Activity of an antimicrobial peptide mimetic against planktonic and biofilm cultures of oral pathogens

Abstract

Antimicrobial peptides (AMPs) are naturally occurring, broad-spectrum antimicrobial agents that have recently been examined for their utility as therapeutic antibiotics. Unfortunately, they are expensive to produce and are often sensitive to protease digestion. To address this problem, we have examined the activity of a peptide mimetic whose design was based on the structure of magainin, exhibiting its amphiphilic structure. We demonstrate that this compound, meta-phenylene ethynylene (mPE), exhibits antimicrobial activity at nanomolar concentrations against a variety of bacterial and Candida species found in oral infections. Since Streptococcus mutans, an etiological agent of dental caries, colonizes the tooth surface and forms a biofilm, we quantified the activity of this compound against S. mutans growing under conditions that favor biofilm formation. Our results indicate that mPE can prevent the formation of a biofilm at nanomolar concentrations. Incubation with 5 nM mPE prevents further growth of the biofilm, and 100 nM mPE reduces viable bacteria in the biofilm by 3 logs. Structure-function analyses suggest that mPE inhibits the bioactivity of lipopolysaccharide and binds DNA at equimolar ratios, suggesting that it may act both as a membrane-active molecule, similar to magainin, and as an intracellular antibiotic, similar to other AMPs. We conclude that mPE and similar molecules display great potential for development as therapeutic antimicrobials.

FIG. 1.

Chemical structure of mPE.

FIG. 2.

Kinetics of bactericidal activity of mPE against S. mutans. A total of 2.5 × 10³ CFU was incubated at 37°C with increasing concentrations of mPE. Aliquots were removed and plated at the times indicated. The reactions were terminated by dilution in PBS. Samples were plated on BHI agar and incubated overnight. Results are presented as the percentage of viable colonies remaining after treatment.

FIG. 3.

Activity of mPE against S. mutans in a biofilm. Bacteria (1 × 10⁵ CFU) were seeded in wells of a 96-well plate in the presence of 1% sucrose. (A) Activity against growing biofilm. After formation of a biofilm, mPE was added at increasing concentrations. Growth in the biofilm was quantified by reading the absorbance at 595 nm, and results are presented as the change in absorbance from day 2 cultures prior to the addition of compound. Controls included no mPE (0) and 500 μg/ml tetracycline. The experiment was performed in triplicate. Error bars equal 1 standard deviation. (B) Bactericidal activity against bacteria in a biofilm. Bacteria were plated in sucrose for 2 days, followed by the addition of mPE. After 24 h, biofilms were disrupted by sonication and plated to measure the remaining viable bacteria. Error bars equal 1 standard deviation. (C) Confocal microscopy analysis of biofilm viability. Biofilms were treated for 24 h with 0 or 50 μg/ml mPE and stained using the LIVE/DEAD kit. Cultures were observed using a confocal laser scanning microscope at 488 nm and 543 nm. Merged images were produced using Zeiss LSM Image Browser software. Cultures are visualized from the side (left panel) and the top (right panel).

FIG. 4.

Inhibition of LPS bioactivity by mPE. RAW 264.7 cells were treated with 100 ng/ml E. coli LPS in the presence of 0, 1.9, or 9.5 mM mPE or 7 nM polymyxin B (PmxB) for 6 h at 37°C. Controls were 9.5 nM mPE or 7 nM polymyxin B alone (no LPS). TNF-α (TNF-a) levels were quantified by ELISA as described in Materials and Methods. Experiments were performed in triplicate. Error bars equal 1 standard error; asterisks indicate statistical significance as measured by a t test (P < 0.0001).

FIG. 5.

DNA binding activity of mPE. A single-stranded GC-rich oligonucleotide (ssDNA) was incubated at 37°C in the absence or presence of mPE at a molar ratio (mPE to DNA) of 1:1, 0.1:1, or 0.01:1, followed by agarose gel electrophoresis. The gel was visualized after ethidium bromide staining and UV irradiation. Double-stranded DNA was created by annealing of the single-stranded oligonucleotide at 65°C in isotonic saline buffer. The double-stranded oligonucleotide was incubated with mPE at a molar ratio of 1:1 (1:1ds) or in buffer alone (ds). The results shown are representative of three experiments. M, molecular marker.