The development of new antimicrobial agents effective against Gram-negative bacteria remains a major challenge in drug discovery. The lasso peptide cloacaenodin has potent antimicrobial activity against multiple strains in the Enterobacter genus, one of the ESKAPE pathogens. Here, we show that cloacaenodin uses a previously uncharacterized TonB-dependent transporter, which we name CloU, to cross the outer membrane (OM) of susceptible bacteria. Inner membrane transport is mediated by the protein SbmA. CloU is distinct from the known OM transporters (FhuA and PupB) utilized by other antimicrobial lasso peptides and thus offers important insight into the spectrum of activity of cloacaenodin. Using knowledge of the transport pathway to predict other cloacaenodin-susceptible strains, we demonstrate the activity of cloacaenodin against clinical isolates of Enterobacter and of a Kluyvera strain. Further, we use molecular dynamics simulations and mutagenesis of CloU to explain the variation in cloacaenodin susceptibility observed across different strains of Enterobacter. This work expands the currently limited understanding of lasso peptide uptake and advances the potential of cloacaenodin as an antibiotic.

中文翻译：

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抗菌套索肽 Cloacaenodin 利用独特的 TonB 依赖性转运蛋白来接触敏感细菌

开发有效对抗革兰氏阴性菌的新型抗菌剂仍然是药物发现中的主要挑战。套索肽阴腔菌素对肠杆菌属（ESKAPE 病原体之一）的多种菌株具有有效的抗菌活性。在这里，我们展示了泄殖腔菌素使用一种以前未表征的 TonB 依赖性转运蛋白（我们将其命名为 CloU）来穿过易感细菌的外膜 (OM)。内膜转运由蛋白质 SbmA 介导。 CloU 与其他抗菌套索肽所利用的已知 OM 转运蛋白（FhuA 和 PupB）不同，因此为了解阴沟里素的活性谱提供了重要的见解。利用转运途径的知识来预测其他泄殖腔菌素敏感菌株，我们证明了泄殖腔菌素对肠杆菌和克鲁维拉菌株的临床分离株的活性。此外，我们使用分子动力学模拟和 CloU 诱变来解释在不同肠杆菌菌株中观察到的泄殖腔菌素敏感性的变化。这项工作扩大了目前对套索肽摄取的有限理解，并提高了阴沟霉素作为抗生素的潜力。