An efficient fusion protein system for expression of Bacillus anthracis protective antigen as immunogenic and diagnostic antigen

Objective:To produce high quantities of recombinant protective antigen(rPA) for human vaccine and diagnosis.Methods:The PA gene was amplified by PCR with pXO1 plasmid as template. The PCR product was cloned into pMAL-c2X vector using the BamH1 and SalI restriction enzymes.The recombinant plasmid was transformed into Escherichia coli DH5 a strain and then screened for transformation.The expression of protective antigen was analyzed by SDS-PAGE and Western blotting after isopropyl β-D-thiogalactopyranoside(IPTG) induction.Results: The full-length PA gene(2.2 kb) was cloned into pMAL vector system.The recombinant vector was confirmed by restriction enzyme and PCR analysis.The expression of cytoplasmic maltose-binding protein-protective(MBP-P) antigen fusion protein was detected by SDS-PAGE and Western blotting,and obtained a 125 kDa protein band,which was similar to expected size of fusion protein.Conclusions:This expression system can be used in the high production of rPA. After purification and immunization studies,the purified rPA may be used in the development of the human recombinant anthrax vaccine and also in diagnosis of anthrax disease.

Role of the Putative Polysaccharide Deacetylase BA1836 from <i>B. anthracis</i>in Spore Development and Germination

Putative and known polysaccharide deacetylases (PDAs) from B. anthracis have key roles in resistance to host lysozyme, stabilization of the cell wall, biogenesis of peptidoglycan (PG) and for neutral polysaccharide modification and attachment to PG. Here we elucidated the physiological role of the putative PDA BA1836 from B. anthracis. The ba1836 gene was expressed upon entrance into the stationary phase of growth and enhanced during the early stages of sporulation. The Δba1836 knockout strain had normal growth rate, did not exhibit any significant alterations in PG pattern of stationary phase cells and was not sensitive to lysozyme, but showed a defect in cell separation. Strikingly, the Δba1836 mutant strain exhibited a severe delay in spore development although mature spores were ultimately developed and had normal morphology. Additionally, digestion of Δba1836 mutant spore PG with mutanolysin produced an almost identical muropeptide pattern compared to peptidoglycan from wild type spores, although the amount of all muropeptides was significantly reduced. Finally, knockout spores exhibited a lower germination rate. To our knowledge, BA1836 has a unique role, among the presently characterized PDAs from B. anthracis, in spore development and germination.

Application of High-Throughput Sequencing: Discovery of Informative SNPs to Subtype <i>Bacillus anthracis</i>

Single Nucleotide Polymorphisms (SNPs) are the most common and abundant genetic variation found in the genome of any living species, from bacteria to humans. In bacterial genotyping, these evolutionarily stable point mutations represent important DNA markers that can be used to elucidate deep phylogenetic relationships among worldwide strains, but also to discriminate closely related strains. With the advent of next generation sequencing (NGS) technologies, affordable solutions are now available to get access to the complete genome sequence of an organism. Sequencing efforts of an increasing number of strains provide an unprecedented opportunity to create comprehensive species phylogenies. In this study, a comparative analysis of 161 genomes of Bacillus anthracis has being conducted to discover new informative SNPs that further resolves B. anthracis SNP-based phylogenetic tree. Nine previously unpublished SNPs that define major groups or sub-groups within the B. anthracis species were selected and developed into SNP discriminative assays. We report here a cost-effective high-resolution melting-based genotyping method for the screening of 27 canonical SNPs that includes these new diagnostic markers. The present assays are useful to rapidly assign an isolate to one sub-lineages or sub-groups and determine its phylogenetic placement on the B. anthracis substructure population.

<i>Bacillus pumilus</i>: Possible Model for the Bioweapon <i>Bacillus anthracis</i>

The misuse of Bacillus anthracis as a bioweapon continues to be a serious concern. Medical personnel and researchers are served well if appropriate non-pathogenic anthrax simulants can be used as countermeasures in preparative planning. While there are several accepted simulants of B. anthracis, the addition of another model organism would be beneficial. This investigation was undertaken to evaluate the suitability of B. pumilus as a simulant for B. anthracis. All organisms were grown on AK Agar #2 to foster sporulation. Optimum conditions for spore formation were determined for B. pumilus as well as for currently used anthrax surrogates B. atrophaeus and B. thuringiensis. Spore dimensions were determined by scanning electron microscopy. Comparative antibody binding studies using commercially available anti-Bacillus antisera were completed with the simulants as well as with a negative control organism, Clostridium sporogenes. We report that B. pumilus sporulated readily (2.9 × 1010 viable spores per plate), had appropriate spore size (1.24 μm × 0.59 μm) and reactivity with anti-Bacillus antibodies. The characteristics of B. pumilus determined in this study suggest this organism represents a novel, suitable model for B. anthracis.

一种荧光碳点R-CDs对炭疽芽孢杆菌标志物的响应检测

采用简单的水解法制备荧光探针R-CDs,高灵敏、可视化地现场检测炭疽芽孢杆菌(Bacillus anthracis)孢子标志物,探索一种荧光碳点R-CDs对炭疽芽孢杆菌标志物的响应检测方法。结果显示,该方法可以保持较好的线性关系,线性相关系数r为0.995 4和0.994 9;利用3σ/k计算方法的检出限(LOD)为48.40 nmol/L。建立了一套操作简便、切实可行的炭疽快速检测技术,能够及时发现问题,有效提高国家进出口制品安全质量,保障人民身体健康,同时具有功能多样化、高效化、应用广泛化等优点,该技术成果转化性强,应用价值和利润值较高,市场推广前景广阔。

Inhibitory effects of nisin-coated multi-walled carbon nanotube sheet on biofilm formation from Bacillus anthracis spores

Multi-walled carbon nanotube（MWCNT） sheet was fabricated from a drawable MWCNT forest and then deposited on poly（methyl methacrylate） film. The film was further coated with a natural antimicrobial peptide nisin. We studied the effects of nisin coating on the attachment of Bacillus anthracis spores, the germination of attached spores, and the subsequent biofilm formation from attached spores. It was found that the strong adsorptivity and the super hydrophobicity of MWCNTs provided an ideal platform for nisin coating. Nisin coating on MWCNT sheets decreased surface hydrophobicity, reduced spore attachment, and reduced the germination of attached spores by 3.5 fold, and further inhibited the subsequent biofilm formation by 94.6% compared to that on uncoated MWCNT sheet. Nisin also changed the morphology of vegetative cells in the formed biofilm.The results of this study demonstrated that the anti-adhesion and antimicrobial effect of nisin in combination with the physical properties of carbon nanotubes had the potential in producing effective anti-biofilm formation surfaces.

Proteomics Reveals that Proteins Expressed During the Early Stage of Bacillus anthracis Infection Are Potential Targets for the Development of Vaccines and Drugs

In this review, we advance a new concept in developing vaccines and/or drugs to target speci?c proteins expressed during the early stage of Bacillus anthracis (an- thrax) infection and address existing challenges to this concept. Three proteins (immune inhibitor A, GPR-like spore protease, and alanine racemase) initially identi?ed by proteomics in our laboratory were found to have di?erential expres- sions during anthrax spore germination and early outgrowth. Other studies of di?erent bacillus strains indicate that these three proteins are involved in either germination or cytotoxicity of spores, suggesting that they may serve as potential targets for the design of anti-anthrax vaccines and drugs.

蜂胶对炭疽杆菌弱毒株的抑菌效果

为了解产自河南省的蜂胶对炭疽杆菌(Bacillus anthracis)弱毒株的抑菌作用,采用琼脂平板扩散法将实验分为pH 5.5～8.5不同组(蜂胶浓度31%),以及在pH 5.5下将31%蜂胶溶液倍比稀释至0.06%(1∶512)及空白组(0%),在每张滤纸片上滴加蜂胶溶液7μL,记录蜂胶的抑菌效果。结果表明,河南省蜂胶在pH 5.5、6.0、6.5、7.0、7.5、8.0、8.5平板上的抑菌环直径依次为(19.16±0.29)、(17.67±0.29)、(14.67±0.58)、(13.67±0.29)、(14.00±0.00)、(13.33±1.15)、(12.00±0.00)mm,蜂胶浓度分别为15.50%、7.75%、3.88%、1.94%、0.97%、0.48%、0.24%、0.12%、0.06%和0%(空白组)的抑菌环直径依次为(19.50±1.80)、(19.67±1.04)、(17.75±0.35)、(15.67±0.58)、(14.00±2.29)、(13.17±1.04)、(11.83±1.53)、(10.83±1.26)、(9.00±0.00)和(0.00±0.00)mm。说明河南蜂胶对炭疽杆菌弱毒株有明显的抑菌活性,对炭疽杆菌的抑菌活性随pH值增加或浓度降低而减小。

Anthrax: A disease of biowarfare and public health importance

Bioterrorism has received a lot of attention in the first decade of this century. Biological agents are considered attractive weapons for bioterrorism as these are easy to obtain, comparatively inexpensive to produce and exhibit widespread fear and panic than the actual potential of physical damage. Bacillus anthracis(B. anthracis), the etiologic agent of anthrax is a Gram positive, spore forming, non-motile bacterium. This is supposed to be one of the most potent BW agents because its spores are extremely resistant to natural conditions and can survive for several decades in the environment. B.anthracis spores enter the body through skin lesion(cutaneous anthrax), lungs(pulmonary anthrax), or gastrointestinal route(gastrointestinal anthrax) and germinate, giving rise to the vegetative form. Anthrax is a concern of public health also in many countries where agriculture is the main source of income including India. Anthrax has been associated with human history for a very long time and regained its popularity after Sept 2001 incidence in United States. The present review article describes the history, biology, life cycle, pathogenicity, virulence, epidemiology and potential of B. anthracis as biological weapon.

Anthrax:an update

Anthrax is a zoonotic disease caused by Bacillus anthracis.It is potentially fatal and highly contagious disease.Herbivores arc the natural host.Human acquire the disease incidentally by contact with infected animal or animal products.In the 18th century an epidemic destroyed approximately half of the sheep in Europe.In 1900 human inhalational anthrax occured sporadically in the United Stales.In 1979 an outbreak of human anthrax occured in Sverdlovsk of Soviet Union.Anthrax continued to represent a world wide presence.The incidence of the disease has decreased in developed countries as a result of vaccination and improved industrial hygiene.Human anthrax clinically presents in three forms,i.e.cutaneous,gastrointestinal and inhalational.About 95%of human anthrax is cutaneous and 5%is inhalational.Gastrointestinal anthrax is very rare(less than 1%).Inhalational form is used as a biological warefare agent. Penicillin,ciprofloxacin(and other quinolones),doxicyclin,ampicillin,imipenem.clindamycin, clarithromycin,vancomycin,chloramphenicol,rifampicin are effective antimicrobials. Antimicrobial therapy for 60 days is recommended.Human anthrax vaccine is available. Administration of anti-protective antigen(PA) antibody in combination with ciprofloxacin produced 90%-100%survival.The combination of CPG-adjuvanted anthrax vaccine adsorbed(AVA) plus dalbavancin significantly improved survival.

Gastrointestinal Anthrax:A Case and Review of Literature

Anthrax which is caused by Bacillus anthracis is typically a disease of herbivores. Spores existing in the skin, meat, hair or mouth and nose of animals are transmitted to humans through contact with a break in the skin, consumption of infected meat or inhalation of spores [1]. Infected uncooked or insufficiently cooked meats cause oropharyngeal and gastrointestinal system (GIS) anthrax. When this infected materials swallowed anthrax spores may cause lesions from the oral cavity to the caecum. The diagnosis of gastrointestinal system (GIS) anthrax is difficult due to insidious clinical progression of the disease and difficulty in the isolation of agent pathogen. Releated symptoms of GIS anthrax are sore throat, neck swelling, diffuculty swallowing, stomach pain, anoreksia, bloody diarrhea, nause, bloody vomiting and fever. Supportive and antibiotic treatments are required. Benzylpenicillin, rifampicin, clindamycin, chloramphenicol, imipenem/cilastatin, or vancomycin can be use for treatment, ciprofloxacin or doxycycline may be added to this treat- ment for serious cases. To emphasize the necessity of taking precautions, an oropharyngeal and intestinal anthrax case due to consumption of infected and insufficiently cooked meat is presented below.

Tb^(3+)-xylenol orange complex-based colorimetric and luminometric dual-readout sensing platform for dipicolinic acid and metal ions

A dual-readout sensing platform based on two signal transduction channels can integrate the unique advantages of each sensing pattern,compensate for the deficiency in the adaptive capacity,and enable a more convincing performance in analytical applications.Here,we introduce a responsive molecule dye,xylenol orange(XO),to combine with lanthanide terbium ions(Tb^(3+)).The resultant Tb^(3+)-XO complex exhibited tunable optical properties and was used as a novel colorimetric and luminometric dual-readout sensing platform for assaying the anthrax biomarker,dipicolinic acid(DPA).In the presence of Tb^(3+),the XO solution underwent a color change from yellow to magenta;however,upon adding DPA,the color changed back to yellow immediately,accompanied by the characteristic luminescence emission of Tb^(3+).Considering the strong affinity between DPA/XO and metal ions,the proposed sensing platform was further employed for the determination and differentiation of certain metal ions using linear discriminant analysis.This convenient dual-readout sensing platform offers several notable features and significantly promotes the application and development of lanthanide-based materials.

Genetic source tracking of an anthrax outbreak in Shaanxi province,China

Background:Anthrax is an acute zoonotic infectious disease caused by the bacterium known as Bacillus anthracis.From 26 July to 8 August 2015,an outbreak with 20 suspected cutaneous anthrax cases was reported in Ganquan County,Shaanxi province in China.The genetic source tracking analysis of the anthrax outbreak was performed by molecular epidemiological methods in this study.Methods:Three molecular typing methods,namely canonical single nucleotide polymorphisms(canSNP),multiple-locus variable-number tandem repeat analysis(MLVA),and single nucleotide repeat(SNR)analysis,were used to investigate the possible source of transmission and identify the genetic relationship among the strains isolated from human cases and diseased animals during the outbreak.Results:Five strains isolated from diseased mules were clustered together with patients’isolates using canSNP typing and MLVA.The causative B.anthracis lineages in this outbreak belonged to the A.Br.001/002 canSNP subgroup and the MLVA15-31 genotype(the 31 genotype in MLVA15 scheme).Because nine isolates from another four provinces in China were clustered together with outbreak-related strains by the canSNP(A.Br.001/002 subgroup)and MLVA15 method(MLVA15-31 genotype),still another SNR analysis(CL10,CL12,CL33,and CL35)was used to source track the outbreak,and the results suggesting that these patients in the anthrax outbreak were probably infected by the same pathogen clone.Conclusions:It was deduced that the anthrax outbreak occurred in Shaanxi province,China in 2015 was a local occurrence.