A propeptide-based biosensor for the selective detection of Vibrio cholerae using an environment-sensitive fluorophore

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Proteases are attractive targets for infectious disease diagnostics. Peptide-based sensors that are cleaved by pathogen proteases can provide a rapid readout of infection. However, identifying peptide substrates specific to a targeted pathogen is a significant challenge. Here, we demonstrate that a structured propeptide domain from a bacterial protease can be repurposed as a protease-activated biosensor of the cholera pathogen Vibrio cholerae. We found that the peptidase inhibitor I9 domain of the secreted V.cholerae protease IvaP is rapidly degraded by V.cholerae, but not by other intestinal bacteria. By conjugating the I9 domain to an environment-sensitive fluorophore, we developed a fluorescent probe that enables the species-specific detection of V.cholerae in mixed bacterial cultures without nonspecific cleavage by other bacteria or intestinal cells. Our findings demonstrate that the IvaP propeptide is sufficient to impart selectivity to a cleavage-based V.cholerae biosensor, suggesting I9 domains could potentially be harnessed for diagnostic applications.