Abstract

Antibiotic-resistant microbial infections are a critical healthcare problem in the U.S. and the world. Improved antimicrobial strategies ? and an efficient mechanism for their ongoing generation to keep pace with microbial evolution ? are sorely needed. Vancomycin-resistant Enterococcus faecium is particularly problematic and is in the CDC's ?Serious Threat? category. Antimicrobial polypeptides (AMPs) offer a compelling class of therapeutics to provide potency, specificity, and hindrance to resistance. Engineered probiotic lactic acid bacteria can provide local delivery of AMPs to overcome barriers to traditional therapeutic delivery. The objective of the proposed studies is to engineer AMPs enterocin A and endolysin ORF9, via an innovative directed evolution platform, for enhanced potency and specificity to combat E. faecium infections. The AMPs will be engineered and evaluated via secretion from two probiotics: Lactococcus lactis and Lactobacillus johnsonii. The objective will be achieved via three aims. (1) Develop a platform for high throughput (~107/day), quantitative selection of potent AMPs as secreted from probiotics. This will be achieved via microdroplet co- encapsulation of probiotic and pathogen along with fluorescence-activated cell sorting. (2) Elucidate efficient evolutionary pathways for AMPs ORF9 lysin and entA to enhance potency and specificity, from a probiotic host, against E. faecium. Innovative combinatorial library designs, including some informed by bioinformatics, will be used and compared. (3) Evaluate engineered probiotic/AMP efficacy against E. faecium in a murine model while elucidating the impacts of potency, host, secretion efficiency, dose, and AMP diversity on E. faecium inhibition and resistance reduction.

Public Health Relevance

We aim to engineer antimicrobial peptides, namely enterocin A and an endolysin, to combat Enterococcus faecium infections. Evolution of enhanced potency and specificity will be achieved through improved selection technologies and innovative combinatorial library designs.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM121777-02
Application #
9406484
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Fabian, Miles
Project Start
2017-01-01
Project End
2020-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost

  Institution

Name
University of Minnesota Twin Cities
Department
Engineering (All Types)
Type
Biomed Engr/Col Engr/Engr Sta
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455