Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706.
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706.
Proc Natl Acad Sci U S A. 2018 May 15;115(20):5271-5276. doi: 10.1073/pnas.1720996115. Epub 2018 Apr 27.
Control and manipulation of bacterial populations requires an understanding of the factors that govern growth, division, and antibiotic action. Fluorescent and chemically reactive small molecule probes of cell envelope components can visualize these processes and advance our knowledge of cell envelope biosynthesis (e.g., peptidoglycan production). Still, fundamental gaps remain in our understanding of the spatial and temporal dynamics of cell envelope assembly. Previously described reporters require steps that limit their use to static imaging. Probes that can be used for real-time imaging would advance our understanding of cell envelope construction. To this end, we synthesized a fluorogenic probe that enables continuous live cell imaging in mycobacteria and related genera. This probe reports on the mycolyltransferases that assemble the mycolic acid membrane. This peptidoglycan-anchored bilayer-like assembly functions to protect these cells from antibiotics and host defenses. Our probe, quencher-trehalose-fluorophore (QTF), is an analog of the natural mycolyltransferase substrate. Mycolyltransferases process QTF by diverting their normal transesterification activity to hydrolysis, a process that unleashes fluorescence. QTF enables high contrast continuous imaging and the visualization of mycolyltransferase activity in cells. QTF revealed that mycolyltransferase activity is augmented before cell division and localized to the septa and cell poles, especially at the old pole. This observed localization suggests that mycolyltransferases are components of extracellular cell envelope assemblies, in analogy to the intracellular divisomes and polar elongation complexes. We anticipate QTF can be exploited to detect and monitor mycobacteria in physiologically relevant environments.
控制和操纵细菌种群需要了解控制生长、分裂和抗生素作用的因素。细胞包膜成分的荧光和化学反应小分子探针可以可视化这些过程，并增进我们对细胞包膜生物合成的了解（例如，肽聚糖的产生）。然而，我们对细胞包膜组装的时空动态的理解仍然存在基本差距。以前描述的报告器需要一些步骤，这些步骤限制了它们在静态成像中的使用。能够用于实时成像的探针将增进我们对细胞包膜结构的理解。为此，我们合成了一种荧光探针，使分枝杆菌和相关属的连续活细胞成像成为可能。该探针报告了组装类脂酸膜的酰基转移酶。这种肽聚糖锚定的双层样组装功能是为了保护这些细胞免受抗生素和宿主防御的侵害。我们的探针，猝灭剂-海藻糖-荧光团（QTF），是天然酰基转移酶底物的类似物。酰基转移酶通过将其正常的转酯化活性转移到水解过程中处理 QTF，这一过程释放出荧光。QTF 能够实现高对比度的连续成像和细胞中酰基转移酶活性的可视化。QTF 表明，酰基转移酶的活性在细胞分裂前增加，并定位于隔膜和细胞两极，尤其是在旧极。这种观察到的定位表明，酰基转移酶是细胞外细胞包膜组装的组成部分，类似于细胞内的分裂体和极伸长复合物。我们预计 QTF 可用于在生理相关环境中检测和监测分枝杆菌。
