Laboratoire Littoral Environnement Sociétés, UMR 6250 CNRS-ULR, Université de La Rochelle, UFR Sciences, Bât. Marie Curie, Avenue Michel Crépeau, 17042 La Rochelle Cedex 01, France.
Antimicrob Agents Chemother. 2011 Mar;55(3):997-1007. doi: 10.1128/AAC.01217-10. Epub 2010 Dec 28.
For the first time, the mechanism of action of microcin L (MccL) was investigated in live bacteria. MccL is a gene-encoded peptide produced by Escherichia coli LR05 that exhibits a strong antibacterial activity against related Enterobacteriaceae, including Salmonella enterica serovars Typhimurium and Enteritidis. We first subcloned the MccL genetic system to remove the sequences not involved in MccL production. We then optimized the MccL purification procedure to obtain large amounts of purified microcin to investigate its antimicrobial and membrane properties. We showed that MccL did not induce outer membrane permeabilization, which indicated that MccL did not use this way to kill the sensitive cell or to enter into it. Using a set of E. coli and Salmonella enterica mutants lacking iron-siderophore receptors, we demonstrated that the MccL uptake required the outer membrane receptor Cir. Moreover, the MccL bactericidal activity was shown to depend on the TonB protein that transduces the proton-motive force of the cytoplasmic membrane to transport iron-siderophore complexes across the outer membrane. Using carbonyl cyanide 3-chlorophenylhydrazone, which is known to fully dissipate the proton-motive force, we proved that the proton-motive force was required for the bactericidal activity of MccL on E. coli. In addition, we showed that a primary target of MccL could be the cytoplasmic membrane: a high level of MccL disrupted the inner membrane potential of E. coli cells. However, no permeabilization of the membrane was detected.
首次在活细菌中研究了微菌素 L（MccL）的作用机制。MccL 是一种由大肠杆菌 LR05 产生的基因编码肽，对相关肠杆菌科，包括肠炎沙门氏菌血清型 Typhimurium 和 Enteritidis，具有很强的抗菌活性。我们首先亚克隆了 McL 遗传系统，以去除不参与 McL 生产的序列。然后，我们优化了 McL 纯化程序，以获得大量纯化的微菌素，以研究其抗菌和膜特性。我们表明 McL 不会诱导外膜通透性，这表明 McL 不会通过这种方式杀死敏感细胞或进入细胞。使用一组缺乏铁载体受体的大肠杆菌和肠炎沙门氏菌突变体，我们证明 McL 的摄取需要外膜受体 Cir。此外，MccL 的杀菌活性依赖于 TonB 蛋白，该蛋白将细胞质膜的质子动力传递到外膜以运输铁载体复合物。使用已知可完全耗散质子动力的羰基氰化物 3-氯苯腙，我们证明了质子动力对 McL 对大肠杆菌的杀菌活性是必需的。此外，我们表明 McL 的一个主要靶标可能是细胞质膜：高水平的 McL 破坏了大肠杆菌细胞的内膜电位。然而，没有检测到膜的通透性。
