Melbourne Dental School - Research Publications

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Author: O'Brien-Simpson, Neil × Author: Attard, Troy × Author: PANTARAT, NAMFON × Start date: 2010 × End date: 2019 ×

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    A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity
    O'Brien-Simpson, NM ; Pantarat, N ; Attard, TJ ; Walsh, KA ; Reynolds, EC ; Castanho, MARB (PUBLIC LIBRARY SCIENCE, 2016-03-17)
    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.
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    Maculatin 1.1 Disrupts Staphylococcus aureus Lipid Membranes via a Pore Mechanism
    Sani, M-A ; Whitwell, TC ; Gehman, JD ; Robins-Browne, RM ; Pantarat, N ; Attard, TJ ; Reynolds, EC ; O'Brien-Simpson, NM ; Separovic, F (AMER SOC MICROBIOLOGY, 2013-08)
    Maculatin 1.1 (Mac1) showed potent activity against Staphylococcus aureus with an MIC of 7 μM. The mode of action of Mac1 was investigated by combining assays with S. aureus cells and lipid vesicles mimicking their membrane composition. A change in Mac1 conformation was monitored by circular dichroism from random coil to ca. 70% α-helix structure in contact with vesicles. Electron micrographs of S. aureus incubated with Mac1 showed rough and rippled cell surfaces. An uptake of 65% of small (FD, 4 kDa [FD-4]) and 35% of large (RD, 40 kDa [RD-40]) fluorescent dextrans by S. aureus was observed by flow cytometry and indicate that Mac1 formed a pore of finite size. In model membranes with both dyes encapsulated together, the full release of FD-4 occurred, but only 40% of RD-40 was reached, supporting the flow cytometry results, and indicating a pore size between 1.4 and 4.5 nm. Finally, solid-state nuclear magnetic resonance showed formation of an isotropic phase signifying highly mobile lipids such as encountered in a toroidal pore structure. Overall, Mac1 is a promising antimicrobial peptide with the potent capacity to form pores in S. aureus membranes.