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.

项目摘要/摘要 炭疽芽孢杆菌孢子是炭疽疾病的传染性颗粒。通过磨损，摄入或 吸入，孢子进入皮下层，胃肠道粘膜或肺泡腔 通过巨噬细胞吞噬，发生发芽并引起皮肤或全身性疾病。而孢子 是传播所必需的，它们的发芽和生长对于发病机理至关重要。但是，由于 作为在环境中的营养细胞，发芽周期，营养生长和孢子的生存率不佳 形成对于炭疽芽孢杆菌的致病生活方式至关重要。全球变暖引起的环境变化 森林砍伐增加了流行地区的季节性炭疽病暴发，并导致了出现 新的人畜共患病原体。解决这些挑战将需要更深入地了解机制 炭疽芽孢杆菌的孢子形成和发芽。针对这些过程将有助于发展 更有效的预防疾病和治疗的策略。 多年来，非致病性土壤细菌枯草芽孢杆菌一直是孢子形成和 孢子形成病原体等病原体等发芽。这些过程中只有少数因素 炭疽芽孢杆菌的特征是；在所有情况下，这些都是枯草芽孢杆菌孢子/发芽的同源物 蛋白质。在初步研究中，我们使用转座子测序（TN-Seq）鉴定了B中的156个孢子形成基因。 炭疽菌。令人惊讶的是，枯草芽孢杆菌孢子不需要这些基因中的77个基因，或者不存在 基因组。该赠款提案旨在利用现代分子方法来识别和表征 炭疽芽孢杆菌中的完整孢子形成和发芽因子。目的是： （1）表征了炭疽芽孢杆菌的完整集。我们将建立一个有序的TN库 使用基因敲除Sudoku方法中插入所有非必需的B.炭疽病基因。然后我们将使用个人 突变体来表征我们鉴定的所有孢子基因。我们将执行定量孢子分析 和荧光显微镜以确定开发的阶段。最后，我们将发起 那些具有强突变表型并且炭疽芽孢杆菌独有的因素的分子表征。 （2）识别并表征炭疽芽孢杆菌中一组全面的发芽因子。我们将表演 我们在枯草芽孢杆菌中成功执行的炭疽芽孢杆菌中的互补发芽TN-seq筛选。 然后，使用有序的敲除藏品，然后我们将验证命中，并表征那些具有最强的命中 炭疽芽孢杆菌独有的发芽缺陷。 如果成功，该建议将确定枯草芽孢杆菌可以作为孢子形成和 炭疽芽孢杆菌中的发芽，将定义该病原体过程中特有的一系列因素 进行机械研究。最后，我们描述的有序淘汰库将提供给 科学界，将催化炭疽芽孢杆菌生物学的所有领域的研究。