Falk Shaun P, Ulijasz Andrew T, Weisblum Bernard
Department of Pharmacology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, USA.
J Biomol Screen. 2007 Dec;12(8):1102-8. doi: 10.1177/1087057107308161.
The previously described Bacillus subtilis reporter strain BAU-102 is capable of detecting cell wall synthesis inhibitors that act at all stages of the cell wall synthesis pathway. In addition, this strain is capable of detecting compounds with hydrophobic/surfactant activity and alternative mechanisms of cell wall disruption. BAU-102 sequesters preformed beta-gal in the periplasm, suggesting leakage of beta-gal as the means by which this assay detects compound activities. A model is proposed according to which beta-gal release by BAU-102 reflects activation of pathways leading to autolysis. The authors also report a simplified high-throughput assay using BAU-102 combined with the fluorogenic substrate N-methylumbelliferyl-beta-D-galactoside as a single reagent. Cell wall inhibitors release beta-gal consistently only after 60 min of incubation, whereas compounds with surfactant activity show an almost immediate release. A high-throughput screen of a 480-compound library of known bioactives yielded 8 compounds that cause beta-gal release. These results validate the BAU-102 assay as an effective tool in antimicrobial drug discovery.
先前描述的枯草芽孢杆菌报告菌株BAU-102能够检测在细胞壁合成途径所有阶段起作用的细胞壁合成抑制剂。此外，该菌株能够检测具有疏水/表面活性剂活性以及细胞壁破坏替代机制的化合物。BAU-102将预先形成的β-半乳糖苷酶隔离在周质中，表明β-半乳糖苷酶的泄漏是该检测方法检测化合物活性的方式。提出了一个模型，根据该模型，BAU-102释放β-半乳糖苷酶反映了导致自溶的途径的激活。作者还报告了一种简化的高通量检测方法，该方法使用BAU-102与荧光底物N-甲基伞形酮基-β-D-半乳糖苷作为单一试剂。细胞壁抑制剂仅在孵育60分钟后才持续释放β-半乳糖苷酶，而具有表面活性剂活性的化合物几乎立即释放。对一个包含480种已知生物活性化合物的文库进行高通量筛选，得到了8种能够导致β-半乳糖苷酶释放的化合物。这些结果验证了BAU-102检测方法作为抗菌药物发现中一种有效工具的有效性。
