Dissecting the Multiple Roles of PqsE in Pseudomonas aeruginosa Virulence by Discovery of Small Tool Compounds.
Dissecting the Multiple Roles of PqsE in Pseudomonas aeruginosa Virulence by Discovery of Small Tool Compounds.
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DOI：
10.1021/acschembio.6b00156
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发表时间：
2016-04
影响因子：
4
通讯作者：
Michael Zender;F. Witzgall;S. Drees;Elisabeth Weidel;C. Maurer;S. Fetzner;W. Blankenfeldt;M. Empting;R. Hartmann
中科院分区：
文献类型：
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作者：
Michael Zender;F. Witzgall;S. Drees;Elisabeth Weidel;C. Maurer;S. Fetzner;W. Blankenfeldt;M. Empting;R. Hartmann
铜绿假单胞菌利用群体感应（QS）作为细胞间通讯系统来协调毒力决定因子的表达。重要的假单胞菌喹诺酮信号（PQS）的生物合成需要pqsABCDE操纵子。在这里，PqsE作为途径特异性硫酯酶，但它也有助于通过未知的机制调节细菌的毒力。在这篇手稿中，我们报告了PqsE抑制剂作为工具化合物的发现，以进一步了解其不同的功能。差示扫描荧光法（DSF）用于筛选片段库，等温滴定量热法（ITC）用作二次过滤。如X射线晶体学所证明的，命中分子结合到活性中心，在基于细胞的测定和体外测定中抑制PqsE的硫酯酶活性。值得注意的是，配体不影响PqsE调节的毒力因子绿脓菌素的水平。这些发现表明PqsE的调节功能与其硫酯酶活性无关，必须在活性中心外编码。这项研究突出了基于片段的筛选工具化合物的发现的潜力。这种方法为复杂的生物系统提供了新的见解，这是敲除研究无法获得的。
Pseudomonas aeruginosa uses quorum sensing (QS) as a cell-to-cell communication system to orchestrate the expression of virulence determinants. The biosynthesis of the important Pseudomonas quinolone signal (PQS) requires the pqsABCDE operon. Here, PqsE acts as a pathway-specific thioesterase, but it also contributes to the regulation of bacterial virulence via an unknown mechanism. In this manuscript, we report the discovery of PqsE inhibitors as tool compounds to gain further insights into its different functions. Differential scanning fluorimetry (DSF) was used to screen a fragment library, and isothermal titration calorimetry (ITC) was employed as a secondary filter. As proven by X-ray crystallography, hit molecules bound to the active center inhibiting PqsE's thioesterase activity in cell-based and in vitro assays. Notably, the ligands did not affect the levels of the PqsE-regulated virulence factor pyocyanin. These findings indicate that the regulatory function of PqsE is not linked to its thioesterase activity and must be encoded outside of the active center. This study highlights the potential of fragment-based screening for the discovery of tool compounds. This approach provided novel insight into complex biological systems, which could not be obtained by knockout studies.