Transpeptidation-mediated Single-Particle Imaging Assay for Sensitive and Specific Detection of Sortase with Dark-Field Optical Microscopy.

Transpeptidation of surface proteins catalyzed by the transpeptidase sortase plays a critical role in the infection process of Gram-positive pathogen. Monitoring sortase activity and screening its inhibitors are of great significance to fundamental understanding of the infection mechanism and pharmaceutical development. Herein, we developed a digital single-particle imaging method to quantify sortase A (SrtA) activity based on transpeptidation-mediated assembly and enumeration of gold nanoparticles (GNPs). The assay utilizes two peptide stands, in which one has the SrtA recognition sequence LPXTG motif while the other carries an oligoglycine nucleophile at the one end and a biotin group at the other. The presence of SrtA enables the ligation of two peptides and allows for the immobilization of streptavidin-functionalized GNPs. Thus, SrtA activity can be quantified by imaging and enumeration of the surface-assembled GNPs at the single-particle level via dark-field microscopy. The single-particle method was highly sensitive to SrtA activity with a low detection limit of 7.9 pM and a wide linear dynamic range from 0.05 to 50 nM. Besides detection of SrtA in complex biological samples such as Gram-positive pathogen lysates, the proposed method was also successfully applied to estimate the half-maximal inhibitory concentration (IC50) values of SrtA inhibitors (curcumin, berberine hydrochloride and quercetin). The present method, combining single-GNP counting and dark-field imaging, provides a facile and novel analytical tool for SrtA activity and its inhibitor screening.