Microbial Delivery of Antimicrobial Peptides for Protection Against Vancomycin-Resistant Enterococcus

Authors: 
Geldart, K., University of Minnesota
Forkus, B., University of Minnesota
Kaznessis, Y. N., University of Minnesota
Antimicrobial peptides (AMPs) are naturally produced by our microflora as a first line of defense against invading species. While numerous probiotic bacterial species are known to produce AMPs targeting different pathogens of interest, native peptide expression is often low or induced under uncontrollable conditions making these probiotics therapeutically unreliable. We harness this natural defense mechanism for the treatment of antibiotic resistant intestinal infections by engineering probiotic bacteria that can be orally administered and will reliably express and secrete AMPs targeting the pathogen of interest in the intestines. We are developing libraries of probiotic species, promoters, secretion systems, and antimicrobial peptides to enable rapid development of new probiotics targeting a wide array of pathogens.1,2,3,4 Herein, we engineer probiotic E. coli Nissle 1917 (EcN) to deliver AMPs targeting vancomycin-resistant Enterococcus (VRE), an opportunistic pathogen that causes thousands of hospital-acquired infections and deaths each year.

We have developed an AMP expression vector for Nissle that uses a strong synthetic promoter to drive the expression of three previously characterized anti-enterococcal AMPs, Enterocin A, Enterocin B, and Hiracin JM79.5 This vector contains the secretion machinery from Microcin V, an E. coli-derived AMP, to enable secretion of the anti-enterococcal peptides.4 We first demonstrate the ability of the probiotic to reduce VR E. faecium and E. faecalis counts in laboratory cultures using both solid and liquid culture activity assays. EcN expressing anti-enterococcal AMPs eliminates over 99% of VR E. faeciumafter one hour of co-culture.

We then test the efficacy of our probiotics in reducing intestinal pathogen counts in mice colonized with VRE via oral administration. Mice administered EcN producing AMPs on average exhibited a 10x reduction in E. faecium fecal counts compared to mice administered no probiotic. This reduction of VRE in the treated versus untreated groups was significant with p = 0.011 based on a Student’s t-test comparing total E. faecium fecal levels over time. This work provides proof-of concept evidence supporting the potential use of AMP-producing probiotic bacteria for the reduction of VRE in the intestines.

Importantly, we demonstrate that this EcN probiotic platform can be readily modified to express alternative peptides targeting a variety of pathogens including Salmonella and diarrheagenic E. coli. Additionally, unlike broad-spectrum traditional antibiotics, the AMPs expressed by these microbes offer species-specific activity. This specificity reduces off-target pressure for resistance development and supports competitive exclusion of the pathogen by the surrounding microbiota.

1. Volzing, ACS Synth Biol. 2013; 2(11):643-50

2. Geldart, Appl Environ Microbiol. 2015; 81(11): 3889–3897.

3. Forkus, Sci Rep. 2017; 7: 40695.

4. Geldart, Pharmaceuticals (Basel). 2016; 9(4): 60.

5. Geldart, Antimicrob Agents Chemother. 2017; AAC.02033-16