An enhancer peptide for membrane-disrupting antimicrobial peptides

Abstract

Background: NP4P is a synthetic peptide derived from a natural, non-antimicrobial peptide fragment (pro-region of nematode cecropin P4) by substitution of all acidic amino acid residues with amides (i.e., Glu --> Gln, and Asp --> Asn).

Results: In the presence of NP4P, some membrane-disrupting antimicrobial peptides (ASABF-alpha, polymyxin B, and nisin) killed microbes at lower concentration (e.g., 10 times lower minimum bactericidal concentration for ASABF-alpha against Staphylococcus aureus), whereas NP4P itself was not bactericidal and did not interfere with bacterial growth at <or= 300 microg/mL. In contrast, the activities of antimicrobial agents with a distinct mode of action (indolicidin, ampicillin, kanamycin, and enrofloxacin) were unaffected. Although the membrane-disrupting activity of NP4P was slight or undetectable, ASABF-alpha permeabilized S. aureus membranes with enhanced efficacy in the presence of NP4P.

Conclusions: NP4P selectively enhanced the bactericidal activities of membrane-disrupting antimicrobial peptides by increasing the efficacy of membrane disruption against the cytoplasmic membrane.

Figure 1

Structure of NP4P. The parent peptide, nematode cecropin P4 pro-piece (P4P), is shown at the top. Inversed letters indicate acidic amino acid residues which were substituted with amides in NP4P. Letters on a grey background represent basic amino acid residues.

Figure 2

Effect of NP4P on bacterial growth. Staphylococcus aureus IFO12732 (A) and Escherichia coli JM109 (B) in the logarithmic phase were suspended in 2 mL of IFO702 medium with or without 300 μg/mL of NP4P. Their optical densities were adjusted to 0.06-0.08 at 600 nm. The bacterial suspension was incubated at 30°C. Bacterial growth was estimated by measuring the change in optical density. All experiments were performed in triplicate. Each data point represents the mean ± SEM.

Figure 3

NP4P enhancement of bactericidal activities of AMPs. The dose-effect curves were determined in the presence of NP4P at various concentrations (0, 2.5, 5, 20, and 100 μg/mL). Bactericidal activities were measured against S. aureus IFO12732 for ASABF-α (A) and against E. coli JM109 to polymyxin B (B). Viability is defined as normalized number of viable cells to the number in the absence of ASABF-α or polymyxin B.

Figure 4

Effect of NP4P on the membrane-disrupting activity of ASABF-α against the cytoplasmic membrane of S. aureus. Disruption of the cytoplasmic membrane was estimated by the increase in fluorescence intensity of diS-C₃-(5). Changes in fluorescence were normalized by the value at the plateau of the dose-response curves. (A) Dose-response curve and (B) dose-bactericidal effect curve of ASABF-α against S. aureus IFO12732. These curves were simultaneously determined. The asterisks indicate that viable cells were not detected. (C) Effect of NP4P on the cytoplasmic membrane. The time courses of fluorescence changes are represented. (D) Effect of NP4P on cytoplasmic membrane disruption by ASABF-α. Dose-response curves were determined in the presence of NP4P at various concentrations (0, 30, and 100 μg/ml). (E) Another assay for NP4P enhancement. NP4P was applied after treatment of 1.28 μg/mL of ASABF-α. The fluorescent change evoked only by ASABF-α is indicated by a dashed line.