Disulfiram Enhances the Activity of Polymyxin B Against Klebsiella pneumoniae by Inhibiting Lipid A Modification

Background: The use of antibiotic adjuvants is a complementary strategy to the development of new antibiotics. The essential role of the ArnA dehydrogenase domain (ArnA_DH) in the addition of 4-amino-L-arabinose (L-Ara4N) to lipid A makes it a potential target in polymyxin adjuvant design. Purpose: This study aimed to identify a dehydrogenase inhibitor that enhances the antibacterial effect of polymyxin B (PB) and to further understand the mechanism of this drug combination. Methods: A susceptible K. pneumoniae strain, ATCC13883, was used to screen a dehydrogenase inhibitor library based on 3-(4,5)dimethylthiazol(-z-y1)-2,5-diphenyltetrazolium bromide (MTT) and chequerboard assays. The protein- and cell-based effects of disulfiram (DSF) on ArnA activity were assessed, and the transcription levels of genes in the arn operon were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Lipid A was isolated, and a structural analysis was performed. The cell wall function was evaluated through membrane integrity and bacterial viability assays. The in vivo antibacterial activity was evaluated using a mouse pulmonary infection model. Results: We screened a dehydrogenase inhibitor library and found that the anti-alcoholism drug DSF significantly enhanced the antibacterial activity of PB in vitro and in vivo. The protein-based enzyme activity assay showed that DSF exerted no direct effect on the dehydrogenase activity of ArnA. Treatment with the combination of DSF and PB but not with PB alone decreased both the transcription level of genes in the arn operon and the modification level of lipid A. DSF also strengthened the disruption of the cell membrane integrity of PB. Moreover, the enhanced PB antibacterial activity was effective against clinical PB-resistant strains. Conclusion: We identified a new drug combination that can be used to reduce the necessary dosage of PB and overcome PB resistance, and this drug combination has good prospects for clinical application.