Role of RepX Protein in Replication/Partitioning of Anthrax Toxin Plasmid pXO1

RepX 蛋白在炭疽毒素质粒 pXO1 复制/分配中的作用

基本信息

批准号：
7843486
负责人：
SALEEM A. KHAN
金额：
$ 22.73万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-15 至 2011-04-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Bacillus anthracis is an important human pathogen that is the etiological agent of anthrax in humans and a potential biological weapon. Two large plasmids, pXO1 and pXO2, play a major role in the virulence of this organism. Gene transfer can frequently occur between B. anthracis and closely related species such as B. cereus and B. thuringiensis, making it likely that the pXO1 and pXO2 plasmids could naturally transfer from B. anthracis into such species that are resistant to antibiotics. Also, the possibility that bioterrorists may introduce the pXO1 and pXO2 plasmids into multiple drug resistant strains to generate "super bioterror agents" cannot be discounted. Although pXO1 is dispensable for the growth of B. anthracis, it is highly stable and rarely lost from growing bacterial cells. Very little is known about the molecular mechanisms involved in the replication and stability of this plasmid. The aims of this proposal are to analyze the replication properties of the anthrax toxin-encoding pXO1 plasmid of B. anthracis. We plan to characterize the minimal replicon of pXO1, including regions involved in plasmid stability and copy number control. We have isolated the replicon of pXO1 and shown that it encodes a novel replication protein, RepX. RepX resembles the FtsZ/tubulin proteins and we have shown that the purified RepX protein is a GTPase that can undergo GTP-dependent dynamic polymerization in vitro. We have also recently shown that RepX polymerizes in vivo in B. anthracis. We hypothesize that RepX may be involved in both the replication and partitioning of the highly stable pXO1 plasmid. Using cell biological approaches with fluorescently labeled proteins and/or pXO1 DNA, we will carry out experiments to study polymerization and localization of RepX in vivo, as well as its co-localization with the pXO1 plasmid and the cell division machinery in vegetatively growing and sporulating B. anthracis cells. By mutational analysis, we will identify regions of RepX that are important for its replication and partitioning activities. Finally, we will utilize "pull-down" assays to identify host- and pXO1-encoded proteins that interact with RepX and may play important roles in pXO1 replication and stability. Our studies could lead to a better understanding of the elements involved in the stable replication and maintenance of the pXO1 plasmid and may contribute to the future development of plasmid-specific co-therapeutic drugs that can reduce the virulence of B. anthracis and related organisms. PUBLIC HEALTH RELEVANCE: The aims of this proposal are to study the role of the RepX protein in the replication and segregation of the anthrax toxin-encoding pXO1 plasmid of Bacillus anthracis. We also plan to study the colocalization of RepX and pXO1 DNA in vivo. We will also investigate the stability and copy number control of pXO1. We plan to identify the functional domains of the RepX protein and identify plasmid- and chromosome-encoded proteins that interact with RepX.

描述（由申请人提供）：炭疽芽孢杆菌是一种重要的人类病原体，是人类炭疽病的病因和潜在的生物武器。两个大质粒PXO1和PXO2在该生物的毒力中起主要作用。基因转移经常发生在炭疽芽孢杆菌和密切相关的物种之间，例如蜡状芽孢杆菌和苏云金芽孢杆菌，因此PXO1和PXO2质粒可能自然地从炭疽芽孢杆菌转移到对抗生素具有抗性的物种中。同样，生物恐怖主义者可以将PXO1和PXO2质粒引入多种抗药性菌株以产生“超级生物异常剂”的可能性不能被打折。尽管PXO1对于炭疽芽孢杆菌的生长是可分配的，但它是高度稳定的，很少因生长细菌细胞而损失。关于该质粒的复制和稳定性所涉及的分子机制知之甚少。该提案的目的是分析炭疽芽孢杆菌的炭疽毒素编码PXO1质粒的复制特性。我们计划表征PXO1的最小复制子，包括涉及质粒稳定性和拷贝数控制的区域。我们已经分离了PXO1的复制子，并表明它编码了一种新型的复制蛋白Repx。 REPX类似于FTSZ/微管蛋白蛋白，我们已经表明，纯化的repx蛋白是一种GTPase，可以在体外在体外经历GTP依赖性动态聚合。我们最近还表明，repx在炭疽芽孢杆菌中的体内聚合。我们假设REPX可能参与了高度稳定的PXO1质粒的复制和分配。使用荧光标记的蛋白质和/或PXO1 DNA的细胞生物学方法，我们将进行实验，以研究REPX在体内的聚合和定位，以及其与PXO1质粒的共定位，并在植物生长和爆发的B. anthracis细胞中与PXO1质粒和细胞分裂机械进行共同定位。通过突变分析，我们将确定REPX的区域，这些区域对于其复制和分区活动很重要。最后，我们将利用“下拉”测定法识别与REPX相互作用的宿主和PXO1编码的蛋白质，并可能在PXO1复制和稳定性中起重要作用。我们的研究可能会更好地理解PXO1质粒稳定复制和维持所涉及的元素，并可能有助于质粒特异性的共同治疗药物的未来发展，这些药物可以减少炭疽芽孢杆菌和相关生物体的毒力。公共卫生相关性：该提案的目的是研究repx蛋白在炭疽芽孢杆菌的炭疽毒素编码PXO1质粒的复制和隔离中的作用。我们还计划研究体内repx和PXO1 DNA的共定位。我们还将研究PXO1的稳定性和拷贝数控制。我们计划鉴定REPX蛋白的功能结构域，并识别与REPX相互作用的质粒和染色体编码的蛋白。