Targeting Replication-Segregation of plasmid pX01 in Bacillus anthracis

炭疽杆菌中质粒 pX01 的靶向复制分离

• 批准号: 7898610
• 负责人: DEBABRATA RAYCHAUDHURI
• 金额: $ 20.63万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-23 至 2012-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7898610
• 关键词: Affect; Anthrax disease; Anti-Bacterial Agents; Bacillus anthracis; Bacillus cereus; Bacillus subtilis; Biochemical; Biological Assay; Breathing; Cell division; Cells; Chemicals; Coupled; DNA-Binding Proteins; Engineering; Epitopes; Escherichia coli; Family member; Filament; GTP Binding; Gene Transfer; Genes; Genetic Screening; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Housing; Human; Hydrolysis; In Vitro; Indium; Life; Maintenance; Maps; Movement; Mutation; Open Reading Frames; Organism; Pathogenesis; Pathogenicity Island; Pharmaceutical Preparations; Plasmids; Play; Positioning Attribute; Proteins; Public Health; Replication Initiation; Replication Origin; Replicon; Role; Sequence Analysis; Site; Structure; Testing; Therapeutic; Transposase; Tubulin; Virulence; anthrax toxin; base; biothreat; chemical genetics; high throughput screening; in vivo; inhibitor/antagonist; novel; pathogen; prokaryotic kingdom; replication initiator protein; segregation; small molecule; small molecule libraries; vector; weapons

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.

描述（由申请方提供）：炭疽芽孢杆菌是一种革兰氏阳性、内孢子形成病原体，可导致人类致命的吸入性炭疽。它最近还作为一种潜在的生物威胁武器而臭名昭著。B的标志。炭疽杆菌的一个重要特征是存在两个携带关键毒力基因的大质粒pXO 1和pXO 2。虽然pXO 1和pXO 2在炭疽病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pXO 1含有一个大的致病性岛，其中包含编码三重炭疽毒素的基因和几种整合酶和转座酶。pXO 1中移动的元件的存在允许基因转移到相关的蜡样芽孢杆菌家族成员。因此，pXO 1天然或工程转移到药物耐受性细菌宿主的可能性提出了一个重大的公共卫生威胁。我们对B中pXO 1维持机制的理解存在差距。炭疽病涉及pXO 1编码的蛋白质与已知的复制起始或由其它质粒加工的分配蛋白质的无关紧要的同源性。最近，pXO 1复制子被克隆，并且发现质粒编码的RepX蛋白对于B中的mini-pXO 1复制是必需的。炭疽病有趣的是，RepX显示出与微管蛋白样细菌FtsZ蛋白的显着同源性，该蛋白在前体王国中广泛保守，并且在细菌细胞分裂中起着重要的细胞骨架作用。在此基础上，据推测，RepX组装成一个GTP调节的细胞骨架结构必不可少的pXO 1复制/分离，并可以利用作为一个新的目标pXO 1消除。本提案的目的是分析RepX是否在体外和体内组装成GTP依赖的蛋白丝，以及RepX组装在pXO 1复制/分离中的作用。另一个目标是设计一种高通量的正向化学遗传筛选，以识别通过拮抗RepX促进pXO 1丢失的小分子。RepX抑制剂将被用作探针来研究RepX和pXO 1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中的一个共同位点，因此可以作为对抗炭疽的双刃剑。