A flow cytometric assay to monitor antimicrobial activity of defensins and cationic tissue extracts
A flow cytometric assay to monitor antimicrobial activity of defensins and cationic tissue extracts
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DOI：
10.1016/j.mimet.2005.08.004
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发表时间：
2006-05-01
影响因子：
2.2
通讯作者：
Stange, EF
中科院分区：
文献类型：
--
作者：
Nuding, S;Fellermann, K;Stange, EF
为了确定活检提取物中防御素和其他抗菌肽的抗菌活性，我们使用膜电位敏感染料双-（1,3-二丁基巴比妥酸）三甲氧醇[DiBAC(4)(3)]评估了流式细胞术方法。通过测量直接靶细胞、细胞质膜和膜电位，本实验能够区分暴露于抗菌肽后的完整的非荧光和去极化荧光细菌。用重组人β -防御素3 （HBD-3）对12种25株细菌进行了流式细胞分析。HBD-3表现出广谱剂量依赖性活性，引起去极化的最小剂量范围为1.25 ~ 150 μ g/ml HBD-3，这取决于所测物种。氯化钠或二硫代苏糖醇的抑菌作用减弱，可通过HBD-3抗体消除。此外，从人体肠道活组织中分离的阳离子提取物显示出对大肠杆菌K12、大肠杆菌ATCC 25922和金黄色葡萄球菌ATCC 25923的强杀菌作用，在150 mM NaCl下对大肠杆菌的杀菌作用减弱，而对金黄色葡萄球菌ATCC 25923的活性在生理盐浓度下不受影响。DTT完全阻断了活组织提取液的杀菌作用。(c) 2005 Elsevier B.V.版权所有
To determine the antibacterial activity of defensins and other antimicrobial peptides in biopsy extracts, we evaluated a flow cytometric method with the membrane potential sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC(4)(3)]. This assay enables us to discriminate intact non-fluorescent and depolarized fluorescent bacteria after exposure to antimicrobial peptides by measurement at the direct target, the cytoplasmic membrane and the membrane potential. The feasibility of the flow cytometric assay was evaluated with recombinant human beta-defensin 3 (HBD-3) against 25 bacterial strains representing 12 species. HBD-3 showed a broad-spectrum dose dependent activity and the minimal dose to cause depolarization ranged from 1.25 to > 15 mu g/ml HBD-3, depending on the species tested. The antibacterial effect was diminished with sodium chloride or dithiothreitol and could be abrogated with a HBD-3 antibody. Additionally, isolated cationic extracts from human intestinal biopsies showed a strong bactericidal effect against Escherichia coli K12, E. coli ATCC 25922 and Slaphylococcus aureus ATCC 25923, which was diminished towards E. coli at 150 mM NaCl, whereas the activity towards S. aureus ATCC 25923 remained unaffected at physiological salt concentrations. DTT blocked the bactericidal effect of biopsy extracts completely. (c) 2005 Elsevier B.V. All rights reserved.