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Directed Mutagenesis of the Rickettsia prowazekii pld Gene Encoding Phospholipase D

机译：立陶宛立克次体编码磷脂酶D的基因的定向诱变

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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.
机译：流行性斑疹伤寒的致病菌是一种专一的胞浆内细菌，这种生活方式对基因操作施加了重大障碍。理解这种独特细菌如何逃避宿主免疫力的关键是诱变选定的基因，这些基因被假定与毒力有关。 R。编码具有磷脂酶D活性的蛋白质的prowazekii pld 基因已经与吞噬体逃逸有关。为了证明立克次体基因的定点敲除诱变的可行性并产生不可逆的疫苗株，我们利用同源重组产生了毒性 R的 pld 突变体。 prowazekii 毒株马德里Evir。使用线性DNA进行转化时，两次交换事件导致用部分缺失的 pld 基因代替了立克次体野生型基因。线性DNA被用于防止潜在的可逆的单交换事件，其导致质粒插入。 Southern印迹和PCR分析用于确认所需突变的存在并证明克隆性。当在组织培养中生长时，突变株和野生株之间没有观察到表型差异，而 pld 突变株在豚鼠模型中显示出减弱的毒力。另外，用该突变株免疫的动物被保护免于随后用强力的Brei1株攻击，表明该转化体可以用作不可逆的减毒疫苗株。这项研究证明了产生定点立枯立克次体基因突变体的可行性，为了解立枯立克次体生物学和进一步预防流行性斑疹伤寒提供了新的工具。

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