Correlating uptake and activity of proline-rich antimicrobial peptides in Escherichia coli
Correlating uptake and activity of proline-rich antimicrobial peptides in Escherichia coli
复制标题
DOI：
10.1007/s00216-017-0496-2
复制
发表时间：
2017-09-01
影响因子：
4.3
通讯作者：
Knappe, Daniel
中科院分区：
文献类型：
--
作者：
Holfeld, Luzia;Hoffmann, Ralf;Knappe, Daniel
抗生素耐药性导致的死亡人数增加需要新型抗生素先导化合物。在不同的有希望的候选类别中，富含脯氨酸的抗微生物肽（PrAMP）由于其细胞内机制而非常有利，即，在依赖于细菌转运蛋白如SbmA和MdtM的主动摄取后，与70S核糖体和DnaK结合。肽内化作为其复杂作用模式的第一步的研究通常依赖于荧光团或放射性标记和使用显微镜、流式细胞术或放射性的定量。在此，应用基于液相色谱的测定法，使用UV吸光度和质谱法定量未标记的内化全长肽及其蛋白水解降解产物（代谢物）。缺乏转运蛋白的敲除突变体显示PrAMP摄取减少，解释了它们对PrAMP的敏感性降低。有趣的是，由细菌蛋白酶产生的主要代谢产物仍然结合到70S核糖体上，这提供了细胞溶质蛋白酶降解作为一种可能的耐药机制不是非常有效的证据。
Increasing death tolls accounted for by antimicrobial drug resistance demand novel antibiotic lead compounds. Among different promising candidate classes, proline-rich antimicrobial peptides (PrAMPs) are very favorable due to their intracellular mechanism, i.e., binding to the 70S ribosome and DnaK, after active uptake relying on bacterial transporters like SbmA and MdtM. Studies on peptide internalization as the first step of their complex mode of action rely typically on fluorophore or radioactive labeling and quantification using microscopy, flow cytometry, or radioactivity. Here, a liquid chromatography based assay was applied to quantify the unlabeled internalized full-length peptides and their proteolytic degradation products (metabolites) using UV absorbance and mass spectrometry. Knockout mutants lacking transporter proteins showed reduced PrAMP uptakes, explaining their reduced susceptibility against PrAMPs. Interestingly, major metabolites produced by bacterial proteases still bound to the 70S ribosome provide evidence that degradation by cytosolic proteases as a possible resistance mechanism is not very efficient.