| Sortase A(SrtA),a membrane-bound cysteine transpeptidase,plays a critical role in different stage of pathogenic process through catalyzing the covalent attachment of surface proteins virulence factors with a sorting motif(LPXTG,where X is any amino acid)to the cell wall of Gram-positive bacteria.The Staphylococcus aureus mutant strains lacking SrtA gene significantly result in the reduced toxicity and infection capability.Thus,SrtA can be regarded as a promising target for the development of anti-virulence drugs,and the accurate detection of SrtA activity is critical for the screening of sortase inhibitors and the development of novel antibacterial agents.The tetracysteine labeling system,which consists of a tetracysteine(TC)tag and a set of biarsenical dyes,is an effective approach for protein labeling.The tetracysteine tag harbors a short peptide sequence of "CCXXCC ",wherein X refers to any amino acid except for cysteine.The binding between the tetracysteine tag and biarsenical dyes is specific with each arsenic atom coordinating to a pair of adjacent cysteines.In comparison with traditional fluorescent proteins,the smaller size of TC motif faces a smaller chance to affect the biological activity and spatial folding of the target protein.In this research,we develop a simple method for sensitive detection of sortase A activity by using transpeptidation-directed intramolecular bipartite tetracysteine display.The sensing system consists of a profluorescent biarsenical dye and two substrate peptides that derive from split avian pancreatic polypeptide(aPP),among which one peptide harbors a C-terminal sorting motif and the other has an N-terminal Gly-Gly,with both containing a di-cysteine motif at opposite end.The presence of SrtA may catalyze two peptides to form a chimeric peptide that can self-assemble to form a hairpin-like structure via split aPP-mediated intramolecular complementation.This unique structure will facilitate the binding of biarsenical dye with two Cys-Cys pairs to generate a strong fluorescence signal,in contrast to almost no fluorescence for the peptide mixture.This assay has significant advantage of simple operation without the involvement of any labeling substrate peptides and washing step,and can be performed using a conventional fluorescence spectrometer.This method can measure SrtA down to10.7 ng/mL,which is 24.7-fold more sensitive than the conventional methods.Moreover,this method can be used to characterize SrtA-targeted inhibitors,providing a new approach for anti-virulence drug discovery. |
