Biophysical Investigations on the Interaction between Antimicrobial Peptides and Bacteria Killed by Cs-137 Irradiation

Abstract

Two major problems arise while investigating the interaction between antimicrobial peptides (AMPs) and bacteria using biophysical techniques: (i) working with living pathogenic bacteria cause safety issues and (ii) some effects might depend on the metabolic activity of the bacteria. To distinguish between AMP-activities depending either on processes in active living bacteria or on interactions between AMPs and passive bacterial components, e.g. membrane permeabilization or DNA binding, one have to kill the bacteria before addition of the AMPs. Most techniques to kill bacteria are not suitable because they pre-damage or modify the bacterial envelope. Therefore, we investigated the usability of Cs-137 irradiation to kill the bacteria. Morphological studies using AFM and TEM showed no or only minor damage using a minimal bactericidal dose (MBD). At a higher dose, the bacterial integrity is damaged. The Zeta-potential of the bacteria is not changed by the irradiation and the ability of the bacteria to stimulate immune cells is not influenced. Binding experiments between AMPs and living bacteria as well as heat killed bacteria using ITC result in complex data because of metabolic activities as well as binding to intracellular components being exposed after heat killing. In contrast, experiments using bacteria killed by irradiation results in typical binding curves. The permeabilization assay based on the binding of Sytox Green to the bacterial DNA demonstrated that the envelopes of most of the irradiated bacteria are still intact. Surprisingly, the two AMPs LL32 (a derivative of the human cathelicidin) and polymyxin B cannot permeabilize the bacterial membrane of the killed bacteria anymore. In summary, biophysical investigations on bacteria killed by Cs-137 irradiation can be used as a tool for a better understanding of the interaction between whole bacteria and AMPs.