(191g) Engineering Probiotic Bacteria for the Delivery of Antimicrobial Peptides to the Intestines

Antibiotic resistant infections have become a severe threat in our healthcare systems worldwide, killing over 700,000 people each year. An increasing number of bacteria have accumulated resistance to nearly all of our current drugs making them more difficult, if not impossible to treat. Without the introduction of new antimicrobial technologies, resistant infections will be competing with cancer as the leading cause of death by 2050.

Antimicrobial peptides (AMPs) are small proteins secreted by plants, animals, fungi, and bacteria as a first line of defense against invading pathogens. Numerous AMPs are known to be active against different pathogens of interest, however, their utility has been limited because they are degraded in the body. This prevents them from reaching the intestinal tract where many resistant infections originate.

Herein, we discuss the development of probiotic bacteria as AMP-delivery vehicles for the reduction of pathogens in the intestines. We engineer probiotic bacteria that can be orally administered, survive passage to the lower intestines, then produce AMPs targeting our pathogen of interest. There is a growing body of evidence suggesting that naturally-occurring AMP production is a primary protective mechanism of many probiotic bacterial species. In our work, we engineer these natural defense systems to produce greater, more controllable quantities of peptides, and to produce new peptides to target a variety of different pathogens.

In the work described here, we engineer probiotic E. coli Nissle 1917 to produce and secrete eight different AMPs targeting diarrheagenic E. coli, Salmonella, and vancomycin-resistant Enterococcus (VRE).¹ To do this, we developed an AMP expression vector for Nissle that employs a powerful synthetic promoter to drive the expression of the desired AMPs. We then add a transporter from the E. coli-derived AMP, Microcin V, and show that we can use this transporter to secrete all eight peptides from Nissle. The flexibility of this system allows us to modularly express numerous AMPs targeting a wide array of pathogens.

Herein, we focus on Nissle engineered to target vancomycin-resistant Enterococcus (VRE). VRE is a flagrant pathogen in hospital environments where it colonizes the intestines of over one in ten patients. In densely-colonized patients, VRE can pass into the bloodstream. Over one in twenty of these infections will result in death. By specifically reducing VRE colonization and expansion, we can prevent these deadly translocation events and limit patient to patient contamination.

We use our modular peptide expression system to simultaneously express three AMPs targeting Enterococcus. When grown in liquid culture with VRE, our engineered Nissle eliminates all pathogen survival with no observed of resistance development. We then show that Nissle administered in drinking water reduces VRE in the intestines of mice by over 90%. We have tested these models using both Enterococcus faecium and Enterococcus faecalis, the two species responsible for nearly all VRE infections. These results demonstrate the practical utility and robustness of our probiotic and provide strong proof of concept evidence for probiotic delivery of AMPs. If successful, AMP-based probiotics would open an entirely new arsenal of treatments against intestinal pathogens.

1. Geldart, K., Forkus, B., McChesney, E., McCue, M. & Kaznessis, Y. pMPES: A Modular Peptide Expression System for the Delivery of Antimicrobial Peptides to the Site of Gastrointestinal Infections Using Probiotics. Pharmaceuticals 9, 60 (2016).