A flow cytometric assay to monitor antimicrobial activity of defensins and cationic tissue extracts - PubMed (original) (raw)

A flow cytometric assay to monitor antimicrobial activity of defensins and cationic tissue extracts

Sabine Nuding et al. J Microbiol Methods. 2006 May.

Abstract

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 [DiBAC4(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 microg/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 Staphylococcus 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.

PubMed Disclaimer

Cited by

Photodynamic Antifungal Activity of Hypocrellin A Against Candida albicans.
Yang Y, Wang C, Zhuge Y, Zhang J, Xu K, Zhang Q, Zhang H, Chen H, Chu M, Jia C. Yang Y, et al. Front Microbiol. 2019 Aug 6;10:1810. doi: 10.3389/fmicb.2019.01810. eCollection 2019. Front Microbiol. 2019. PMID: 31447816 Free PMC article.
Intestinal manipulation affects mucosal antimicrobial defense in a mouse model of postoperative ileus.
Stein K, Hieggelke L, Schneiker B, Lysson M, Stoffels B, Nuding S, Wehkamp J, Kikhney J, Moter A, Kalff JC, Wehner S. Stein K, et al. PLoS One. 2018 Apr 13;13(4):e0195516. doi: 10.1371/journal.pone.0195516. eCollection 2018. PLoS One. 2018. PMID: 29652914 Free PMC article.
Synergetic Inactivation Mechanism of Protocatechuic Acid and High Hydrostatic Pressure against Escherichia coli O157:H7.
Hao J, Lei Y, Gan Z, Zhao W, Shi J, Jia C, Sun A. Hao J, et al. Foods. 2021 Dec 8;10(12):3053. doi: 10.3390/foods10123053. Foods. 2021. PMID: 34945604 Free PMC article.
Reduction of disulphide bonds unmasks potent antimicrobial activity of human β-defensin 1.
Schroeder BO, Wu Z, Nuding S, Groscurth S, Marcinowski M, Beisner J, Buchner J, Schaller M, Stange EF, Wehkamp J. Schroeder BO, et al. Nature. 2011 Jan 20;469(7330):419-23. doi: 10.1038/nature09674. Nature. 2011. PMID: 21248850
Reduced mucosal antimicrobial activity in Crohn's disease of the colon.
Nuding S, Fellermann K, Wehkamp J, Stange EF. Nuding S, et al. Gut. 2007 Sep;56(9):1240-7. doi: 10.1136/gut.2006.118646. Epub 2007 Apr 24. Gut. 2007. PMID: 17456510 Free PMC article.

A flow cytometric assay to monitor antimicrobial activity of defensins and cationic tissue extracts - PubMed (original) (raw)