title: Genetically programmable pathogen sense and destroy.
abstract: Pseudomonas aeruginosa (P. aeruginosa) is a major cause of urinary tract and nosocomial infections. Here, we propose and demonstrate proof-of-principle for a potential cell therapy approach against P. aeruginosa. Using principles of synthetic biology, we genetically modified E. coli to specifically detect wild type P. aeruginosa (PAO1) via its quorum sensing (QS) molecule, 3OC 12 HSL. Engineered E. coli sentinels respond to the presence of 3OC 12 HSL by secreting CoPy, a novel pathogen-specific engineered chimeric bacteriocin, into the extracellular medium using the flagellar secretion tag FlgM. Extracellular FlgM-CoPy is designed to kill PAO1 specifically. CoPy was constructed by replacing the receptor and translocase domain of Colicin E3 with that of Pyocin S3. We show that CoPy toxicity is PAO1 specific, not affecting sentinel E. coli or the other bacterial strains tested. In order to define the system's basic requirements and PAO1-killing capabilities, we further determined the growth rates of PAO1 under different conditions and concentrations of purified and secreted FlgM-CoPy. The integrated system was tested by co-culturing PAO1 cells, on semisolid agar plates, together with engineered sentinel E. coli, capable of secreting FlgM-CoPy when induced by 3OC 12 HSL. Optical microscopy results show that the engineered E. coli sentinels successfully inhibit PAO1 growth.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Gupta
authors.forename: Eran E
authors.initials: EE
authors.lastname: Bram
authors.forename: Ron
authors.initials: R
authors.lastname: Weiss
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001430:Bacteriocins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D053038:Quorum Sensing / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001430:Bacteriocins; D011993:Recombinant Fusion Proteins; C071266:FlgM protein, Bacteria