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.

当前有许可炭疽疫苗的关键保护性抗原（PA）蛋白在天然生物的无菌菌株中可实现。虽然已在炭疽疫苗的产生中使用了替代表达系统（例如，大肠杆菌），但仍有几个原因继续使用炭疽芽孢杆菌。在当前的报告期间，我们与NIDDK的生物技术核心单元合作，探索了我们先前开发的蛋白酶缺陷菌株BH500的进一步提高蛋白质的产量。 RNA-seq在菌株BH500产生重组PA（RPA）或包含空表达载体的菌株上进行，目的是鉴定可能指向基因和途径的应力反应，这些反应可能会在遗传上修饰以增强蛋白质的产生。细菌是在生物反应器中生长的，例如在大规模蛋白质生产中使用的细菌。在生长阶段，在三个阶段收集样品，并生成RNA-seq数据。在观察到的差异中，表达RPA的菌株的转录增加了SIGL的转录，编码RNA聚合酶Sigma因子54的基因和编码一般应力转录Sigma因子B的SIGBB。除了观察到的许多其他表达变化，观察到的许多其他变化，对于发现外细胞外伴侣CSAA和PRSA的巨大变化是特别有趣的。 PRSA基因编码具有脯氨酸肽异构酶活性的折叠酶，细胞表面脂蛋白。该活性对于允许脯氨酸异构化需要在革兰氏阳性细胞壁上分泌时正确折叠蛋白质。先前的证据表明，RPA产生的主要局限性不是在蛋白质表达水平上，而是以分泌机制的能力，其中折叠酶是关键成分。因此，增加折叠酶的量可能会提高RPA产量。实际上，这已经显示出其他革兰氏阳性细菌的表现。因此，我们计划修改BH500应变以表达更多的折叠酶。所得的宿主菌株也可能在生产异源（即非细菌）蛋白的生产中有用，可用于其他各种正在进行的项目。 在2019财政年度的其他工作中，我们扩展了对炭疽芽孢杆菌中心转录激活剂ATXA的分析。开发了用于生产和纯化大肠杆菌蛋白质的方法。产生了突变的蛋白质，这些蛋白质在其他组已被证明是磷酸化的两个组氨酸残基中发生了变化。蛋白质的生物物理表征已成为未来研究的基础，以确定蛋白质如何识别其控制的基因。