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

项目总结/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，孢子进入皮下层，胃肠道粘膜或肺泡腔， 被巨噬细胞吞噬，经历发芽，并引起皮肤或全身疾病。虽然孢子 是传播所必需的，它们的萌发和生长对发病至关重要。但由于 作为营养细胞在环境中生存不良，循环萌发、营养生长和孢子 形成对于B的致病生活方式是必不可少的。炭疽病全球变暖导致的环境变化 和森林砍伐增加了流行地区的季节性炭疽疫情， 新的人畜共患病病原体。应对这些挑战将需要更深入地了解这些机制 的B。炭疽孢子形成和萌发。以这些进程为目标，将促进 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的模型， 孢子萌发形成病原体如B。炭疽病只有少数因素参与了这些过程 已在B中表征。炭疽菌;在所有情况下，这些都是B的同源物。枯草芽孢杆菌孢子形成/萌发 proteins.在初步研究中，我们使用转座子测序（Tn-seq）来鉴定B中的156个孢子形成基因。 炭疽病引人注目的是，其中77个基因不是B所必需的。枯草芽孢杆菌孢子形成或不存在于其 基因组这项拨款提案旨在利用现代分子方法来识别和表征 在B中具有完整的孢子形成和萌发因子。炭疽病其目标是： (1)刻画B的完备集。炭疽孢子形成因子我们将建立一个有序的Tn库， 在所有非必需的B中插入。使用敲除数独方法对炭疽菌基因进行测序。我们将使用个人 突变体来表征我们鉴定的所有孢子形成基因。我们将进行定量孢子形成试验 和荧光显微镜以确定发育被阻断的阶段。最后，我们将启动 对具有强突变表型并且是B所特有的那些因子的分子表征。炭疽病 (2)在B中识别和表征一组全面的发芽因素。炭疽病我们将执行 B中的互补萌发Tn-seq筛选。我们在B实验中成功的炭疽病。枯草杆菌。 然后，我们将使用有序敲除集合来验证命中，并表征具有最强命中的命中 B特有的发芽缺陷。炭疽病 如果成功的话，这个建议将确定B。枯草芽孢杆菌可以作为孢子形成的模型， 在B中萌发。炭疽病，并将确定一个大的一套因素独特的过程中，这种病原体， 进行机械学研究。最后，我们所描述的有序敲除文库将可用于 科学界，并将促进B的所有领域的研究。炭疽生物学

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.