Molecular mechanism of topoisomerase poisoning by the peptide antibiotic albicidin.

The peptide antibiotic albicidin is a DNA topoisomerase inhibitor with low-nanomolar bactericidal activity towards fluoroquinolone-resistant Gram-negative pathogens. However, its mode of action is poorly understood. We determined a 2.6 Å resolution cryoelectron microscopy structure of a ternary complex between Escherichia coli topoisomerase DNA gyrase, a 217 bp double-stranded DNA fragment and albicidin. Albicidin employs a dual binding mechanism where one end of the molecule obstructs the crucial gyrase dimer interface, while the other intercalates between the fragments of cleaved DNA substrate. Thus, albicidin efficiently locks DNA gyrase, preventing it from religating DNA and completing its catalytic cycle. Two additional structures of this trapped state were determined using synthetic albicidin analogues that demonstrate improved solubility, and activity against a range of gyrase variants and E. coli topoisomerase IV. The extraordinary promiscuity of the DNA-intercalating region of albicidins and their excellent performance against fluoroquinolone-resistant bacteria holds great promise for the development of last-resort antibiotics.

Transcription activation by the resistance protein AlbA as a tool to evaluate derivatives of the antibiotic albicidin.

The rising numbers of fatal infections with resistant pathogens emphasizes the urgent need for new antibiotics. Ideally, new antibiotics should be able to evade or overcome existing resistance mechanisms. The peptide antibiotic albicidin is a highly potent antibacterial compound with a broad activity spectrum but also with several known resistance mechanisms. In order to assess the effectiveness of novel albicidin derivatives in the presence of the binding protein and transcription regulator AlbA, a resistance mechanism against albicidin identified in Klebsiella oxytoca, we designed a transcription reporter assay. In addition, by screening shorter albicidin fragments, as well as various DNA-binders and gyrase poisons, we were able to gain insights into the AlbA target spectrum. We analysed the effect of mutations in the binding domain of AlbA on albicidin sequestration and transcription activation, and found that the signal transduction mechanism is complex but can be evaded. Further demonstrating AlbA's high level of specificity, we find clues for the logical design of molecules capable of avoiding the resistance mechanism.