Identification and characterization of a comprehensive set of factors required for sporulation and germination in Bacillus anthracis

炭疽芽孢杆菌孢子形成和萌发所需的一系列综合因素的鉴定和表征

• 批准号: 10510204
• 负责人: DAVID Z RUDNER
• 金额: $ 25.42万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10510204
• 关键词: Address; Agriculture; Alveolar; Animals; Anthrax disease; Applications Grants; Area; Bacillus anthracis; Bacillus anthracis spore; Bacillus cereus; Bacillus subtilis; Bacteria; Biological Assay; Biology; Bioterrorism; Cells; Collection; Communities; Cytology; Defect; Deforestation; Development; Developmental Process; Disease Outbreaks; Engineering; Environment; Fluorescence Microscopy; Genes; Genome; Germination; Global Warming; Growth; Heat Losses; Human; Impairment; Incubated; Individual; Ingestion; Inhalation; Knock-out; Libraries; Life Style; Manuals; Methods; Modeling; Modernization; Molecular; Molecular Genetics; Mucous Membrane; Mutation; Organism; Pathogenesis; Pathogenicity; Phenotype; Process; Proteins; Reproduction spores; Research; Resistance; Soil; Starvation; Sterilization; System; Systemic disease; Time; Virulence Factors; Work; Zoonoses; anthrax toxin; capsule; combinatorial; disorder prevention; environmental change; gastrointestinal; macrophage; medical schools; member; mutant; next generation sequencing; particle; pathogen; skin disorder; subcutaneous; transmission process; transposon sequencing; virtual

PROJECT SUMMARY/ABSTRACT Bacillus anthracis spores are the infectious particles for the disease anthrax. Through abrasion, ingestion, or inhalation, spores access subcutaneous layers, gastrointestinal mucosa, or alveolar cavities where they are phagocytosed by macrophages, undergo germination, and cause cutaneous or systemic disease. While spores are required for transmission, their germination and outgrowth are critical for pathogenesis. However, owing to poor survival as vegetative cells in the environment, the cycle of germination, vegetative growth, and spore formation is essential for the pathogenic lifestyle of B. anthracis. Environmental changes due to global warming and deforestation have increased seasonal anthrax outbreaks in endemic areas and have led to the emergence of new zoonotic pathogens. Addressing these challenges will require a deeper understanding of the mechanisms of B. anthracis sporulation and germination. Targeting these processes will facilitate the development of strategies for more effective disease prevention and treatment. For years, the non-pathogenic soil bacterium Bacillus subtilis has served as the model for sporulation and germination of spore forming pathogens like B. anthracis. Only a handful of factors involved in these processes have been characterized in B. anthracis; in all cases these were homologs of B. subtilis sporulation/germination proteins. In preliminary studies, we used transposon sequencing (Tn-seq) to identify 156 sporulation genes in B. anthracis. Strikingly, 77 of these genes are either not required for B. subtilis sporulation or are not present in its genome. This grant proposal seeks to leverage modern molecular approaches to identify and characterize the complete set of sporulation and germination factors in B. anthracis. The aims are: (1) Characterize the complete set of B. anthracis sporulation factors. We will build an ordered library of Tn insertions in all nonessential B. anthracis genes using the Knockout Sudoku method. We will then use individual mutants to characterize all the sporulation genes we identified. We will perform quantitative sporulation assays and fluorescence microscopy to determine the stage at which development is blocked. Finally, we will initiate molecular characterization of those factors that have strong mutant phenotypes and are unique to B. anthracis. (2) Identify and characterize a comprehensive set of germination factors in B. anthracis. We will perform a complementary germination Tn-seq screen in B. anthracis that we have successfully performed in B. subtilis. Using the ordered knock out collection, we will then validate the hits and characterize those with the strongest germination defect that are unique to B. anthracis. If successful, this proposal will establish the extent to which B. subtilis can serve as a model for sporulation and germination in B. anthracis and will define a large set of factors unique to processes in this pathogen that will be subjected to mechanistic study. Finally, the ordered knockout library we describe will be made available to the scientific community and will catalyze research in all areas of B. anthracis biology.

项目概要/摘要 炭疽芽孢杆菌孢子是炭疽病的传染性颗粒。通过磨损、摄入或 吸入后，孢子进入皮下层、胃肠粘膜或肺泡腔 被巨噬细胞吞噬、发芽并引起皮肤或全身疾病。当孢子 是传播所必需的，它们的发芽和生长对于发病机制至关重要。然而，由于 营养细胞在环境中的存活率、发芽、营养生长和孢子的周期较差 形成对于炭疽芽孢杆菌的致病生活方式至关重要。全球变暖导致的环境变化 森林砍伐增加了流行地区季节性炭疽病的爆发，并导致了炭疽病的出现 新的人畜共患病病原体。应对这些挑战需要更深入地了解其机制 炭疽芽孢杆菌孢子形成和发芽。以这些过程为目标将有助于开发 更有效的疾病预防和治疗策略。 多年来，非致病性土壤细菌枯草芽孢杆菌一直作为孢子形成和生长的模型。 形成孢子的病原体如炭疽芽孢杆菌的萌发。这些过程只涉及少数因素 已在炭疽芽孢杆菌中进行了表征；在所有情况下，这些都是枯草芽孢杆菌孢子形成/萌发的同源物 蛋白质。在初步研究中，我们使用转座子测序（Tn-seq）鉴定了 B. 炭疽病。引人注目的是，其中 77 个基因要么不是枯草芽孢杆菌孢子形成所必需的，要么不存在于其孢子中。 基因组。该拨款提案旨在利用现代分子方法来识别和表征 炭疽芽孢杆菌中完整的孢子形成和萌发因子。目标是： (1) 表征全套炭疽杆菌孢子形成因子。我们将建立一个 Tn 的有序库 使用敲除数独方法插入所有非必需炭疽芽孢杆菌基因。然后我们将使用单独的 突变体来表征我们确定的所有孢子形成基因。我们将进行定量孢子形成测定 和荧光显微镜以确定发育受阻的阶段。最后我们将发起 具有强突变表型并且是炭疽芽孢杆菌特有的那些因子的分子特征。 (2) 鉴定并表征炭疽芽孢杆菌中一套全面的发芽因子。我们将表演 我们已在枯草芽孢杆菌中成功进行了炭疽芽孢杆菌的互补发芽 Tn-seq 筛选。 使用有序淘汰集合，我们将验证命中并表征最强的命中 炭疽芽孢杆菌特有的发芽缺陷。 如果成功，该提案将确定枯草芽孢杆菌可以在多大程度上作为孢子形成和 炭疽芽孢杆菌中的发芽，并将定义该病原体过程中特有的一大组因素，这些因素将 进行机理研究。最后，我们描述的有序敲除库将提供给 科学界并将促进炭疽杆菌生物学所有领域的研究。