Molecular Genetics and Pathogenesis of Anthrax

炭疽病的分子遗传学和发病机制

基本信息

批准号：
8336252
负责人：
Stephen Leppla
金额：
$ 73.12万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8336252
关键词：
Adopted Affect Animals Anthrax Vaccines Anthrax disease Anti-Infective Agents Antigens Area Attenuated Bacillus (bacterium)Bacillus anthracis Bacteria Biochemical Biochemical Genetics Cell division Cells Chromosomes Complex DNA biosynthesis Data Development Disease Escherichia coli Family Gene Proteins Genes Genetic Genetic Recombination Genomics Human Immune response Individual Infection Intellectual Property Knowledge Licensing Location Maintenance Methods Molecular Genetics Pathogenesis Peptide Hydrolases Phase Plasmids Play Population Preparation Process Production Proteins Proteomics Recombinant Proteins Recombinants Reporting Role Saccharomyces cerevisiae System Testing Toxin Variant Virulence Virulent Work anthrax lethal factor anthrax toxin base camelysin combat daughter cell defined contribution edema factor genetic analysis genetic element genetic manipulation improved in vivo killings knockout gene pathogen protein degradation segregation tool vaccine candidate

项目摘要

In 2011, we continued to delete additional secreted proteases from B. anthracis. Strains lacking multiple proteases were used to define the contributions of individual proteases to the degradation of proteins in the secretome. In this study we showed that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the attenuated B. anthracis Ames 35 variant strain, are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). This allowed the role of each protease in the degradation of the B. anthracis toxin components and ALO to be determined. In the final strain constructed, which lacks six proteases, the levels of the anthrax toxin components and ALO in the supernatant were significantly increased and remained stable over 24 h. For general use as a host for the production of recombinant proteins, the strain was cured of pXO1 and made permanently sporulation deficient. This strain, designated BH460, has proven useful in the preparation of a number of recombinant proteins. As an example, BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or E. coli hosts. BH460 is therefore recommended as an effective host strain for recombinant protein production, typically yielding greater than 10 mg pure protein per liter of culture. Work is underway to obtain intellectual property protection and to license this strain to several commercial entities. In a separate project, we continued analyses of the genes and sequences that are needed for replication and maintenance of the key virulence plasmid pXO1, which encodes all three anthrax toxin proteins. In previous work we identified and isolated the region of pXO1 that is responsible for plasmid replication, and thereby defined a minireplicon that was different from one reported by others. It has now become clear through the work of others that the minireplicon we defined is universally conserved among the large family of similar bacillus species plasmids, validating our identification. However, DNA replication alone does not assure that every daughter cell receives a copy of pXO1 when cell division occurs. Thus, we seek to understand the mechanism that causes accurate segregation of plasmids between daughter cells, an active process that assures plasmid maintenance in the bacterial population. Decreased segregational stability of the pXO1 minireplicon in comparison with the native pXO1 was observed in B. anthracis during repeated passage at 37C. This is likely to result from the absence of a functional plasmid maintenance system within the minireplicon region. During the current reporting period of 2011, we adopted the Saccharomyces cerevisiae Flp-FRT recombination system to the genetic manipulation of B. anthracis. Using this system along with the Cre-loxP system, two distinct areas responsible for pXO1 maintenance were identified on the plasmid. The exact locations of these new genetic elements are being determined by constructing and testing additional deletions. This knowledge will provide targets for anti-infective agents - those that do not directly kill the pathogen but instead render it less virulent, thereby allowing host immune responses to effectively combat it.

2011 年，我们继续从炭疽芽孢杆菌中删除额外的分泌蛋白酶。使用缺乏多种蛋白酶的菌株来确定单个蛋白酶对分泌蛋白组中蛋白质降解的贡献。在这项研究中，我们发现炭疽杆菌 Ames 35 减毒株产生的炭疽毒素 O (ALO) 和三种炭疽毒素蛋白、保护性抗原 (PA)、致死因子 (LF) 和水肿因子 (EF)由于蛋白酶的作用，在稳定期开始时完全降解。使用改进的Cre-loxP基因敲除系统依次删除编码六种蛋白酶（InhA1、InhA2、camlysin、TasA、NprB和MmpZ）的基因。这使得可以确定每种蛋白酶在炭疽芽孢杆菌毒素成分和 ALO 降解中的作用。在构建的最终菌株中，缺乏六种蛋白酶，上清液中炭疽毒素成分和ALO的水平显着增加，并在24小时内保持稳定。为了一般用作重组蛋白生产的宿主，该菌株被消除了 pXO1 并使其永久孢子形成缺陷。该菌株被命名为 BH460，已被证明可用于制备多种重组蛋白。例如，BH460 用于生产重组 EF，而以前很难从炭疽芽孢杆菌中获得重组 EF。与先前从炭疽芽孢杆菌或大肠杆菌宿主纯化的任何 EF 相比，由 BH460 产生的 EF 蛋白具有最高的体内效力。因此，推荐 BH460 作为重组蛋白生产的有效宿主菌株，通常每升培养物产量大于 10 毫克纯蛋白。目前正在努力获得知识产权保护并将该菌株许可给多个商业实体。在另一个项目中，我们继续分析复制和维持关键毒力质粒 pXO1 所需的基因和序列，该质粒编码所有三种炭疽毒素蛋白。在之前的工作中，我们鉴定并分离了 pXO1 负责质粒复制的区域，从而定义了一个与其他人报道的不同的微型复制子。现在通过其他人的工作已经清楚，我们定义的微型复制子在类似芽孢杆菌属质粒的大家族中普遍保守，验证了我们的鉴定。然而，DNA 复制本身并不能确保细胞分裂时每个子细胞都会收到 pXO1 的副本。因此，我们试图了解导致子细胞之间质粒精确分离的机制，这是确保细菌群体中质粒维持的主动过程。在 37°C 重复传代期间，在炭疽芽孢杆菌中观察到 pXO1 微型复制子与天然 pXO1 相比分离稳定性降低。这可能是由于微型复制子区域内缺乏功能性质粒维持系统造成的。 2011年报告期内，我们采用酿酒酵母Flp-FRT重组系统对炭疽芽孢杆菌进行遗传操作。使用该系统和 Cre-loxP 系统，在质粒上鉴定出了负责 pXO1 维持的两个不同区域。这些新遗传元件的确切位置正在通过构建和测试额外的缺失来确定。这些知识将为抗感染药物提供靶点——那些药物不会直接杀死病原体，而是降低其毒性，从而使宿主免疫反应能够有效地对抗病原体。