Phage-protease-peptide: a novel trifecta enabling multiplex detection of viable bacterial pathogens

Abstract

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 tobacco etch virus (TEV) protease; (ii) expression of TEV protease by Escherichia 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 h after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (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.

Fig. 1

Phage-Protease-Peptide Multiplex Detection Scheme. (a) Phage specific to a bacterial target, is bioengineered to carry a gene for a specific protease. Protease acts on a specific peptide resulting in a unique fluorescent signal or peptide product. (b) Phage and corresponding peptides are added to a single sample. Signal is produced only if corresponding bacterial target is present in sample to produce the protease.

Fig. 2

Diagram of DNA Constructs. (a) Our designed TEV protease gene cassette. (b) Genome of T7 Select® 415-1 indicating 10B capsid protein and insertion site. (c) Genome of T7tev with the insertion of our gene cassette. (d) Genome of T7control with insertion of the S•Tag™ control.

Fig. 3

Confirming TEV protease product. Ratio of fluorescent signal in lysate samples to that in an LB negative control over time. Plaque 15 and 41 are phage confirmed by PCR to carry the TEV protease gene cassette, Plaque C1 carries the S•Tag™ insert.

Fig. 4

TEV protease activity from varying starting cell concentrations. Ratio of fluorescent signal in T7_tev lysates (with starting cell concentrations of 10⁶, 10⁷, 10⁸, and 10⁹ CFU/mL) to that of a control with no cells, over time.

Fig. 5

Fluorescent detection of E. coli via T7-induced TEV protease activity. Fluorescent signal intensity of E.coli positive samples and a negative (Control) over time.

Fig. 6

MALDI-TOF detection of E. coli via T7-induced TEV protease activity. Spectra of representative E.coli positive sample (a) and negative control (b). The peaks corresponding to TEV-L, TEV-S, and REF-L are labeled.