(620y) Development of a Lactococcal Vector for Antimicrobial Peptide Delivery in the Gut

The rise in antibiotic resistant pathogens has sparked an urgent need for new antimicrobial treatment options. Antimicrobial peptides (AMPs) are small proteins that exhibit micromolar to nanomolar activity against many pathogens of interest; however, their utility has been limited by their degradation in the gastrointestinal (GI) tract. We propose to overcome this delivery challenge by engineering probiotic bacteria that can survive passage through the upper GI tract and produce the desired AMPs at the site of infection. We are currently developing systems to target Vancomycin-resistant Enterococcus faecium and Vancomycin-resistant Enterococcus faecalis as these pathogens pose serious threats in health care. In our previous work, we engineered a powerful AMP expression system for Lactococccus lactis that was able to reduce in vitro counts of Vancomycin-resistant Enterococcus faecium (VRE) by approximately four orders of magnitude.¹ The work described here aims to build on this system by improving on three fronts; survivability of the probiotic in the gut, potency of the peptides, and decreased occurrence of resistance.

It is well-known that bile tolerance is a primary factor impacting bacterial survival in the gut. Consequently, we have now included a heterologous bile-resistance component in our AMP expression vector. We are currently testing these genes under different promoters to identify the most effective configuration to confer bile resistance both in vitro and in vivo. To improve the potency of our AMP expression system, we are exploring new AMPs exhibiting powerful activity both alone and in combination with our current peptides. Previously, we produced Enterocin A, Enterocin P, and Hiracin JM79.¹ In the work presented here, we demonstrate the successful production and secretion of Enterocin B from L. lactis using a fusion of Enterocin B with the lactococcal signal peptide Usp45. We are now testing the efficacy of these peptides individually and in combination to identify synergistic activity. To hinder resistance to our peptides, we are pursuing combinations of AMPs that rely on orthogonal targets; a method commonly used with traditional antibiotics to slow the development of resistance. We are thus working to identify peptide combinations that are not only effective at low concentrations, but also combinations that exhibit the rarest occurrence of resistance.

1. Geldart, K., Borrero, J., & Kaznessis, Y. N. (2015). A Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Against Antibiotic-Resistant Enterococcus faecium. Applied and Environmental Microbiology, 81(11), 3889–3897. doi:10.1128/AEM.00227-15