Abstract
The ever increasing multidrug-resistance of clinically important pathogens and the lack of novel antibiotics have resulted in a true antibiotic crisis where many antibiotics are no longer effective. Further complicating the treatment of bacterial infections are antibiotic-tolerant persister cells. Besides being responsible for the recalcitrant nature of chronic infections, persister cells greatly contribute to the observed antibiotic tolerance in biofilms and even facilitate the emergence of antibiotic resistance. Evidently, eradication of these persister cells could greatly improve patient outcomes and targeting persistence may provide an alternative approach in combatting chronic infections. We recently characterized 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009), a novel anti-persister molecule capable of directly killing persisters from both Gram-negative and Gram-positive pathogens. SPI009 potentiates antibiotic activity in several in vitro and in vivo infection models and possesses promising anti-biofilm activity. Strikingly, SPI009 restores antibiotic sensitivity even in resistant strains. In this study, we investigated the mode of action of this novel compound using several parallel approaches. Genetic analyses and a macromolecular synthesis assays suggest that SPI009 acts by causing extensive membrane damage. This hypothesis was confirmed by liposome leakage assay and membrane permeability studies, demonstrating that SPI009 rapidly impairs the bacterial outer and inner membranes. Evaluation of SPI009-resistant mutants, which only could be generated under severe selection pressure, suggested a possible role for the MexCD-OprJ efflux pump. Overall, our results demonstrate the extensive membrane-damaging activity of SPI009 and confirm its clinical potential in the development of novel anti-persister therapies.
Highlights
Modifying existing antibiotic scaffolds upon emergence of resistance has proven a successful strategy to extend a drug class’ utility in the past
Functional enrichment analysis of upand downregulated genes (Figure 2B), combined with network analysis (Supplementary Figure S2) revealed a first group of SPI009 upregulated genes to be involved in antibacterial efflux and multidrug resistance, suggesting the increased efforts of the cell to protect itself against SPI009
Since the natural membrane permeability of different bacterial species contributes to their intrinsic antibiotic resistance (Breidenstein et al, 2011), we investigated whether this affected the activity of SPI009
Summary
Modifying existing antibiotic scaffolds upon emergence of resistance has proven a successful strategy to extend a drug class’ utility in the past. Contributing to the difficult treatment of bacterial infections is the presence of persister cells which constitute a small but important fraction of phenotypic variants tolerant to treatment with high doses of antibiotics (Lewis, 2010). Their occurrence in many bacterial pathogens combined with a demonstrated link between persistence and the recalcitrant nature of chronic bacterial infections renders persisters a serious threat to immunocompromised patients and effective anti-persister treatments are much needed (Fauvart et al, 2011; Zhang, 2014; Fisher et al, 2017). The current need for novel antibacterials active against Gram-negative species, together with the unique characteristic of SPI009 to kill both normal and persister cells, prompted us to further investigate the mode of action of this compound
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