The PhoQ-activating potential of antimicrobial peptides contributes to antimicrobial efficacy and is predictive of the induction of bacterial resistance

Abstract

Antimicrobial peptides (AMPs) are among the leading candidates to replace antibiotics which have been rendered ineffective by the evolution of resistant bacterial strains. Concerns do exist, however, that the therapeutic administration of AMPs may also select for resistant strains but with much more dire consequences, as these peptides represent an endogenous and essential component of host immune defense. The recent demonstration that AMPs function as ligands for the bacterial sensory kinase PhoQ for the initiation of virulence and adaptive responses lends credence to these concerns. While the ability to serve as PhoQ ligands suggests that the therapeutic administration of AMPs could (i) exacerbate infections by promoting bacterial virulence and (ii) select resistant mutants by encouraging adaptive behaviors, it also provides a rational basis for AMP selection and optimization. Here, we demonstrate that derivatives of a representative AMP have differential abilities to serve as PhoQ ligands and that this correlates with the ability to induce bacterial adaptive responses. We propose that PhoQ-activating potential is a logical parameter for AMP optimization and introduce a novel strategy for the treatment of minimal bactericidal concentration data that permits the discrimination and quantification of the contributions of PhoQ-activating potential and direct antimicrobial activity to net antimicrobial efficiency.

FIG. 1.

Relative contributions of changes in DAA and tendencies to activate PAPs toward the change in net peptide efficiency of a peptide derivative compared to that of the parent peptide. Peptides with negative scores indicate more effective antimicrobials. Error bars show standard deviations.

FIG. 2.

Activation of PhoPQ by AMPs. The abilities of Bac2A peptide derivatives to initiate the activation of a PhoPQ-dependent reporter were examined by the incubation of bacteria in the presence of sublethal (1 μg/ml) concentrations of the indicated peptides, as well as 1 mM magnesium. The values reported represent the averages of the results for three replicate experiments. The activation and repression of PhoPQ by low (50 μM) and high (5 mM) magnesium concentrations, respectively, are also reported. Error bars show standard deviations.

FIG. 3.

Verification that calculated PAPs accurately predict the ability of a peptide to serve as a PhoQ ligand. The close correlation between calculated and observed PAPs is demonstrated by a linear regression plot.

FIG. 4.

Activation of AMP responses following exposure to sublethal concentrations of AMPs. The abilities of peptide derivatives to induce adaptive responses were determined by preincubation of bacteria for 4 h in the presence of sublethal concentrations of peptides prior to standard MBC assays. The values reported represent the averages of the results of three repeated experiments. Error bars show standard deviations. All assays were performed in the presence of 1 mM Mg²⁺.