Design and delivery of novel antimicrobial peptides (AMPs) targeted towards specific microbial pathogens.
Targeted therapies selectively eliminate a pathogen without disrupting the native microbiota. Traditional antibiotics, in contrast, destroy the native microbiota along with pathogens, causing adverse health outcomes for the patient. In my research, antimicrobial peptides (AMPs) were synthesized by fusing a guide-peptide that makes them selective towards a target pathogen. Staphylococcus aureus was chosen as a preliminary pathogen and a previously published guiding peptide (A12C) was selected. A12C was fused to AMPs, eurocin and plectasin, and the guided-AMPs (gAMPs) were expressed in E. coli. The gAMPs showed strong selective inhibition of S. aureus in vitro but were significantly less toxic towards several off-target bacteria. This selective bactericidal effect was observed in both planktonic culture and bacterial film formations. To optimize the in vivo delivery of gAMPs through oral route, I used engineered Lactococcus lactis, a probiotic bacterium and native resident of the human stomach flora. Helicobacter pylori, a main causal factor for peptic ulcers and gastric cancer, was the target pathogen. I targeted the VacA protein, an important virulence factor of H. pylori, with a guide peptide from a portion of Multimerin-1 (MM1), a human receptor for VacA. Three different AMPs, each fused to the MM1 guide, were tested. In vitro, co-culture of the engineered probiotic expressing gAMPs strongly inhibited H. pylori while being significantly less toxic to off-target bacteria. In vivo tests in mice were completed by introducing the H. pylori and engineered probiotic by oral gavage. Probiotics delivering gAMPs as a therapy reduced the H. pylori stomach titer by 1860-fold compared to untreated infected mice. As a prophylactic, gAMP probiotics effectively inhibited H. pylori colonization of the stomach. Microbiome analysis showed that the recovery or preservation of taxonomic diversity of the stomach microbiota was much greater with the use of gAMP probiotics than with AMP probiotics or antibiotics. My research shows that guided AMPs can be a novel and useful approach for combating pathogens without endangering the natural microbial flora and that bioengineered probiotics can be used to secrete gAMPs at the site of infection. Given the wealth of AMPs and potential guide peptides, both natural and synthetic, this approach can be adapted to develop a diverse array of chimeric guided AMPs and can be cloned into probiotics to create a safe and effective alternative to conventional chemical antibiotics.