Targeting Replication-Segregation of plasmid pX01 in Bacillus anthracis

描述(申请人提供)：炭疽芽孢杆菌是一种革兰氏阳性、内孢子形成的病原体，可导致人类致命的吸入性炭疽病。它最近还因潜在的生物治疗武器而臭名昭著。炭疽杆菌的一个特征是存在两个携带关键毒力基因的大质粒pXO1和pXO2。虽然pXO1和pXO2在炭疽病的发病机制中发挥了重要作用，但这些质粒的复制、分离和拷贝数控制的机制仍然缺乏特征。质粒pXO1含有一个大的致病岛，其中含有编码三方炭疽毒素的基因以及几个整合酶和转座酶。PXO1中可移动元件的存在允许基因转移到相关的蜡状芽孢杆菌家族成员。因此，将pXO1自然或工程转移到耐药细菌宿主的可能性对公共卫生构成了重大威胁。我们对炭疽杆菌中pXO1维持机制的理解存在差距，这与pXO1编码的蛋白与已知的复制起始蛋白或其他质粒所阐述的分割蛋白的同源性不高有关。最近，pXO1复制子被克隆，并发现在炭疽杆菌中编码的RepX蛋白是微小pXO1复制所必需的。有趣的是，RepX与微管蛋白样细菌FtsZ蛋白有显著的同源性，FtsZ蛋白在原核生物王国中广泛保守，在细菌细胞分裂中起着重要的细胞骨架作用。在此基础上，假设RepX组装成GTP调控的细胞骨架结构，对pXO1的复制/分离是必不可少的，并可作为消除pXO1的新靶点。这项建议的目的是分析RepX在体外和体内是否组装成依赖GTP的蛋白质细丝，以及RepX组装在pXO1复制/分离中的作用。另一个目标是设计一种高通量、正向化学遗传筛选，以识别通过拮抗RepX来促进pXO1丢失的小分子。RepX抑制剂将被用作探针来研究RepX和pXO1在体内的动力学，并可能构成潜在的治疗线索。抑制剂的一个子集可能针对RepX和FtsZ中的一个共同部位，因此可以作为对抗炭疽病的双刃剑。

DESCRIPTION (provided by applicant): Bacillus anthracis is a Gram-positive, endospore-forming pathogen, which causes lethal inhalation anthrax in humans. It has also gained recent notoriety as a potential biothreat weapon. A hallmark of B. anthracis is the presence of two large plasmids pXO1 and pXO2 that carry the key virulence genes. Although pXO1 and pXO2 play major roles in the pathogenesis of anthrax, mechanisms of replication, segregation, and copy number control of these plasmids remain poorly characterized. Plasmid pXO1 contains a large pathogenicity island that houses genes encoding the tripartite anthrax toxin and several intregrases and transposases. The presence of mobile elements in pXO1 allows gene transfer to related Bacillus cereus family members. Therefore, the possibility of natural or engineered transfer of pXO1 to drug-resisant bacterial hosts presents a significant public health threat. The gap in our understanding of the mechanism of pXO1 maintenance in B. anthracis relates to the insignificant homology of pXO1-encoded proteins to known replication initiation or partitioning proteins elaborated by other plasmids. Recently, the pXO1 replicon was cloned and the plasmid-encoded RepX protein was found to be essential for mini-pXO1 replication in B. anthracis. Intriguingly, RepX shows significant homology to the tubulin-like bacterial FtsZ protein, which is widely conserved in the Prokaryotic Kingdom, and plays an essential cytoskeletal role in bacterial cell division. On this basis, it is hypothesized that RepX assembles into a GTP-regulated cytoskeletal structure essential for pXO1 replication/segregation and can be exploited as a novel target for pXO1 elimination. The goal of this proposal is to analyze if RepX assembles into GTP-dependent protein filaments in vitro and in vivo, and the role of RepX assembly in pXO1 replication/segregation. Another goal is to devise a high-throughput, forward chemical genetic screen to identify small molecules that will promote pXO1 loss by antagonizing RepX. The RepX inhibitors will be used as probes to study RepX and pXO1 dynamics in vivo and may constitute potential therapeutic leads. A subset of inhibitors may target a common site in RepX and FtsZ, and could thus serve as double-edged swords against anthrax.