Identification of Bacillus anthracis strains in China

As part of a project to establish a reference strain collection, phenotypic, biochemical and genetic analyses were carried out on 84 strains of Bacillus anthracis, 81 of them of Chinese origin from various sources. Particular differences from reports on isolates of other origins were the possession of fimbriae and single polar flagella with consequent motility in 77, self-agglutination by 64 and failure to ferment maltose in 60 of the Chinese strains. The findings were considered to be of significance to identification, classification and evolution of B. anthracis.     

In Vitro and In Vivo Analysis of RTK Inhibitor Efficacy and Identification of Its Novel Targets in Glioblastomas

Treatment for glioblastoma consists of radiotherapy and temozolomide-based chemotherapy. However, virtually all patients recur, leading to a fatal outcome. Receptor tyrosine kinase (RTK)-targeted therapy has been the focus of attention in novel treatment options for these patients. Here, we compared the efficacy of imatinib, sunitinib, and cediranib in glioblastoma models. In the present work, the biologic effect of the drugs was screened by viability, cell cycle, apoptosis, migration, and invasion in vitro assays or in vivo by chick chorioallantoic membrane assay. Intracellular signaling was assessed by Western blot and the RTK targets were identified using phospho-RTK arrays. The amplified status of KIT, PDGFRA, and VEGFR2 genes was assessed by quantitative polymerase chain reaction. In a panel of 10 glioblastoma cell lines, we showed that cediranib was the most potent. In addition, cediranib and sunitinib synergistically sensitize the cells to temozolomide. Cediranib efficacy was shown to associate with higher cytostatic and unique cytotoxic effects in vitro and both antitumoral and antiangiogenic activity in vivo, which could associate with its great capacity to inhibit mitogen-activated protein kinase (MAPK) and AKT pathways. The molecular status of KIT, PDGFRA, and VEGFR2 did not predict glioblastoma cell responsiveness to any of the RTK inhibitors. Importantly, phospho-RTK arrays revealed novel targets for cediranib and sunitinib therapy. In conclusion, the novel targets found may be of value as future biomarkers for therapy response in glioblastoma and lead to the rational selection of patients for effective molecular targeted treatment.     

Characterization of Bacillus anthracis Persistence In Vivo

Pulmonary exposure to Bacillus anthracis spores initiates inhalational anthrax, a life-threatening infection. It is known that dormant spores can be recovered from the lungs of infected animals months after the initial spore exposure. Consequently, a 60-day course antibiotic treatment is recommended for exposed individuals. However, there has been little information regarding details or mechanisms of spore persistence in vivo. In this study, we investigated spore persistence in a mouse model. The results indicated that weeks after intranasal inoculation with B. anthracis spores, substantial amounts of spores could be recovered from the mouse lung. Moreover, spores of B. anthracis were significantly better at persisting in the lung than spores of a non-pathogenic Bacillus subtilis strain. The majority of B. anthracis spores in the lung were tightly associated with the lung tissue, as they could not be readily removed by lavage. Immunofluorescence staining of lung sections showed that spores associated with the alveolar and airway epithelium. Confocal analysis indicated that some of the spores were inside epithelial cells. This was further confirmed by differential immunofluorescence staining of lung cells harvested from the infected lungs, suggesting that association with lung epithelial cells may provide an advantage to spore persistence in the lung. There was no or very mild inflammation in the infected lungs. Furthermore, spores were present in the lung tissue as single spores rather than in clusters. We also showed that the anthrax toxins did not play a role in persistence. Together, the results suggest that B. anthracis spores have special properties that promote their persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence.     

过表达CodY蛋白的苏云金芽胞杆菌基因工程菌YBT-881-L1的构建及特性鉴定

为研究革兰氏阳性菌中的全局性转录调控因子CodY在苏云金芽胞杆菌中的作用机制,利用苏云金芽胞杆菌野生株YBT-881,构建了过表达CodY蛋白的基因工程菌YBT-88 1-L1,并对此工程菌的特性进行了研究.结果表明,YBT-881-L1和野生株的生长曲线以及伴胞晶体形态无明显差异.SDS-PAGE及质谱分析发现,野生株中沉默的cy2Ac4基因在工程菌中被激活,并产生大量的Cry2Ac4蛋白.生物测定结果表明,重组菌株YBT-881 -Ll较野生株对鳞翅目害虫棉铃虫的杀虫活性明显增强.     

Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization

Background  

Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are closely related members of the B. cereus-group of bacilli. Suppressive subtractive hybridization (SSH) was used to identify specific chromosomal sequences unique to B. anthracis.     

Identification of Linear Epitopes in Bacillus anthracis Protective Antigen Bound by Neutralizing Antibodies

Protective antigen (PA), the binding subunit of anthrax toxin, is the major component in the current anthrax vaccine, but the fine antigenic structure of PA is not well defined. To identify linear neutralizing epitopes of PA, 145 overlapping peptides covering the entire sequence of the protein were synthesized. Six monoclonal antibodies (mAbs) and antisera from mice specific for PA were tested for their reactivity to the peptides by enzyme-linked immunosorbent assays. Three major linear immunodominant B-cell epitopes were mapped to residues Leu¹⁵⁶ to Ser¹⁷⁰, Val¹⁹⁶ to Ile²¹⁰, and Ser³¹² to Asn³²⁶ of the PA protein. Two mAbs with toxin-neutralizing activity recognized two different epitopes in close proximity to the furin cleavage site in domain 1. The three-dimensional complex structure of PA and its neutralizing mAbs 7.5G and 19D9 were modeled using the molecular docking method providing models for the interacting epitope and paratope residues. For both mAbs, LeTx neutralization was associated with interference with furin cleavage, but they differed in effectiveness depending on whether they bound on the N- or C-terminal aspect of the cleaved products. The two peptides containing these epitopes that include amino acids Leu¹⁵⁶–Ser¹⁷⁰ and Val¹⁹⁶–Ile²¹⁰ were immunogenic and elicited neutralizing antibody responses to PA. These results identify the first linear neutralizing epitopes of PA and show that peptides containing epitope sequences can elicit neutralizing antibody responses, a finding that could be exploited for vaccine design.Bacillus anthracis is a Gram-positive, facultatively anaerobic, rod-shaped bacterium that secretes a variety of toxins, including anthrax toxin. This toxin is made up of three proteins as follows: protective antigen (PA),³ edema factor (EF), and lethal factor (LF). Like other binary toxins, anthrax toxin follows the same pattern where distinct subunits are involved in the different steps at which it can act. The B subunit (PA) is involved in receptor binding and cellular internalization into the cytoplasm, whereas the A subunit (EF and/or LF) bears the enzymatic activity (1). Anthrax can occur naturally in animals by spore transmission via ingestion, inhalation, or an open skin wound, but it can also be a result of bioterrorism and biological warfare (2).PA is the component of the currently licensed anthrax vaccine that elicits protective antibodies. Recent studies have demonstrated that a strong humoral response to truncated recombinant PA contributes to a protective immune response to anthrax (3, 4). Accordingly, there is considerable interest in ascertaining the location and immunogenicity of B-cell epitopes on the PA molecule. The development of numerous monoclonal antibodies (mAbs) to different epitopes on the PA molecule that influence PA functions, in conjunction with epitope mapping, has provided an opportunity to study the fine antigenic structure of PA. Most of these epitopes have been defined in mice (5–8), in macaques (9), in rabbits (10), as well as in vaccinated humans (11). Consequently, the epitopes described thus far are localized to three discrete regions within the PA. These regions correspond to the 2β2–2β3 loop of domain 2, the domain 3-domain 4 boundary, and the small loop of domain 4. The 2β2–2β3 loop of domain 2 is responsible for mediating membrane insertion, and many neutralizing murine mAbs target this region (5, 8). The boundary between domains 3 and 4, which does not have a known functional activity, has been suggested as a region recognized by polyclonal antibodies from vaccinated humans and rabbits (6, 12). The cellular receptor binding region is localized to the small loop of domain 4, and this region has been described to be recognized by two neutralizing mAbs (7, 9). With the exception of a neutralizing mAb that bound to PA₂₀ (13), no B-cell epitopes have been reported in domain 1. Therefore, identification of further dominant antigenic epitopes is pivotal for understanding the minimal immunogenic region of PA that will allow for precise direction of potent immune responses.Two mAbs to PA have been reported previously by our laboratory, one known as 7.5G binds to domain 1 and can neutralize the cytotoxic activity of lethal toxin (LeTx) (13). The other, mAb 10F4, binds to domain 4 and has weak neutralizing activity. In addition, we now describe four new anti-PA mAbs, of which only one neutralizes LeTx. The characterization of the B-cell epitopes in PA recognized by protective and nonprotective mAbs is important to better understand the antigenic structure of this toxin, and such information is potentially useful for the design of vaccines and passive immune therapies against B. anthracis. Because PA has been identified as an effective subunit vaccine, we wanted to identify the specific epitopes that provide the protection and use them as immunogens. Using mAbs and 145 overlapping peptides covering the entire sequence of PA, we identify the first linear neutralizing epitopes in domain 1 of PA, and we demonstrate that two peptides containing epitopes in domain 1 were capable of inducing strong LeTx-neutralizing antibody responses.     

Identification and characterization of Bacillus anthracis by multiplex PCR on DNA chip

Bacillus anthracis can be identified by detecting virulence factor genes located on two plasmids, pXO1 and pXO2. Combining multiplex PCR with arrayed anchored primer PCR and biotin-avidin alkaline phosphatase indicator system, we developed a qualitative DNA chip method for characterization of B. anthracis, and simultaneous confirmation of the species identity independent of plasmid contents. The assay amplifies pag gene (in pXO1), cap gene (in pXO2) and Ba813 gene (a B. anthracis specific chromosomal marker), and the results were indicated by an easy-to-read profile based on the color reaction of alkaline phosphatase. About 1 pg of specific DNA fragments on the chip wells could be detected after PCR. With the proposed method, the avirulent (pXO1+/2-, pXO1-/2+ and pXO1-/2-) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria were unambiguously identified, while the genera other than Bacillus gave no positive signal.     

Identification of Bacillus anthracis by polyclonal antibodies against extracted vegetative cell antigens

The extractable protein antigens EA1 and EA2 of Bacillus anthracis were prepared from electrophoresis transblots of SDS extracts of vegetative bacteria of the Sterne strain. Hyperimmune guinea-pig antiserum against EA2 failed to react with B. anthracis cells in immunofluorescence (IF) tests. Guinea-pig antiserum against EA1 (anti-EA1) reacted strongly in IF tests with non-encapsulated vegetative cell of 10 of 12 strains of B. anthracis and with cells of strains of B. cereus and B. thuringiensis. The unreactive B. anthracis strains were delta-Vollum-1B-1 and Texas. Encapsulated cells of B. anthracis stained poorly except for small bright regions. Absorption of anti-EA1 with cells of B. cereus NCTC 8035 and NCTC 9946 removed activity towards all B. cereus strains tested, but only partly reduced cross-reaction with B. thuringiensis strains. Absorption of anti-EA1 with B. thuringiensis 4041 removed activity towards this strain and B. cereus strains. Evidence is produced that B. thuringiensis cells grown on nutrient agar possess more cross-reacting antigens than cells grown in nutrient broth. The reaction of anti-EA1 with Bacillus spores immobilized in clumps on microscope slides was attributed to contaminating vegetative debris because well-separated individual spores failed to react. A rapid IF test was developed allowing identification of B. anthracis sampled from overnight cultures on blood plates. When sodium dodecyl sulphate extracts of B. anthracis vegetative cells were analysed on immunoblots (Western blots) by reaction with anti-EA1, a number of bands were visualized in addition to the expected 91 kiloDalton EA1 band. Prior absorption of anti-EA1 with B. cereus or B. thuringiensis cells resulted in the disappearance of most or all of the brands in blots of these species, but had less effect on blots of the B. anthracis strains. All six B. anthracis strains that were blotted including delta-Vollum-1B-1 and Texas, could thus be distinguished from B. cereus and B. thuringiensis by their differential reaction with unabsorbed and absorbed anti-EA1.     

Competition by Estrogens for Catecholamine Receptor Binding In Vitro

Abstract: We have examined the ability of various steroids to compete for high-affinity binding of ³H-labeled ligands to catecholamine receptors in membranes prepared from rat cerebral cortex, striatum, and anterior pituitary. Ligands employed were: [³H]WB4101, [³H]prazosin, [³H]yohimbine, and [³H]clonidine (alpha-noradrenergic); [³H]dihydroalprenolol (beta-noradrenergic); [³H]spiperone and [³H]ADTN (dopaminergic). Only the 17β estrogens were effective and only binding of [³H]spiperone and [³H]ADTN in striatum and [³H]WB4101 and [³H]prazosin in cerebral cortex was reduced. Thus putative dopaminergic and alpha₁-noradrenergic sites alone appear to recognize estrogens. A slight competitive effect on [³H]spiperone binding to anterior pituitary membranes was also observed. Among the 17β estrogens tested, the most effective in all cases was the catechol estrogen 2-hydroxyestradiol (2-OHE₂). The ability of 2-OHE₂ (IC₅₀= 20–30 μM) to inhibit ligand binding to alpha₁ receptors was comparable to that of norepinephrine (IC₅₀= 10–20 μM), whereas for dopamine receptors in striatum and pituitary 2-OHE₂ was an order of magnitude less effective than dopamine (IC₃₀= 12 μM) in reducing binding of ³H ligands. Estradiol-17β and 2-hydroxyestrone were also able to inhibit binding, but the order of steroid potency was different for alpha₁ and dopaminergic receptors. Progesterone, testosterone, and corticosterone were without effect in all cases. These results show that there is specificity of steroid interactions with catecholamine receptors in the brain, both in terms of steroid structure and receptor type. The possible relevance of these interactions to neuroendocrine function is discussed.     

In vitro amino acid requirements of Bacillus anthracis cells

In the process of batch cultivation in a synthetic medium B. anthracis cells actively consume free amino acids. The maximum amino acid consumption per unit of biomass occurs at the exponential phase of growth, but the consumption of serine is maintained at a high level also during the stationary phase. At the same time the consumption of proline by both vaccine and virulent strains is insignificant at the stationary phase of growth. In contrast to B. anthracis virulent strains, vaccine strains have been shown to consume no lysine, histidine, arginine and tryptophan.