expensive than sheep blood agar, for samples with high numbers of bacteria, they can be used to isolate B. anthracis with proper training and experience and with the knowledge that there are limitations to each media.     

炭疽杆菌水肿因子基因的克隆与序列测定

采用聚合酶链反应（ＰＣＲ）从炭疽芽孢杆菌减毒株ＹＢ１中扩增其水肿因子（ＥＦ）的编码区基因,将其克隆至ｐＧＥＭ－Ｔ载体中,并分步测定其序列。序列测定表明,该基因长２ ３０１ ｂｐ,编码７６７个氨基酸,与已报道的Ｓｔｅｒｎｅ标准株的ＥＦ基因完全一致。     

炭疽芽孢杆菌分子分型研究进展

随着分子生物学的发展,分子分型技术被广泛应用于鉴别炭疽芽孢杆菌菌株间的遗传相关性和流行病学特征。我们就近年来常用的炭疽芽孢杆菌分子分型方法的优缺点和研究进展进行综述。     

Formaldehyde gas inactivation of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials

AIMS: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using formaldehyde gas. METHODS AND RESULTS: B. anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to approx. 1100 ppm formaldehyde gas for 10 h. Formaldehyde exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with B. subtilis (galvanized metal and painted wallboard paper) and G. stearothermophilus (industrial carpet and painted wallboard paper). Formaldehyde gas inactivated>or=50% of the biological indicators and spore strips (approx. 1x10(6) CFU) when analyzed after 1 and 7 days. CONCLUSIONS: Formaldehyde gas significantly reduced the number of viable spores on both porous and nonporous materials in which the two surrogates exhibited similar log reductions to that of B. anthracis on most test materials. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using formaldehyde gas.     

The osmoprotectants glycine and its methyl derivatives prevent the thermal inactivation of protective antigen of Bacillus anthracis

Protective antigen (PA) is the main immunogenic constituent of all vaccines against anthrax. It is known to lose its biological activity even at 37 degrees C. Its thermolabile nature has, thus, remained a cause of concern as even transient exposure of the vaccine to higher temperature could compromise its efficacy. Various types of cosolvent excipients have been used to stabilize a number of proteins with variable success. However, no comprehensive and systematic study to stabilize anthrax PA molecule using this approach has ever been undertaken. We have carried out a systematic study on the effect of osmoprotectants like glycine and its methyl derivatives, sarcosine, dimethylglycine, and betaine, on the thermostability of PA. The thermal stability of PA was found to be highly sensitive to pH with maxima at pH 7.9. All the cosolvent additives used were able to enhance the thermal stability of PA as inferred from an increase in T(1/2) values, the temperature at which 50% activity was retained during short-term incubation. Glycine was found to be the best stabilizer, while the ability of its methyl derivatives to stabilize PA decreased with an increase in the number of substituted methyl groups suggesting perturbation of hydrophobic interactions. On extended incubation at 40 degrees C the half-life of PA thermal inactivation increased more than four times in the presence of glycine. Thus, glycine could be used as an effective stabilizer to enhance the shelf life of recombinant vaccine against anthrax.