Department of Biological Sciences, Ohio University, Athens, Ohio, USA.
Department of Biological Sciences, Ohio University, Athens, Ohio, USA
Infect Immun. 2018 Oct 25;86(11). doi: 10.1128/IAI.00379-18. Print 2018 Nov.
The cyclophilin PpiB is an intracellular peptidyl prolyl isomerase (PPIase) that has previously been shown to contribute to secreted nuclease and hemolytic activity. In this study, we investigated the contribution of PpiB to virulence. Using a murine abscess model of infection, we demonstrated that a mutant is attenuated for virulence. We went on to investigate the mechanism through which PpiB protein contributes to virulence, in particular the contribution of PpiB PPIase activity. We determined the amino acid residues that are important for PpiB PPIase activity and showed that a single amino acid substitution (F64A) completely abrogates PPIase activity. Using purified PpiB F64A protein , we showed that PPIase activity only partially contributes to Nuc refolding and that PpiB also possesses PPIase-independent activity. Using allelic exchange, we introduced the F64A substitution onto the chromosome, generating a strain that produces enzymatically inactive PpiB. Analysis of the PpiB F64A strain revealed that PPIase activity is not required for hemolysis of human blood or virulence in a mouse. Together, these results demonstrate that PpiB contributes to virulence via a mechanism unrelated to prolyl isomerase activity.
亲环素 PpiB 是一种细胞内肽基脯氨酰顺反异构酶（PPIase），先前已被证明有助于分泌核酸酶和溶血活性。在这项研究中，我们研究了 PpiB 对毒力的贡献。我们使用了一种感染的鼠脓肿模型，证明突变体的毒力减弱。我们接着研究了 PpiB 蛋白有助于毒力的机制，特别是 PpiB PPIase 活性的贡献。我们确定了对 PpiB PPIase 活性很重要的氨基酸残基，并表明单个氨基酸取代（F64A）完全消除了 PPIase 活性。使用纯化的 PpiB F64A 蛋白，我们表明 PPIase 活性仅部分有助于 Nuc 重折叠，并且 PpiB 还具有 PPIase 非依赖性活性。通过等位基因交换，我们将 F64A 取代引入 染色体，生成产生酶失活 PpiB 的菌株。对 PpiB F64A 菌株的分析表明，PPIase 活性不是溶血人血或在小鼠中引起毒力所必需的。这些结果表明，PpiB 通过与脯氨酰异构酶活性无关的机制促进了毒力。
