High-throughput generation of small antibacterial peptides with improved activity

Abstract

Cationic antimicrobial peptides are able to kill a broad variety of Gram-negative and Gram positive bacteria and thus are good candidates for a new generation of antibiotics to treat multidrug-resistant bacteria. Here we describe a high-throughput method to screen large numbers of peptides for improved antimicrobial activity. The method relies on peptide synthesis on a cellulose support and a Pseudomonas aeruginosa strain that constitutively expresses bacterial luciferase. A complete substitution library of 12-amino-acid peptides based on a linearized variant (RLARIVVIRVAR-NH(2)) of the bovine peptide bactenecin was screened and used to determine which substitutions at each position of the peptide chain improved activity. By combining the most favorable substitutions, we designed optimized 12-mer peptides showing broad spectrum activities with minimal inhibitory concentrations (MIC) as low as 0.5 microg/ml against Escherichia coli. Similarly, we generated an 8-mer substituted peptide that showed broad spectrum activity, with an MIC of 2 microg/ml, against E. coli and Staphylococcus aureus.

Using Intrinsic X-ray Absorption Spectral Differences To Identify and Map Peptides and Proteins

“…Sample Sources and Preparation. Sub6 (RWWKI-WVIRWWR-NH 2 ), 27 an optimized variant of Bac2A, which in turn is a linearized version of bactenecin, a naturally occurring bovine peptide, 28 was synthesized by P. Henklein using 9-fluorenylmethyl carbamate (fMOC) solid-phase synthesis. Indolicidin (ILPWKWPWWPWRR-NH 2 ) was synthesized by P. Owen at the Peptide Synthesis Facility, Biomedical Research Centre, UBC, using tertiary butyloxycarbonyl (tBOC) solidphase synthesis.…”

Use of luminescent bacteria for rapid screening and characterization of short cationic antimicrobial peptides synthesized on cellulose using peptide array technology

“…Since purity and quantity of the peptides synthesized on cellulose may vary, all results achieved using this method need to be confirmed with peptides produced by conventional solid-phase peptide synthesis on resin. It is worth noting that antibacterial activity assays with soluble peptides usually confirm the activity of peptides synthesized on cellulose 8,10 . However, the correct design and preparation of the assay are critical to the success of the method.…”

“…Screening large numbers of peptides using peptide synthesis on resin is labor intense and expensive. At only approximately 1% of the cost per peptide compared to the conventional peptide synthesis on resin, the synthesis of arrays of peptides on cellulose using SPOT technology represents a powerful tool for characterizing or screening large numbers of peptides for antimicrobial activity [6][7][8][9] . Using SPOT synthesis, up to 1,000 peptides per 20 Â 29-cm 2 cellulose sheet can be synthesized in a highly parallel and addressable manner.…”

“…Direct testing of killing activity is possible but cumbersome, and both the sensitivity and dynamic range can be limited. Therefore we adapted a luminescence inhibition assay that has been used previously for a variety of bacteria and antimicrobial agents [12][13][14][15][16] , can be performed in small volumes and is rapid (giving a readout of activity within 30 min or less), sensitive and quantitative 8,15 . This assay relies on the genes expressed from a luxCDABE cassette.…”

Inhibition of phospho-MurNAc-pentapeptide translocase (MraY) by nucleoside natural product antibiotics, bacteriophage ϕX174 lysis protein E, and cationic antibacterial peptides

“…Many synthetic variants of cationic antimicrobial peptides have been generated by Hancock and co-workers, who have observed that Arg and Trp predominate in high activity peptide sequences [56][57][58], including that of clinical candidate MX226 (Omiganan) [57]. Other short synthetic peptides containing Arg-Trp have also been reported to show high levels of antimicrobial activity [59,60], even dipeptide derivatives based upon Trp-Arg [61].…”