Antimicrobial peptides (AMPs) are promising therapeutics against antibiotic-resistant pathogens, but they are difficult to administer at the site of infection due to poor stability. To address this issue, we developed components of a novel, cell-based, drug delivery platform for AMPs in which a therapeutic microbe is engineered to specifically detect a pathogen of interest and effectively release AMPs at high, local concentrations in response.
We focused the majority of our project on developing a system to produce normally host-toxic AMPs in high quantities in our model therapeutic microbe Escherichia coli (E. coli.) using a protein cage system called an encapsulin (Enc) nanocompartment system. We successfully produced an AMP called HBCM2 (HB) that is effective in killing our model pathogen, Pseudomonas aeruginosa (P. aeruginosa), in E. coli by fusing it to an engineered, non-cage forming Enc protein, which prevented its toxicity to the E. coli. This engineered Enc protein could then be digested by a specific protease (TEV protease) for release of HB when desired. In addition to the HB production system, we engineered a preliminary E. coli capable of sensing P. aeruginosa via its specific quorum-sensing molecule, which then produces an HB peptide, and releases it via self-lysis in response. Our work creates a foundation to fully characterize the system in future studies. Our results demonstrate the power of synthetic biology to confer novel functions on cells for biomanufacturing and therapeutic applications.
This study supported and leveraged the Lawrence Livermore National Laboratory's core competency in bioscience and bioengineering and supports the Laboratory's chemical and biological security mission area. To advance our research, we constructed and tested novel synthetic biology components that can be used for the future development of therapeutic microbes as drug delivery systems, a novel countermeasure strategy against emerging antibiotic-resistant pathogen threats. The protease-sensitive encapsulin system we developed is broadly applicable to the biomanufacturing of AMPs, which are of direct relevance to medicinal, biodefense, and forensic mission-related studies at Livermore. The synthetic biology outcomes of this project helped establish a new synthetic biology research area at Livermore and advanced the Laboratory's portfolio of novel countermeasure tools against biosecurity threats.
Lee, T-H., et al. 2018. "Improved Expression of Host-Toxic Antimicrobial Peptides in Escherichia Coli Using An Encapsulin Nanocompartment System." 2018 Synthetic Biology: Engineering, Evolution & Design (SEED) Conference, Scottsdale, AZ, June 2018. LLNL-POST-752026.
——— . 2019. "Expression and Purification of Highly Active Antimicrobial Peptide HBCM2 from Escherichia Coli Using an Encapsulin Nanocompartment System." 2019 Synthetic Biology: Engineering, Evolution & Design (SEED) Conference, New York, NY, June 2019. LLNL-POST-772916.
——— . 2019. "Engineered Bacterial Compartmentalization Systems for Enhanced Toxic Protein Production." Invited talk, Boston College Department of Chemistry Seminar Series, Chestnut Hill, MA, April 2019. LLNL-PRES-771279.
——— . 2019. "Production of Antimicrobial Peptides Using Protein-Cage Carrier Proteins." Invited talk, PEGS 2019: Protein Expression System Engineering, Boston, MA, April 2019. LLNL-PRES-770537.
——— . 2019. "Understanding and Designing Microbial Systems Using Synthetic Biology." PLS External Review Committee Meeting, Livermore, CA, June 2019. LLNL-PRES-777038.
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