Phage Protease Peptide: A novel trifecta enabling multiplex detection of viable bacterial pathogens

摘要

Bacteriophages represent rapid, readily targeted, and easily produced molecular probes for the detection of bacterial pathogens. Molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Despite their host-specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. We propose a proof-of-principal phage-based scheme to enable multiplex detection. Our scheme involves bioengineering bacteriophage to carry a gene for a specific protease, which is expressed during infection of the target cell. Upon lysis, the protease is released to cleave a reporter peptide, and the signal detected. Here we demonstrate the successful: i) modification of T7 bacteriophage to carry TEV protease; ii) expression of TEV protease by E. coli following infection by our modified T7, an average of 2000 units of protease per phage are produced during infection; and iii) proof-of-principle detection of E. coli in 3 hours after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for MALDI-TOF MS analysis. This proof-of-principle can be translated to other phage-protease-peptide combinations to enable multiplex bacterial detection, and readily adopted on multiple platforms, like MALDI-TOF MS or fluorescent readers, commonly found in labs.