Systematic Approach to Optimizing Specifically Targeted Antimicrobial Peptides against Streptococcus mutans

Abstract

Previously we reported a novel strategy of "targeted killing" through the design of narrow-spectrum molecules known as specifically targeted antimicrobial peptides (STAMPs) Construction of these molecules requires the identification and the subsequent utilization of two conjoined yet functionally independent peptide components: the targeting and killing regions. In this study, we sought to design and synthesize a large number of STAMPs targeting Streptococcus mutans, the primary etiologic agent of human dental caries, in order to identify candidate peptides with increased killing speed and selectivity compared with their unmodified precursor antimicrobial peptides (AMPs). We hypothesized that a combinatorial approach, utilizing a set number of AMP, targeting, and linker regions, would be an effective method for the identification of STAMPs with the desired level of activity. STAMPs composed of the Sm6 S. mutans binding peptide and the PL-135 AMP displayed selectivity at MICs after incubation for 18 to 24 h. A STAMP where PL-135 was replaced by the B-33 killing domain exhibited both selectivity and rapid killing within 1 min of exposure and displayed activity against multispecies biofilms grown in the presence of saliva. These results suggest that potent and selective STAMP molecules can be designed and improved via a tunable "buildingblock" approach.(Pathogenic microorganisms have been a continuous source of human suffering and mortality throughout the course of human history and have spurred the clinical development of novel therapeutics. Even today, the overall burden of infectious disease remains high, constituting a leading (and rising) cause of death worldwide (16,18). The conventional medical response to bacterial infections, administration of small-molecule antibiotics, has become less effective against emerging pathogens due to the evolution of drug resistance stemming in part from the misuse of antibiotics (13). Additionally, antibiotics and oral antiseptics currently in use to treat mucosal infections eliminate pathogens and bystander bacteria alike, an outcome that can be associated with negative clinical consequences (15, 17). Therefore, there is an unmet medical need to develop novel, narrow-spectrum therapeutics capable of maintaining the protective benefits of the normal microflora during treatment.Our strategy for creating novel, selective antibacterial agents is based on the addition of a targeting peptide to an existing broadspectrum antimicrobial peptide (AMP), thereby generating a specifically targeted antimicrobial peptide (STAMP) selective for a particular bacterial species or strain. A completed STAMP consists of conjoined but functionally independent targeting and killing regions, separated by a small flexible linker, all within a linear peptide sequence. The STAMP targeting region drives enhancement of antimicrobial activity by increasing binding to the surface of a targeted pathogen, utilizing specific determinants such as overall membrane hydrophobicity, charge, and/or pheromone recept...

Selective Membrane Disruption: Mode of Action of C16G2, a Specifically Targeted Antimicrobial Peptide

“…After the supernatants with excess dye were removed from biofilms, they were visualized by fluorescence microscopy (Leica DMI6000B microscope and Leica DFC340FX camera). To quantify the level of SYTOX green, green pixels were selected from each image utilizing GIMP software (http://www.gimp.org), adjusted for any background green fluorescence, and expressed as fluorescence intensity units (15,20).…”

New trends in peptide-based anti-biofilm strategies: a review of recent achievements and bioinformatic approaches

“…C-terminal alcohol (Sang and Blecha 2008;Duclohier 2010) . (Eckert et al 2006a;He et al 2010) . Chemically synthesized with high-yield in vitro (Eckert et al 2006a) .…”

“…Chemically synthesized with high-yield in vitro (Eckert et al 2006a) . Bacteria (Eckert et al 2006a;He et al 2010) . Increased killing potency, selectivity, and kinetics (Eckert et al 2006a) Synthetic antimicrobial peptidomimetics (SAMPs) .…”

Non-conventional therapeutics for oral infections

“…100,101 Other approaches include the modification of natural peptides to generate peptides with more favorable efficacy/toxicity profiles. 102 Alternatively, peptide mimetics have been designed and synthesized which retain the biological activity of an AMP but are advantageous as regards production costs, possess favorable therapeutic index and show stability under physiological conditions.…”