Molecular Genetics and Pathogenesis of Anthrax

炭疽病的分子遗传学和发病机制

• 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
• 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

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其他分泌的蛋白酶。缺乏多种蛋白酶的菌株被用来定义单个蛋白酶对分泌组中蛋白质降解的贡献。在这项研究中，我们表明，雌雀巢蛋白（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）（EF）（EF）是由protease easease ecation ot e Cation ot e Prote separe sensation of Prot ot e Cation ot e Cation ot e prote osease san n nastary sep n n n persation of Prote ecepe os ecepe of Prote eaperease ease eacees ecations prote sepease san s n nastary B. anthacis Ames 35变异菌株的完全去分析的。改进的Cre-loxP基因基因敲除系统被依次删除编码六个蛋白酶（INHA1，INHA2，Camelysin，camelysin，tasa，nprB和MMPZ）的基因。这允许每个蛋白酶在炭疽芽孢杆菌毒素成分和ALO的降解中的作用。在缺少六个蛋白酶的最终菌株中，上清液中炭疽毒素成分和ALO的水平显着增加，并且在24小时内保持稳定。为了一般用作重组蛋白的生产宿主，将菌株固化为PXO1，并永久性地孢子不足。该菌株被指定为BH460，已被证明可用于制备许多重组蛋白。例如，使用BH460来产生重组EF，以前很难从炭疽芽孢杆菌中获得。 BH460产生的EF蛋白具有先前从炭疽芽孢杆菌或大肠杆菌宿主纯化的任何EF的体内效力。因此，建议将BH460作为重组蛋白质产生的有效宿主应变，通常每升培养物产生超过10 mg的纯蛋白质。正在进行的工作以获得知识产权保护并向多个商业实体许可。 在一个单独的项目中，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，该质粒质粒PXO1编码所有三种炭疽毒素蛋白。 在先前的工作中，我们确定并隔离了负责质粒复制的PXO1区域，从而定义了一种与其他报道的Minireplicon不同。现在，通过其他人的工作已经清楚地表明，我们所定义的Minireplicon在类似的芽孢杆菌物种质粒的大家庭中普遍保守，从而验证了我们的鉴定。但是，单独的DNA复制不能确保在细胞分裂发生时每个子细胞都会收到PXO1的副本。 因此，我们试图理解导致质粒在子细胞之间准确隔离的机制，这是一个积极的过程，可确保细菌种群中的质粒维持。 与天然PXO1相比，PXO1 Minireplicon的分离稳定性降低了，在37°C的重复通道中观察到炭疽芽孢杆菌中的天然PXO1。这可能是由于Minireplicon区域内没有功能性质粒维持系统而造成的。在2011年目前的报告期间，我们采用了酿酒酵母FLP-FRT重组系统，以对植物链球菌的遗传操纵。使用该系统以及CRE-LoxP系统，在质粒上确定了两个负责PXO1维护的区域。这些新遗传元素的确切位置是通过构建和测试其他缺失来确定的。这些知识将为抗感染剂提供靶标 - 那些不直接杀死病原体而是使其具有较低的毒力，从而允许宿主免疫反应有效地对抗它。