Antimicrobial Lasso Peptide Cloacaenodin Utilizes a Unique TonB-Dependent Transporter to Access Susceptible Bacteria

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.针对革兰氏阴性菌的新型抗菌剂研发仍是药物发现领域的重大挑战。套索肽（lasso peptide）cloacaenodin对肠杆菌属（Enterobacter，ESKAPE病原体之一）的多种菌株具有强效抗菌活性。本研究表明，cloacaenodin通过一种此前未被表征的TonB依赖性转运蛋白（TonB-dependent transporter，我们将其命名为CloU）穿过易感细菌的外膜（outer membrane, OM）。内膜转运则由蛋白SbmA介导。CloU与其他抗菌套索肽所使用的已知外膜转运蛋白FhuA和PupB存在显著差异，因此为阐明cloacaenodin的抗菌谱提供了重要见解。我们利用该转运通路的相关知识预测其他对cloacaenodin易感的菌株，证实了cloacaenodin对肠杆菌临床分离株以及克吕沃尔氏菌属（Kluyvera）菌株的抗菌活性。此外，我们通过分子动力学模拟和CloU的诱变实验，解释了不同肠杆菌菌株对cloacaenodin易感程度的差异。本研究拓展了当前对套索肽摄取机制的有限认知，并提升了cloacaenodin作为抗生素的应用潜力。