(353f) Engineering Gene Expression Systems for Bacterial Delivery of Antimicrobial Peptides

The rising issue of antibiotic resistant infection demands the immediate development of novel antibiotics to combat the world’s evolving pathogens. Antimicrobial peptides (AMPs) are naturally produced by many eukaryotic and prokaryotic cells as a defense against surrounding bacteria. Despite their promise as an alternative to traditional antibiotics, AMPs remain unusable for most infections largely because of their rapid degradation in the body. Here we propose to overcome the delivery challenge of AMPs by producing AMPs at the site of infection using probiotic lactic acid bacteria. We are currently developing systems to fight both Enterococcus faecalis and Enterococcus faecium as these bacteria pose a significant and escalating threat in hospital infections. In this work, we describe a chloride-inducible expression system which shows promise for the delivery of AMPs. In our co-culture inhibition studies we have shown that Lactococcus lactis producing the three AMPs enterocin A, enterocin P, and hiracin JM79 under the chloride-inducible promoter is capable of immediately decreasing E. faecium counts by over three orders of magnitude using human physiological chloride concentrations. This expression system is beneficial in that it will be induced by the body’s own conditions and can be used to combat a wide range of pathogens.

We propose that this powerful promoter can also be used in conjunction with pathogen-inducible promoters to create expression systems with high protein production induced only in the presence of the pathogen of interest. This is achievable because of the reliance of the chloride-inducible system on the activator protein GadR. We propose to produce GadR using the E. faecalis-inducible promoter, PrgX-PrgQ such that in the presence of E. faecalis, GadR will be produced, and the chloride-inducible promoter will be activated to produce AMPs. This system would thus use the chloride-inducible promoter to amplify the pathogen signal to create a highly active, but infection-specific expression system.  Obtaining both specific and broad range expression systems is essential for bacterial delivery of AMPs to a variety of infections. The successful development of this delivery technology could open the gates for a much needed new class of antibiotics.