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A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity.

ABSTRACT: We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy.

SUBMITTER: O'Brien-Simpson NM

PROVIDER: S-EPMC4795541 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity.

O'Brien-Simpson Neil M NM Pantarat Namfon N Attard Troy J TJ Walsh Katrina A KA Reynolds Eric C EC

PloS one 20160317 3

We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pat ...[more]

PMID: 26986223

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Project description:Background: Cell-membrane expressing enzymes such as ADAM (a disintegrin and metalloproteinase) superfamily members are thought to be key catalysts of vital cellular functions. To directly measure these enzymes and determine their association with particular cells and functions, individual-cell membrane-bound enzyme activity assays are required, but unavailable. Methods: We developed two such assays, using a fluorescence resonance energy transfer (FRET) peptide substrate (FPS) and flow cytometry. One assay measured live-cell natural processing of FPS and binding of its fluorescent product onto individual-cell membrane-bound enzymes. The other assay measured processing of specifically-bound and glutaraldehyde-crosslinked FPS, and consequent generation of its coupled fluorescent product onto individual-cell membrane-bound enzymes. Results: Confocal-microscopy imaging indicated that proteolytic processing of FPS selectively occurred on and labeled cell membrane of individual cells. The new assays measured specific increases of cell-associated FPS fluorescent product in substrate-concentration-, temperature- and time-dependent manners. A large proportion of processed FPS fluorescent products remained cell-associated after cell washing, indicating their binding to cell-membrane expressing enzymes. The assays measured higher levels of cell-associated FPS fluorescent product on wild-type than ADAM10-knockout mouse fibroblasts and on human monocytes than lymphocytes, which correlated with ADAM10 presence and expression levels on cell membrane, respectively. Furthermore, the enzyme activity assays could be combined with fluorescent anti-ADAM10 antibody staining to co-label and more directly associate enzyme activity and ADAM10 protein levels on cell membrane of individual cells. Conclusions: We report on two novel assays for measuring cell-membrane anchored enzyme activity on individual cells, and their potential use to directly study specific biology of cell-surface-expressing proteases.

Dataset Information

A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity.

Publications

A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity.

Similar Datasets

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