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

项目摘要/摘要 炭疽芽胞是炭疽病的侵染性颗粒。通过磨损、摄取或 吸入、孢子进入皮下层、胃肠道粘膜或肺泡腔 巨噬细胞吞噬细胞，萌发，引起皮肤或全身疾病。而孢子 是传播所必需的，它们的萌发和生长是致病的关键。然而，由于 在环境、萌发周期、营养生长和孢子中作为营养细胞存活不良 炭疽杆菌致病生活方式的形成是必不可少的。全球变暖导致的环境变化 森林砍伐增加了流行地区的季节性炭疽暴发，并导致了 新的人畜共患病病原体。应对这些挑战需要对这些机制有更深入的了解。 炭疽芽胞杆菌的产孢量和萌发率。以这些进程为目标将有助于开发 更有效地预防和治疗疾病的战略。 多年来，非致病土壤细菌枯草芽孢杆菌一直是产孢子和 像炭疽杆菌这样的孢子形成病原体的萌发。这些过程中只涉及少数几个因素 在炭疽杆菌中具有特征；在所有情况下，这些都是枯草杆菌产孢量/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)鉴定了芽胞杆菌的156个产孢子基因。 炭疽病。引人注目的是，其中77个基因要么不是枯草杆菌产孢所必需的，要么不存在于其 基因组。这项拨款提案寻求利用现代分子方法来识别和表征 炭疽杆菌成套产孢子和萌发因子。目标是： (1)炭疽芽胞形成因子的全套鉴定。我们将建立一个有序的TN库 使用敲除数独方法在所有非必需的炭疽杆菌基因中插入。然后，我们将使用个人 突变体来表征我们鉴定的所有产孢子基因。我们将进行定量的产孢量分析 和荧光显微镜来确定发育受阻的阶段。最后，我们将发起 具有强烈突变表型且为炭疽杆菌所特有的因子的分子特征。 (2)鉴定和鉴定炭疽菌中的一系列萌发因子。我们将表演 我们已经在枯草杆菌中成功地进行了炭疽杆菌的互补萌发TN-seq筛选。 使用有序的敲击集合，然后我们将验证命中并确定具有最强命中的命中 炭疽杆菌特有的萌发缺陷。 如果成功，这项提议将在多大程度上建立枯草杆菌作为产孢子和 在炭疽杆菌中的萌发，并将定义一大套在这种病原体的过程中独有的因素，这些因素将是 受机械论研究的影响。最后，我们描述的有序淘汰库将提供给 并将促进炭疽杆菌生物学各个领域的研究。

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.