(72g) Discovery of Novel Plantaricin-423 Analogs From Synthetic Oligonucleotide Libraries

Excessive use of antibiotics in human and animal health care for the treatment and prevention of bacterial infections led to a rapid increase in antibiotic-resistant pathogenic bacteria. Antimicrobial peptides (AMPs) belong to a class of natural microbicidal molecules with lower propensity for inducing drug resistance; hence, they present significant potential to be used as alternative drugs to conventional antibiotics. By generating AMP libraries, one can study a large number of candidates for their activities simultaneously in a timely manner. However, current methodologies for the construction of AMP libraries present many limitations when it comes to sequence design, peptide length, or the library size. We have previously reported a novel technology for the construction of custom peptide libraries, where fully defined collections of in silico-designed peptides are encoded into DNA oligonucleotides and expressed in a cellular host for rapid screening of active molecules. We now present an application of this method to discovery of novel Plantaricin-423 analogs. Plantaricin-423 (Pln-423) is a 37-amino acid Class II-a bacteriocin and it displays bactericidal activities against many foodborne pathogens and spoilage bacteria. A custom mutant library coding for 12,000 unique Pln-423 analogs was constructed in Escherichia coli and screened against Listeria innocua and several other gram positive bacteria. Analysis of the selected clones by both Sanger-sequencing and high-throughput 454 pyrosequencing resulted in identification of more than 20 novel sequences with greater activities. Further investigation of the sequencing data revealed the specific mutations that are responsible for the change in antimicrobial activity of the wild-type peptide.  The data generated in this study can be used for the construction of secondary libraries to optimize the anti-listerial activity of Pln-423 mutants or broaden their range of activity against other gram positive bacteria.  Therefore, this study successfully demonstrates the great potential of our method for AMP discovery and development.