Abstract
The circularized bacteriocin enterocin AS-48 produced by Enterococcus sp. exhibits antibacterial activity through membrane disruption. The membrane-penetrating activity of enterocin AS-48 has been attributed to a specific alpha-helical region on the circular peptide. Truncated, linearized forms containing these domains have been shown to preserve limited bactericidal activity. We utilized the amino acid sequence of the active helical domain of enterocin AS-48 to perform a homology-based search of similar sequences in other bacterial genomes. We identified similar domains in three previously uncharacterized AS-48-like bacteriocin genes in Clostridium sordellii, Paenibacillus larvae, and Bacillus xiamenensis. Enterocin AS-48 and homologs from these bacterial species were used as scaffolds for the design of a minimal peptide library based on the active helical domain of each bacteriocin sequence. 95 synthetic peptide variants of each scaffold peptide, designated Syn-enterocin, Syn-sordellicin, Syn-larvacin, and Syn-xiamensin, were designed and synthesized from each scaffold sequence based on defined biophysical parameters. A total of 384 total peptides were assessed for antibacterial activity against Gram-negative and Gram-positive bacteria. Minimal Inhibitory Concentrations (MICs) as low as 15.6 nM could be observed for the most potent peptide candidate tested, with no significant cytotoxicity to eukaryotic cells. Our work demonstrates for the first time a general workflow of using minimal domains of natural bacteriocin sequences as scaffolds to design and rapidly synthesize a library of bacteriocin-based antimicrobial peptide variants for evaluation.
Highlights
New chemical and biological scaffolds for the design of antibiotic compounds have become a highly prioritized area of research, especially as novel pandemics and secondary bacterial infections become a wider global concern (Rosenberg and Goldblum, 2006)
Previous studies demonstrated that the antimicrobial activity of full-length enterocin AS-48 can largely be attributed to cationic helices four and five (Montalbán-López et al, 2008)
Since linearized forms containing this region have been shown to be active and capable of optimization, we hypothesized that homologous regions in similar enterocin AS-48-like natural sequences could serve as a scaffold for peptide sequence optimization
Summary
New chemical and biological scaffolds for the design of antibiotic compounds have become a highly prioritized area of research, especially as novel pandemics and secondary bacterial infections become a wider global concern (Rosenberg and Goldblum, 2006). Bacteriocins are highly diverse in structure and function, the most fundamental division of bacteriocins, based on structure, is into class I (modified) and class II (unmodified) types (Cotter et al, 2013; Alvarez-Sieiro et al, 2016) In both cases, bacteriocins are believed to undergo the cleavage of a leader sequence from the core peptide domain. Using a naturally occurring thiopeptide as a scaffold, the compound LFF57 was designed by using traditional medicinal chemistry and structure activity relationship approaches and is in clinical trials (Lamarche et al, 2012; Mullane et al, 2015) Despite these significant advances, bacteriocins are still highly underrepresented as template sources for the design of linear AMPs
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