Understanding the mechanisms and kinetics of membrane damage is of interest to researchers in several overlapping fields of biology. In this study, we describe the development and validation of a simple 32PO43- release radioassay used to track nanometer-scale damage to the bacterial cell membrane. Nanoscale membrane damage will result in the release of small cytoplasmic molecules, such as amino acids, sugars, and osmolytes. Our radioassay tracks the release of these molecules using the release of cytoplasmic 32PO43- as a proxy. Our assay can both detect 32PO43- release and track release kinetics in the order of minutes. We demonstrate the use of our radioassay using A. baumannii treated with colistin and Ω76: two agents known to cause membrane damage. Our assay tracks greater membrane damage in A. baumannii treated with both these agents, compared to an untreated control. Our assay fills a niche that is not covered by traditional 51Cr release radioassays and fluorescent staining techniques. Furthermore, our assay can potentially be used to track membrane damage in other membrane systems such as lipid vesicles, animal cells, and organelles.
Keywords: antibiotics; antimicrobial peptides; colistin; membrane damage; radioassay.