Mechanism of action of the arylomycin antibiotics and effects of signal peptidase I inhibition

Abstract

Clinically approved antibiotics inhibit only a small number of conserved pathways that are essential for bacterial viability, and the physiological effects of inhibiting these pathways have been studied in great detail. Likewise, characterizing the effects of candidate antibiotics that function via novel mechanisms of action is critical for their development, which is of increasing importance due to the ever-growing problem of resistance. The arylomycins are a novel class of natural-product antibiotics that act via the inhibition of type I signal peptidase (SPase), which is an essential enzyme that functions as part of the general secretory pathway and is not the target of any clinically deployed antibiotic. Correspondingly, little is known about the effects of SPase inhibition or how bacteria may respond to mitigate the associated secretion stress. Using genetically sensitized Escherichia coli and Staphylococcus aureus as model organisms, we examine the activity of arylomycin as a function of its concentration, bacterial cell density, target expression levels, and bacterial growth phase. The results reveal that the activity of the arylomycins results from an insufficient flux of proteins through the secretion pathway and the resulting mislocalization of proteins. Interestingly, this has profoundly different effects on E. coli and S. aureus. Finally, we examine the activity of arylomycin in combination with distinct classes of antibiotics and demonstrate that SPase inhibition results in synergistic sensitivity when combined with an aminoglycoside.

Fig 1

Structure of arylomycin A-C₁₆. Other members of the arylomycin family of natural-product antibiotics are defined by different fatty acid lipid tails or modifications of the central biphenyl core (51).

Fig 2

Viability of exponentially growing E. coli PAS0260 at normal density (A) and high density (B) and exponentially growing S. aureus PAS8001 at normal density (C) and high density (D) in the presence of arylomycin A-C₁₆ at concentrations 2-fold (open squares) or 8-fold (open triangles) above the MIC compared to that of the vehicle control (open diamonds). A 3-log kill is used as the definition of bactericidal activity and is represented by dashed lines.

Fig 3

Viability of stationary-phase cultures of arylomycin-susceptible E. coli and S. aureus strains. Stationary-phase cultures of E. coli (A) and S. aureus (B) were washed and diluted into PBS and treated with vehicle controls (open diamonds) or with arylomycin A-C₁₆ at concentrations 2-fold (open squares) or 8-fold (open triangles) above the respective MICs. Cultures of S. aureus whose growth had been arrested by the addition of tetracycline were treated with vehicle controls (open diamonds) or with arylomycin A-C₁₆ at 8 times the MIC (open triangles) (C). A 3-log kill is used as the definition of bactericidal activity and is represented by dashed lines.