Targeting Replication-Segregation of plasmid pX01 in Bacillus anthracis

描述（由申请人提供）：炭疽芽孢杆菌是一种革兰氏阳性，孢子内形成的病原体，可导致人类致命的吸入性炭疽。它最近也因潜在的生物威胁武器而臭名昭著。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。尽管px1和pXO2在炭疽的发病机制中起着重要作用，但这些质粒的复制、分离和拷贝数控制机制仍不清楚。质粒pXO1含有一个大的致病性岛，其中包含编码炭疽三联毒的基因和几个整合酶和转座酶。pXO1中存在的可移动元件允许基因转移到相关的蜡样芽孢杆菌家族成员。因此，pXO1自然或工程转移到耐药细菌宿主的可能性构成了重大的公共卫生威胁。我们对炭疽芽孢杆菌pXO1维持机制的理解存在空白，这与pXO1编码蛋白与其他质粒制备的已知复制起始或分配蛋白的同源性无关。最近，人们克隆了pXO1复制子，并发现编码RepX蛋白的质粒在炭疽芽孢杆菌中对迷你pXO1复制至关重要。有趣的是，RepX与微管蛋白样细菌FtsZ蛋白具有显著的同源性，该蛋白在原核生物王国中广泛保守，并在细菌细胞分裂中起重要的细胞骨架作用。在此基础上，假设RepX组装成gtp调控的pXO1复制/分离所必需的细胞骨架结构，可以作为pXO1消除的新靶点。本研究的目的是分析RepX是否在体外和体内组装成gtp依赖性蛋白丝，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的前向化学遗传筛选，以识别通过拮抗RepX促进px1丢失的小分子。RepX抑制剂将被用作研究体内RepX和pXO1动力学的探针，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的共同位点，因此可以作为对抗炭疽的双刃剑。

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.