Improving Localization of AMP Delivery By Engineering Pathogen-Binding Commensal Bacteria
- Conference: Translational Medicine and Bioengineering Conference
- Year: 2017
- Proceeding: 2nd Bioengineering & Translational Medicine Conference
- Group: Poster Submissions
- Time: Saturday, October 28, 2017 - 6:30pm-7:30pm
We propose to improve localization of AMP delivery by engineering the probiotic cell surface to âdisplayâ VRE-binding ligands. Motivated by the known strain specificity of endolysin binding domains, we identified potential secreted endolysins that selectively bind VRE, found on VRE-specific lytic viruses. We queried the BLAST NR database to differentiate the catalytic and binding domains of endolysins. S. cerevisiae yeast were engineered to display these endolysins â both as complete proteins and as the isolated binding domains â for directed evolution of specificity and affinity towards select VRE strains using magnetic and flow cytometric selections with biotinylated VRE. Because the parameter space is too large to test all possible mutations, the binding paratope will be identified via mutational mapping strategies. The paratope will then be mutated via a focused combinatorial library design to conserve intramolecularly critical residues while enabling functional diversity at complementarity-determining sites.
In parallel, we are engineering Lactococcus lactis for cell surface display of engineered endolysins. To do this, we are utilizing existing secretion mechanisms (Sec1) in L. lactis to secrete and attach endolysins to the cell surface. Our approach is to add a conserved cell wall anchor sequence (LPXTG) to the binder, which enables covalent conjugation to the cell wall via the sortase enzyme. After the binder is expressed on L. lactis, the efficacy of AMP delivery method will be compared to L. lactis without binder expression.