A synthetic congener modeled on a microbicidal domain of thrombin- induced platelet microbicidal protein 1 recapitulates staphylocidal mechanisms of the native molecule

Abstract

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a staphylocidal peptide released by activated platelets. This peptide initiates its microbicidal activity by membrane permeabilization, with ensuing inhibition of intracellular macromolecular synthesis. RP-1 is a synthetic congener modeled on the C-terminal microbicidal alpha-helix of tPMP-1. This study compared the staphylocidal mechanisms of RP-1 with those of tPMP-1, focusing on isogenic tPMP-1-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains for the following quantitative evaluations: staphylocidal efficacy; comparative MIC; membrane permeabilization (MP) and depolarization; and DNA, RNA, and protein synthesis. Although the proteins had similar MICs, RP-1 caused significant killing of ISP479C (<50% survival), correlating with extensive MP (>95%) and inhibition of DNA and RNA synthesis (>90%), versus substantially reduced killing of ISP479R (>80% survival), with less MP (55%) and less inhibition of DNA or RNA synthesis (70 to 80%). Interestingly, RP-1-induced protein synthesis inhibition was equivalent in both strains. RP-1 did not depolarize the cell membrane and caused a relatively short postexposure growth inhibition. These data closely parallel those previously reported for tPMP-1 against this strain set and exemplify how synthetic molecules can be engineered to reflect structure-activity relationships of functional domains in native host defense effector molecules.

FIG. 1.

Staphylocidal activities of RP-1 and tPMP-1 on ISP479C and ISP479R at an initial inoculum of 10⁷ CFU/ml. Logarithmic-phase S. aureus cells were exposed to either 25 (□), 50 (), or 100 () μg/ml of RP-1 or 2 μg/ml (▪) of tPMP-1. The mean percentage (±SD) of S. aureus survival versus controls was calculated from at least three independent experiments.

FIG. 2.

S. aureus MP by RP-1 and tPMP-1. Panels A and B show the fluorescence distribution (FL-1 units) versus the number of S. aureus ISP479C (A) and ISP479R (B) cells treated with 50 μg/ml RP-1 over 0 (—), 60 ( · · · ), and 120 (- - - - ) minutes. Arrows in panels A and B represent the extent of fluorescence of ISP479C and ISP479R at 120 min of RP-1 (50 μg/ml) exposure, respectively. Panel C shows the percent MP by RP-1 at 50 μg/ml on ISP479C (○) and ISP479R (□) and RP-1 at 100 μg/ml on ISP479R (▪) versus controls (±SD). Panel D shows the percent MP of ISP479C (○) and ISP479R (□) caused by tPMP-1 (2 μg/ml) versus controls. Each experiment was done in triplicate.

FIG. 3.

Effect of RP-1 on DNA synthesis in S. aureus ISP479C and ISP479R. Panel A (ISP479C) and panel B (ISP479R) present the actual [methyl-³H]thymidine incorporation kinetic curves (○, untreated control; ▴, novobiocin; and ⧫, RP-1). CPM, counts per minute. Panel C (ISP479C) and panel D (ISP479R) show the percent DNA synthesis inhibition versus untreated controls over 120 min of incubation (□, 30 min; ▩, 60 min; , 90 min; and ▪, 120 min). Novobiocin was used as a positive control DNA synthesis inhibitor.

FIG. 4.

Effect of RP-1 on protein synthesis in S. aureus ISP479C and ISP479R. Panel A (ISP479C) and panel B (ISP479R) present the actual [4,5-³H]leucine incorporation kinetic curves (○, untreated control; ▴, novobiocin; and ⧫, RP-1). Panel C (ISP479C) and panel D (ISP479R) show the percent protein synthesis inhibition versus untreated controls over 120 min of exposure (□, 30 min; ▩, 60 min; , 90 min; and ▪, 120 min). Tetracycline was used as a positive control protein synthesis inhibitor.