Molecular Genetics and Pathogenesis of Anthrax

炭疽病的分子遗传学和发病机制

• 批准号: 10014139
• 负责人: Stephen Leppla
• 金额: $ 48.56万
• 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/10014139
• 关键词: Affect; Animals; Anthrax Vaccines; Anthrax disease; Antigens; Bacillus (bacterium); Bacillus anthracis; Bacteria; Biochemical; Biochemical Genetics; Bioreactors; Biotechnology; Cell Wall; Cell surface; Cells; Chromosomes; Complement Factor B; Complex; DNA-Directed RNA Polymerase; Data; Development; Disease; Escherichia coli; Future; Gene Proteins; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genomics; Gram-Positive Bacteria; Growth; Histidine; Human; Individual; Infection; Isomerase; Lipoproteins; Maintenance; Methods; Molecular Chaperones; Molecular Genetics; Mutate; National Institute of Diabetes and Digestive and Kidney Diseases; Organism; Pathogenesis; Pathway interactions; Peptide Hydrolases; Peptides; Phase; Plasmids; Play; Process; Production; Proline; Proteins; Proteomics; Recombinants; Reporting; Role; Sampling; Sigma Factor; Stress; System; Toxin; Transcription Coactivator; Vaccine Production; Virulence; Work; anthrax toxin; biological adaptation to stress; biophysical properties; expression vector; extracellular; genetic manipulation; improved; pathogen; pathogenic bacteria; protein degradation; protein expression; protein folding; protein purification; tool; transcriptome sequencing; vaccine candidate

Production of the key Protective Antigen (PA) protein of the current licensed anthrax vaccine is achieved in avirulent strains of the native organism, Bacillus anthracis. While alternative expression systems (e.g., E. coli) have been used in anthrax vaccine production, there are several reasons that B. anthracis continues to be used. In the current reporting period, we collaborated with the Biotechnology Core Unit of NIDDK to explore ways to further enhance yields of proteins from the protease-deficient strain BH500, which we previously developed. RNA-seq was performed on strain BH500 producing recombinant PA (rPA) or containing an empty expression vector, with the objective of identifying stress responses that could point to genes and pathways that might be genetically modified to enhance protein production. The bacteria were grown in a bioreactor like that used in large scale protein production. Samples were collected at three stages during the growth phase and RNA-seq data generated. Among the observed differences, the strain expressing rPA had increased transcription of sigL, the gene encoding RNA polymerase sigma factor 54, and sigB, encoding the general stress transcription sigma factor B. Among many other changes in expression observed, it was particularly interesting to find large changes in the extracellular chaperones csaA and prsA. The prsA genes encode foldases, cell-surface lipoproteins that have proline-peptide isomerase activity. This activity is essential to allow the proline isomerization needed to properly fold proteins as they are secreted through the Gram-positive cell wall. Prior evidence had suggested that a principal limitation on production of rPA did not lie at the level of protein expression but in the capacity of the secretion machinery, of which the foldases are key components. It follows that increasing the amount of the foldases may enhance rPA yields. In fact, this has been shown for other Gram-positive bacteria. Therefore we plan to modify our BH500 strain to express more of the foldases. The resulting host strains may also be useful in production of heterologous (i.e., non-bacterial) proteins for use in various other ongoing projects. In other work during fiscal year 2019, we extended our analysis of the central transcriptional activator of B. anthracis, AtxA. Methods were developed for production and purification of the protein from E. coli. Mutated proteins were produced that are altered in two histidine residues that other groups have shown to be phosphorylated. Biophysical characterization of the proteins has been done as a basis for future studies to identify how the protein recognizes the genes that it controls.

目前获得许可的炭疽疫苗的关键保护性抗原（PA）蛋白的生产是在天然有机体炭疽杆菌的无毒菌株中实现的。虽然替代表达系统（例如大肠杆菌）已用于炭疽疫苗生产，但炭疽芽孢杆菌继续使用有几个原因。在本报告期内，我们与NIDDK生物技术核心部门合作，探索进一步提高我们之前开发的蛋白酶缺陷菌株BH500的蛋白质产量的方法。 RNA-seq 对产生重组 PA (rPA) 或含有空表达载体的菌株 BH500 进行，目的是识别可能指向基因和途径的应激反应，这些基因和途径可能经过基因改造以增强蛋白质产量。这些细菌在类似于大规模蛋白质生产中使用的生物反应器中生长。在生长阶段的三个阶段收集样本并生成 RNA-seq 数据。在观察到的差异中，表达 rPA 的菌株的 sigL（编码 RNA 聚合酶 sigma 因子 54 的基因）和 sigB（编码一般应激转录 sigma 因子 B）的转录增加。在观察到的许多其他表达变化中，特别有趣的是发现细胞外伴侣 csaA 和 prsA 发生较大变化。 prsA 基因编码折叠酶，即具有脯氨酸肽异构酶活性的细胞表面脂蛋白。当蛋白质通过革兰氏阳性细胞壁分泌时，这种活性对于正确折叠蛋白质所需的脯氨酸异构化至关重要。先前的证据表明，rPA 产生的主要限制并不在于蛋白质表达水平，而在于分泌机制的能力，而折叠酶是分泌机制的关键组成部分。由此可见，增加折叠酶的量可能会提高 rPA 产量。事实上，其他革兰氏阳性细菌也已证明了这一点。因此，我们计划修改我们的 BH500 菌株以表达更多的折叠酶。所得宿主菌株也可用于生产异源（即非细菌）蛋白质，用于各种其他正在进行的项目。 在 2019 财年的其他工作中，我们扩展了对炭疽芽孢杆菌中央转录激活因子 AtxA 的分析。开发了从大肠杆菌中生产和纯化蛋白质的方法。产生的突变蛋白的两个组氨酸残基发生了改变，而其他研究组已证明这两个组氨酸残基已被磷酸化。蛋白质的生物物理表征已作为未来研究的基础，以确定蛋白质如何识别其控制的基因。