Su Tiantian, He Jing, Li Ningna, Liu Shiheng, Xu Sujuan, Gu Lichuan
State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China.
Front Microbiol. 2020 May 13;11:760. doi: 10.3389/fmicb.2020.00760. eCollection 2020.
A glycosyl hydrolase produced by , PslG, has become a promising candidate for biofilm treatment because of its ability to inhibit and disperse biofilms by disrupting exopolysaccharide matrix at nanomolar concentrations. However, as a protein, PslG used for treatment may be degraded by the ubiquitous proteases (of which trypsin-like serine proteases are a major group) secreted by human cells. This would lead to an insufficient effective concentration of PslG. Here, based on the result of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and structural analysis, we generate a PslG mutant (K286A/K433S) with greatly enhanced trypsin resistance. This measure raises IC (the concentration of trypsin that can degrade 50% of protein in 30 min at 37°C) from 0.028 mg mL of the wild-type PslG to 0.283 mg mL of PslG . In addition, biofilm inhibition assay shows that PslG is much more efficient than wild-type PslG in the presence of trypsin. This indicates that PslG is a better biofilm inhibitor than wild-type PslG in clinical use where trypsin-like proteases widely exist.
由 产生的一种糖基水解酶PslG，因其能够在纳摩尔浓度下通过破坏胞外多糖基质来抑制和分散生物膜，已成为生物膜治疗的一个有前景的候选物。然而，作为一种蛋白质，用于治疗的PslG可能会被人体细胞分泌的普遍存在的蛋白酶（其中胰蛋白酶样丝氨酸蛋白酶是主要类别）降解。这将导致PslG的有效浓度不足。在此，基于液相色谱 - 串联质谱（LC-MS/MS）结果和结构分析，我们生成了一种胰蛋白酶抗性大大增强的PslG突变体（K286A/K433S）。这一措施将IC（在37°C下30分钟内可降解50%蛋白质的胰蛋白酶浓度）从野生型PslG的0.028 mg/mL提高到PslG 的0.283 mg/mL。此外，生物膜抑制试验表明，在存在胰蛋白酶的情况下，PslG 比野生型PslG效率高得多。这表明在胰蛋白酶样蛋白酶广泛存在的临床应用中，PslG 是比野生型PslG更好的生物膜抑制剂。
