The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of .

The myxobacterial secondary metabolite carolacton inhibits growth of and kills biofilm cells of the caries- and endocarditis-associated pathogen at nanomolar concentrations. Here, we studied the response to carolacton of an strain that lacked the outer membrane protein TolC. Whole-genome sequencing of the laboratory strain TolC revealed the integration of an insertion element, IS, at the locus and a close phylogenetic relationship to the ancient K-12. We demonstrated via transcriptome sequencing (RNA-seq) and determination of MIC values that carolacton penetrates the phospholipid bilayer of the Gram-negative cell envelope and inhibits growth of TolC at similar concentrations as for streptococci. This inhibition is completely lost for a C-9 () epimer of carolacton, a derivative with an inverted stereocenter at carbon atom 9 [() → ()] as the sole difference from the native molecule, which is also inactive in and , suggesting a specific interaction of native carolacton with a conserved cellular target present in bacterial phyla as distantly related as and . The efflux pump inhibitor (EPI) phenylalanine arginine β-naphthylamide (PAβN), which specifically inhibits AcrAB-TolC, renders susceptible to carolacton. Our data indicate that carolacton has potential for use in antimicrobial chemotherapy against Gram-negative bacteria, as a single drug or in combination with EPIs. Strain TolC has been deposited at the DSMZ; together with the associated RNA-seq data and MIC values, it can be used as a reference during future screenings for novel bioactive compounds. The emergence of pathogens resistant against most or all of the antibiotics currently used in human therapy is a global threat, and therefore the search for antimicrobials with novel targets and modes of action is of utmost importance. The myxobacterial secondary metabolite carolacton had previously been shown to inhibit biofilm formation and growth of streptococci. Here, we investigated if carolacton could act against Gram-negative bacteria, which are difficult targets because of their double-layered cytoplasmic envelope. We found that the model organism is susceptible to carolacton, similar to the Gram-positive , if its multidrug efflux system AcrAB-TolC is either inactivated genetically, by disruption of the gene, or physiologically by coadministering an efflux pump inhibitor. A carolacton epimer that has a different steric configuration at carbon atom 9 is completely inactive, suggesting that carolacton may interact with the same molecular target in both Gram-positive and Gram-negative bacteria.