Broad-spectrum antibiotic activity of the arylomycin natural products is masked by natural target mutations

Abstract

Novel classes of broad-spectrum antibiotics are needed to treat multidrug-resistant pathogens. The arylomycin class of natural products inhibits a promising antimicrobial target, type I signal peptidase (SPase), but upon initial characterization appeared to lack whole-cell activity against most pathogens. Here, we show that Staphylococcus epidermidis, which is sensitive to the arylomycins, evolves resistance via mutations in SPase and that analogous mutations are responsible for the natural resistance of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. We identify diverse bacteria lacking these mutations and demonstrate that most are sensitive to the arylomycins. The results illustrate that the arylomycins have a broad-spectrum of activity and are viable candidates for development into therapeutics. The results also raise the possibility that naturally occurring resistance may have masked other natural product scaffolds that might be developed into therapeutics.

Figure 1. Chemical composition of the arylomycin class of natural product antibiotics

Arylomycin A₂ has the substituent pattern (R₁ = H, R₂ = H, R₃ = H, R₄ = isoC₁₁) and Arylomycin C₁₆ has the substituent patter (R₁ = H, R₂ = H, R₃ = H, R₄ = nC₁₅).

Figure 2. Growth rates and arylomycin C₁₆ sensitivities of E. coli strains harboring the indicated amino acid at SPase residue 84

Horizontal bars indicate standard deviation of growth rates from three independent experiments. MICs varied less than 2-fold between experiments. His (MIC 4 μg/ml) and Phe (MIC 2 μg/ml) variants have a temperature sensitive phenotype and are therefore not shown. For Pro²⁹, the MIC exceeds the detection limit of 256 μg/ml. See also Figure S1.

Figure 3. Physical and biochemical evidence for the proposed mechanism of arylomycin resistance

(A) Crystal structure of E. coli SPase in complex with arylomycin A₂ (PDB ID 1T7D) (Paetzel et al., 2004). Hydrogen-bonds observed in the crystal structure are shown in green, while the potential hydrogen bond prevented by Pro⁸⁴ is shown in red. Equilibrium binding affinities of arylomycin for Pro- and Ser- variants of E. coli (B) and S. aureus (C) SPases. Data points and bars represent average values and standard deviations within a single experiment. K_D values shown are the average of three independent experiments. See also Figure S2.

Figure 4. Distribution of SPase with Pro²⁹ (or Pro³¹) residues based on bacterial 16S rRNA phylogenies

Phylogenies of Gram-positive Firmicutes and Actinobacteria and Gram-negative Proteobacteria, Bacteroidetes, and Verrucomicrobia/Chlamydiae based on 16S rRNA sequences. Species are color coded by phylogenetic class (inner color band). The number of SPases and the presence or absence Pro at the relevant positions is indicated by the length and color(s) of the outer color band. Scale bar corresponds to a 10% divergence in 16S rRNA sequence. See also Figure S3 and S4, and Table S1.