College of Oceanography, Hohai University, Nanjing, 210098, P.R.China.
Paul G. Allen School for Global Animal Health, Washington State University, Pullman, WA, 99164, USA.
Sci Rep. 2017 Feb 16;7:42529. doi: 10.1038/srep42529.
Microcin PDI inhibits a diversity of pathogenic Escherichia coli through the action of an effector protein, McpM. In this study we demonstrated that expression of the inhibitory phenotype is induced under low osmolarity conditions and expression is primarily controlled by the EnvZ/OmpR two-component regulatory system. Functional, mutagenesis and complementation experiments were used to empirically demonstrate that EnvZ is required for the inhibitory phenotype and that regulation of mcpM is dependent on binding of the phosphorylated OmpR to the mcpM promoter region. The phosphorylated OmpR may recognize three different binding sites within this promoter region. Site-directed mutagenesis revealed that the McpM precursor peptide includes two leader peptides that undergo sequential cleavage at positions G17/G18 and G35/A36 during export through the type I secretion system. Competition assays showed that both cleaved products are required for the PDI phenotype although we could not distinguish loss of function from loss of secretion in these assays. McpM has four cysteines within the mature peptide and site-directed mutagenesis experiments demonstrated that the first two cysteines are necessary for McpM to inhibit susceptible cells. Together these data combined with previous work indicate that MccPDI is unique amongst the microcins that have been described to date.
微菌素 PDI 通过效应蛋白 McpM 抑制多种致病性大肠杆菌。在本研究中，我们证明在低渗透压条件下诱导抑制表型的表达，并且表达主要由 EnvZ/OmpR 双组分调节系统控制。功能、突变和互补实验用于经验性地证明 EnvZ 是抑制表型所必需的，并且 mcpM 的调节依赖于磷酸化 OmpR 与 mcpM 启动子区域的结合。磷酸化的 OmpR 可能在该启动子区域识别三个不同的结合位点。定点突变揭示，McpM 前体肽包含两个前导肽，在通过 I 型分泌系统进行输出时，在位置 G17/G18 和 G35/A36 处经历顺序切割。竞争测定表明，两种切割产物都需要 PDI 表型，尽管我们无法在这些测定中区分功能丧失和分泌丧失。McpM 在成熟肽内有四个半胱氨酸，定点突变实验表明，前两个半胱氨酸对于 McpM 抑制易感细胞是必需的。这些数据与以前的工作一起表明，MccPDI 在迄今为止描述的微菌素中是独特的。
