Targeted killing of Streptococcus mutans by a pheromone-guided "smart" antimicrobial peptide

Abstract

Within the repertoire of antibiotics available to a prescribing clinician, the majority affect a broad range of microorganisms, including the normal flora. The ecological disruption resulting from antibiotic treatment frequently results in secondary infections or other negative clinical consequences. To address this problem, our laboratory has recently developed a new class of pathogen-selective molecules, called specifically (or selectively) targeted antimicrobial peptides (STAMPs), based on the fusion of a species-specific targeting peptide domain with a wide-spectrum antimicrobial peptide domain. In the current study, we focused on achieving targeted killing of Streptococcus mutans, a cavity-causing bacterium that resides in a multispecies microbial community (dental plaque). In particular, we explored the possibility of utilizing a pheromone produced by S. mutans, namely, the competence stimulating peptide (CSP), as a STAMP targeting domain to mediate S. mutans-specific delivery of an antimicrobial peptide domain. We discovered that STAMPs constructed with peptides derived from CSP were potent against S. mutans grown in liquid or biofilm states but did not affect other oral streptococci tested. Further studies showed that an 8-amino-acid region within the CSP sequence is sufficient for targeted delivery of the antimicrobial peptide domain to S. mutans. The STAMPs presented here are capable of eliminating S. mutans from multispecies biofilms without affecting closely related noncariogenic oral streptococci, indicating the potential of these molecules to be developed into "probiotic" antibiotics which could selectively eliminate pathogens while preserving the protective benefits of a healthy normal flora.

FIG. 1.

Selective killing activity of C16G2 against S. mutans. S. mutans, S. sanguinis, and S. gordonii planktonic cells were exposed to 25 μM of the STAMP C16G2, or its untargeted parent antimicrobial peptide G2, for 1 min. Surviving CFU per milliliter were detected and compared. Data represent averages of the results of at least three independent experiments.

FIG. 2.

Inhibitory activity of G2 and C16G2 against single-species biofilms. S. gordonii (A), S. sanguinis (B), and S. mutans (C) monoculture biofilms were grown and then exposed for 1 min to a 25 μM concentration of STAMP or a STAMP component (as indicated in the figure), washed, and regrown with fresh medium. Biofilm recovery was monitored over time by OD₆₀₀. Data represent averages of the results from three independent experiments.

FIG. 3.

C16G2 activity against S. mutans within a multispecies biofilm. Mixed cultures of S. mutans, S. sanguinis, and S. gordonii were allowed to form a biofilm in saliva and were then exposed to 25 μM C16G2, CSP_C16, or G2. After being washed, the biofilms were allowed to recover in fresh medium-saliva. The regrowth of the biofilm over time was monitored by measuring absorbance at OD₆₀₀, while the health of the S. mutans within the biofilm was measured by luciferase activity (RLU production). The data were plotted as RLU/OD₆₀₀ and represent averages of the results of at least three independent experiments.

FIG. 4.

Activity of M8G2 against oral bacteria in biofilms. S. mutans (A) or S. sanguinis (B) single-species biofilms were mock treated or exposed to 25 μM M8G2 (as specified in the figure). After removal of the STAMP and the addition of fresh medium, biofilm recovery was monitored over time by monitoring absorbance at OD₆₀₀. The data represent the averages of the results of three independent experiments.

FIG. 5.

Biofilm-inhibitory activity of S6L3-33 and S6L3-33-containing STAMPs. Single-species biofilms of S. mutans (A) or S. sanguinis (B) were treated with M8-33, C16-33, or S6L3-33 alone (as specified in the figure) for 1 min. After agent removal and stringent washing, the regrowth of the biofilms was tracked over 4 h by measuring absorbance at OD₆₀₀ after the addition of fresh medium. The data represent average values obtained from the results of at least three independent assays.