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Sequence-Specific Targeting of Bacterial Resistance Genes Increases Antibiotic Efficacy

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Targeting the genes that encode for the AcrAB-TolC efflux pump complex increases antibiotic susceptibility.

(A) Cartoon representation of the AcrAB-TolC efflux system based on available crystal structures (PDB IDs: AcrA-2f1m, AcrB-2dhh, and TolC-1ek9). IM: Inner Membrane; OM: Outer Membrane. (B) PPMOs are antisense molecules that bind to complementary mRNAs and sterically interfere with their translation. Silencing resistance-conferring genes with this strategy leads to antibiotic susceptibility. (C) We have engineered three separate PPMOs in order to target the acrA (blue), acrB (magenta), and tolC (green) genes. These PPMOs target gene regions that span the start codons of the transcribed mRNA. Alignment of the acrA, acrB, and tolC genes of different bacterial genera demonstrate that the PPMO sequences, designed for E. coli, are also complementary in other pathogens. The overlapping nucleotides between the gene sequences and the PPMOs are highlighted in color. The PPMO sequences are homologous in Klebsiella pneumoniae and Salmonella enterica genes but have limited homology to the remaining bacterial species. (D) Growth of bacteria is quantified by calculating the area under the curve (AUC), which is simply integrating OD600 from 0 to 24 h (Materials and Methods). Area under the black (circles) and cyan lines (triangles) correspond to the growth of the wild type and acrA deletion E. coli strains, respectively, in a subinhibitory dose of clindamycin. (E) (Left) Dose response curves as a function of clindamycin concentration. Dose response curves are generated using the AUC values. Curves are labeled as untreated wild type E. coli (black lines, empty circles), with 10 μM control-PPMO (grey lines, filled circles), with 10 μM acrA-PPMO (top panel, blue lines, filled squares), E. coli with acrA deletion (top panel, cyan lines, empty triangles), with 10 μM acrB-PPMO (middle panel, magenta lines, filled squares), E. coli with acrB deletion (middle panel, pink lines, empty triangles), with 10 μM tolC-PPMO (bottom panel, dark green lines, filled squares), and E. coli with tolC deletion (bottom panel, light green lines, empty triangles). The horizontal dashed lines represent 95% growth inhibition, while the vertical lines represent the MIC value for WT. (Right) Sample OD600 versus time growth curves at the conditions shown within the grey shaded areas on the dose response curves (Left). Each line is interpolated, integrated and the AUC is normalized to the wild type growth in the absence of clindamycin. Dose response curves and corresponding MIC values for all 11 antibiotics may be found in S4 Fig and S2 Table, respectively.

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doi: https://doi.org/10.1371/journal.pbio.1002552.g002