(500e) Pmpes: A Modular Peptide Expression Vector for the Production of Antimicrobial Peptides from Probiotic E. coli

Antimicrobial peptides (AMPs) are a diverse class of small proteins with antibacterial activity against a wide array of pathogens. AMPs have long been used in the food industry as preservatives and are currently being explored for use in the medical realm. Unfortunately, AMP delivery has been a challenge; AMPs may be toxic when administered intravenously, or they quickly degrade in the stomach and the gastrointestinal (GI) tract upon oral administration.

To overcome this challenge, we are engineering probiotic bacteria to produce and secrete AMPs inside host GI tracts. AMPs are chosen or engineered to target medically-relevant pathogens, which reside inside GI tracts, such as Salmonella and Vancomycin-resistant enterococcus (VRE).^1â??3 These probiotics are safe to administer orally, are bile-resistant and will reach the site of infection, where they will eliminate the targeted pathogen.

A goal of ours is to develop a library of antibiotic probiotics that can target a wide variety of GI tract pathogens. The development of a bacterial vector that can powerfully express and secrete a wide array of AMPs would be an important breakthrough in order to efficiently engineer this library of probiotics for AMP delivery. However, the issue of secretion poses a major challenge in implementing and testing new peptides from a single bacterial production system. In most naturally-occurring bacterial AMP production systems, each AMP has its own dedicated secretion machinery. For our application, it becomes challenging to construct unique secretion machineries for each new peptide. These systems often comprise several large, poorly characterized proteins. This translates to additional, extensive molecular biology research before they can be employed. Additionally, simultaneous expression of multiple large secretion systems could quickly become a burden on the producer organism.

We have created a powerful AMP expression vector, pMPES, which employs a single previously-characterized E. coli AMP secretion system to produce and secrete a wide-array of AMPs from probiotic E. coli Nissle 1917. To achieve this, we have remodeled a Microcin V production plasmid, pHK22, so as to create a vector that contains the entire MicV secretion machinery. This plasmid also contains ProTeOn+, a powerful synthetic promoter system we have built and an AMP molecular cloning site.⁴ We refer to this vector as pMPES (Modular Peptide Expression System).

In this presentation, we will describe the development of this system and will demonstrate that using this vector, we are able to produce and secrete several AMPs of diverse structure, function, and origin from Nissle 1917. Thus far, we have tested secretion of E. coli-derived Microcin V, Microcin N, and Microcin L and of Enterococcus-derived Enterocin A, Enterocin P, Hiracin JM79, and Enterocin B. With the exception of Enterocin B, sufficient quantities of all peptides were secreted so as to allow detectable inhibition of the target pathogen. This new expression system enables the rapid development and testing of single or multiple peptide constructs against any given pathogen of interest.

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2. Borrero, J., Chen, Y., Dunny, G. M. & Kaznessis, Y. N. Modified Lactic Acid Bacteria Detect and Inhibit Multiresistant Enterococci. ACS Synth. Biol. (2014). doi:10.1021/sb500090b

3. Volzing, K., Borrero, J., Sadowsky, M. J. & Kaznessis, Y. N. Antimicrobial peptides targeting Gram-negative pathogens, produced and delivered by lactic acid bacteria. ACS Synth. Biol. 2, 643â??50 (2013).

4. Volzing, K., Biliouris, K. & Kaznessis, Y. N. proTeOn and proTeOff, New Protein Devices That Inducibly Activate Bacterial Gene Expression. ACS Chem. Biol. 6, 1107â??1116 (2011).