In a loss-of-viability screen of small molecules against methicillin-resistant Staphylococcus aureus (MRSA) USA300, we found a small molecule, designated DNAC-2, which has an MIC of 8 μg ml−1. DNAC-2 is a quinolinol derivative that is bactericidal at 2X MIC. Macromolecular synthesis assays at 2 × MIC of DNAC-2 revealed inhibition of DNA, cell wall, RNA and protein synthesis within fifteen to thirty minutes of treatment when compared to the untreated control. Transmission electron microscopy of DNAC-2-treated cells revealed a significantly thicker cell wall and impaired daughter cell separation. Exposure of USA300 cells to 1 × MIC of DNAC-2 resulted in mislocalization of PBP2 away from the septum in an FtsZ-independent manner. In addition, membrane localization with FM4–64, as well as depolarization study with DiOC2 and lipophilic cation TPP+ displayed membrane irregularities and rapid membrane depolarization, respectively, in DNAC-2-treated cells vs -untreated control. However, DNAC-2 exhibited almost no toxicity toward eukaryotic membranes. Notably, DNAC-2 drives energy generation toward substrate level phosphorylation and the bacteria become more sensitive to DNAC-2 under anaerobic conditions. We propose that DNAC-2 affects USA300 by targeting the membrane, leading to partial membrane depolarization and subsequently affecting aerobic respiration and energy-dependent functional organization of macromolecular biosynthetic pathways. The multiple effects may have the desirable consequence of limiting the emergence of resistance to DNAC-2.
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We thank NERCE-BEID for facilitating this research, SRI Biosciences under the auspices of NIH Product Development Services for the pharmacokinetic and toxicity analyses, Louisa Howard for processing the TEM samples and imaging. HGS received support by the German Centre of Infection Research (DZIF). NERCE-BEID was funded by NIH grant U54AI057159. Work in the AC laboratory was partially funded by COBRE (NIH) # P30GM106394 and the Pfeiffer Foundation. MGP laboratory was funded by ERC-2012-StG-310987 grant from the European Research Council and JMM was supported by fellowship SFRH/BD/71993/2010.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on The Journal of Antibiotics website