Directed Mutagenesis of the Rickettsia prowazekii pld Gene Encoding Phospholipase D
Directed Mutagenesis of the Rickettsia prowazekii pld Gene Encoding Phospholipase D
复制标题
DOI：
10.1128/iai.00395-09
复制
发表时间：
2009-08-01
影响因子：
3.1
通讯作者：
Wood, David O.
中科院分区：
文献类型：
--
作者：
Driskell, Lonnie O.;Yu, Xue-jie;Wood, David O.
普氏立克次体是流行性斑疹伤寒的病原体，是一种专性的胞质内细菌，这种生活方式对基因操作施加了显著的障碍。理解这种独特的细菌如何逃避宿主免疫的关键是对被假设参与毒力的选定基因的突变。Prowazekii的PLD基因编码一种具有磷脂酶D活性的蛋白质，与吞噬小体逃逸有关。为了证明定点敲除突变立克次体基因的可行性和产生不可逆转的疫苗株，我们利用同源重组产生了普氏立克次体马德里Evir强毒株的PLD突变体。利用线性DNA进行转化，双交换事件导致立克次体野生型基因被部分缺失的PLD基因取代。线形DNA被用来防止潜在的可逆单交叉事件导致的质粒插入。Southern杂交和聚合酶链式反应分析证实了所需突变的存在，并证明了克隆的存在。虽然在组织培养中没有观察到突变株和野生型菌株之间的表型差异，但在豚鼠模型中，PLD突变株显示出减弱的毒力。此外，用突变株免疫的动物可以抵抗强毒株Blinl的后续攻击，这表明该转化子可能成为一种不可逆转的减毒疫苗株。本研究证实了获得定点突变立克次体基因的可行性，为进一步了解立克次体生物学和进一步研究斑疹伤寒的预防提供了新的工具。
Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligately intracytoplasmic bacterium, a lifestyle that imposes significant barriers to genetic manipulation. The key to understanding how this unique bacterium evades host immunity is the mutagenesis of selected genes hypothesized to be involved in virulence. The R. prowazekii pld gene, encoding a protein with phospholipase D activity, has been associated with phagosomal escape. To demonstrate the feasibility of site-directed knockout mutagenesis of rickettsial genes and to generate a nonrevertible vaccine strain, we utilized homologous recombination to generate a pld mutant of the virulent R. prowazekii strain Madrid Evir. Using linear DNA for transformation, a double-crossover event resulted in the replacement of the rickettsial wild-type gene with a partially deleted pld gene. Linear DNA was used to prevent potentially revertible single-crossover events resulting in plasmid insertion. Southern blot and PCR analyses were used to confirm the presence of the desired mutation and to demonstrate clonality. While no phenotypic differences were observed between the mutant and wild-type strains when grown in tissue culture, the pld mutant exhibited attenuated virulence in the guinea pig model. In addition, animals immunized with the mutant strain were protected against subsequent challenge with the virulent Breinl strain, suggesting that this transformant could serve as a nonrevertible, attenuated vaccine strain. This study demonstrates the feasibility of generating site-directed rickettsial gene mutants, providing a new tool for understanding rickettsial biology and furthering advances in the prevention of epidemic typhus.