Department of Chemistry, Shanghai Stomatological Hospital, State Key Laboratory of Molecular Engineering of Polymers and Institute of Biomedical Sciences, Fudan University, Shanghai, 200433, PR China.
Department of Neurosurgery, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, 200062, PR China.
Biosens Bioelectron. 2021 Apr 15;178:113003. doi: 10.1016/j.bios.2021.113003. Epub 2021 Jan 15.
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 (IC) 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.
表面蛋白的转肽作用由转肽酶 sortase 催化，在革兰氏阳性病原体的感染过程中起着关键作用。监测 sortase 的活性并筛选其抑制剂对于深入了解感染机制和药物开发具有重要意义。本文开发了一种基于转肽介导的金纳米粒子（GNPs）组装和计数的数字单颗粒成像方法来定量 SrtA （sortase A）活性。该测定方法利用两个肽支架，其中一个具有 SrtA 识别序列 LPXTG 基序，另一个在一端带有寡甘氨酸亲核基团，在另一端带有生物素基团。SrtA 的存在使得两个肽的连接成为可能，并允许链霉亲和素功能化的 GNPs 的固定化。因此，可以通过暗场显微镜对表面组装的 GNPs 进行成像和计数来定量 SrtA 活性。该单颗粒方法对 SrtA 活性具有很高的灵敏度，检测限低至 7.9 pM，线性动态范围从 0.05 到 50 nM。除了检测革兰氏阳性病原体裂解物等复杂生物样品中的 SrtA 外，该方法还成功地用于估计 SrtA 抑制剂（姜黄素、盐酸小檗碱和槲皮素）的半数最大抑制浓度（IC）值。本方法结合单颗粒计数和暗场成像，为 SrtA 活性及其抑制剂筛选提供了一种简单新颖的分析工具。
