Molecular Probes for the Determination of Subcellular Compound Exposure Profiles in Gram-Negative Bacteria,ACS Infectious Diseases

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Molecular Probes for the Determination of Subcellular Compound Exposure Profiles in Gram-Negative Bacteria
ACS Infectious Diseases ( IF 4.0 ) Pub Date : 2018-05-30 00:00:00 , DOI: 10.1021/acsinfecdis.8b00093
Benjamin Spangler ₁ , Dustin Dovala ₁ , William S. Sawyer ₁ , Katherine V. Thompson ₁ , David A. Six ₁ , Folkert Reck ₁ , Brian Y. Feng ₁

Affiliation

The Gram-negative cell envelope presents a formidable barrier to xenobiotics, and achieving sufficient compound exposure inside the cell is a key challenge for the discovery of new antibiotics. To provide insight on the molecular determinants governing compound exposure in Gram-negative bacteria, we developed a methodology leveraging a cyclooctyne-based bioorthogonal probe to assess compartment-specific compound exposure. This probe can be selectively localized to the periplasmic or cytoplasmic compartments of Gram-negative bacteria. Once localized, the probe is used to test azide-containing compounds for exposure within each compartment by quantifying the formation of click-reaction products by mass spectrometry. We demonstrate this approach is an accurate and sensitive method of determining compartment-specific compound exposure profiles. We then apply this technology to study the compartment-specific exposure profiles of a small panel of azide-bearing compounds with known permeability characteristics in Gram-negative bacteria, demonstrating the utility of the system and the insight it is able to provide regarding compound exposure within intact bacteria.

中文翻译：

测定革兰氏阴性细菌中亚细胞化合物暴露特征的分子探针

革兰氏阴性细胞包膜对异源生物构成了强大的屏障，而在细胞内实现足够的化合物暴露是发现新抗生素的关键挑战。为了提供有关控制革兰氏阴性细菌中化合物暴露的分子决定因素的见解，我们开发了一种方法，该方法利用基于环辛炔的生物正交探针评估隔室特异性化合物的暴露。该探针可以选择性地定位于革兰氏阴性细菌的周质或细胞质区室。定位后，该探针可用于通过质谱定量点击反应产物的形成来测试含叠氮化物的化合物在每个隔室内的暴露情况。我们证明了这种方法是一种准确而灵敏的方法，可以确定特定于车厢的化合物的暴露曲线。

更新日期：2018-05-30

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