A New Natural Product Analog of Blasticidin S Reveals Cellular Uptake Facilitated by the NorA Multidrug Transporter

Abstract

The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics.

Keywords: antibiotic resistance; chemical genomics; natural products; transporters.

FIG 1

Structures of blasticidin S (1) and P10 (2).

FIG 2

In vitro inhibition of translation by blasticidin S and P10. Translation activity in the presence of antibiotics relative to the expression level in the absence of compounds was determined by expression of a luciferase reporter in an E. coli cell extract. Error bars represent the range of duplicate assays. The positive (chloramphenicol [50 μM]) and negative (DMSO) controls inhibited translation by 99% and 0%, respectively.

FIG 3

Genetic map of the determinants of blasticidin S and P10 resistance in S. aureus. Scaffolds resulting from genomic assembly of the four parent strains of S. aureus are represented in the outer ring, with size marked in kilobases. Mutant strains are displayed in the inner rings and are ordered and colored by the average increase in MIC of blasticidin S and P10 relative to the wild-type levels (strain names are abbreviated such that P10^r-1 corresponds to P1). The positions of mutations are marked for each strain, with a square denoting large deletions, a triangle denoting gene inactivations (frameshift or stop codon), and a circle representing the remainder. Dark markers represent unique mutations, while light markers represent identical polymorphisms observed in multiple strains. Gene symbols corresponding to mutations (except deletions) are displayed around the outer side of the assembly track.

FIG 4

The effect of norA on resistance and cellular uptake for blasticidin S (BlaS), P10, and norfloxacin (NOR). (A) Disk diffusion assay showing zones of inhibition surrounding paper disks loaded with antibiotic overlaid on bacterial cultures of S. aureus TCH1516 WT (panel i), TCH1516 P10^r-16 (ΔnorA) (panel ii), TCH1516 P10^r-16::norA (norA complementation) exposed to blasticidin S (panel iii), TCH1516 WT (panel iv), TCH1516 P10^r-16 (panel v), TCH1516 P10^r-16::norA exposed to P10 (panel vi), TCH1516 WT (panel vii), TCH1516 P10^r-16 (panel viii), and TCH1516 P10^r-16::norA exposed to norfloxacin (panel ix). (B) E. coli NEB5α (left panel) and NEB5α::norA (right panel) exposed to blasticidin S (panel i), P10 (panel ii), and norfloxacin (panel iii). (C) Quantitation of the concentration of blasticidin, P10, and norfloxacin in cell lysates of E. coli NEB5α transformed with a control plasmid or the norA gene, exposed to each antibiotic; error bars represent standard deviations of results from three replicates.

FIG 5

The functions of genes associated with significant levels of susceptibility or resistance to blasticidin S (circles) and P10 (triangles). Significant genes were selected with a P_adj cutoff value of <0.01. Points are colored according to gene product function (Table S10). The top six most significant hits for P10 with a P_adj value of ≪10⁻⁸, encoding the subunits and regulator of an ABC superfamily membrane transport complex, were omitted for clarity.