Exploration of Antibacterial Mechanisms of Cold Atmospheric Plasma (CAP): Effects on cell membranes during responses to oxidative stress

Plasma is a complex mixture which contains electrons, charged particles, reactive species, ions, UV radiation and electromagnetic fields. CAP has been shown to possess huge potential in antibacterial and anticancer applications and is considered as an effective alternative to traditional antibiotics and chemotherapy in killing multi-drug-resistant bacteria and cancer cells. However, the underlying mechanisms are not clear. Reactive oxygen and nitrogen species (RONS) are proposed to play important roles. One important target of CAP and RONS are cell membranes that fulfill important functions and prevent certain RONS from entering the cell. Our study aims to investigate how CAP kills bacteria depending on conditions and how cell membrane impairment is involved in these effects. We used the kINPen plasma source, which can ionize argon gas between its internal electrodes and delivers its reactive “cocktail” to the targets. E. coli and S. simulans were used as representative Gram-negative and Gram-positive bacteria to assess the antibacterial efficiency. The impairment of the membrane was characterized in both bacterial membranes and liposomes. Our results show that short-lived RONS dominate the effects in killing bacteria, that acidity enhanced it, while exponentially growing bacteria were more susceptible to direct CAP. Alterations in membrane packing depended on the properties of membranes and the used probes, as well as on the acidity of suspensions. In Gram-positive bacteria, the membrane potential was dissipated, membrane proteins were oxidized, and membrane pores were formed. In Gram-negative bacteria, the permeabilization was induced in the outer membrane by both short-and long-lived RONS while no pore formation for larger molecules was observed at the inner membrane. Lipid hydroperoxides were only formed in poly-unsaturated LUVs and were further accumulated after long-term post-incubation. Oxidized lipids with truncated acyl chains were produced in all LUVs at a similar amount and were further accumulated in poly-unsaturated LUVs. Membrane permeabilization in LUV was obtained very rapidly and independently of lipid unsaturation probably due to the formation of truncated oxidized lipids while permeabilization happened also upon longer incubation periods with polyunsaturated lipids probably because of the appearance of oxidized adducts such as lipid hydroperoxides. In reconstituted models, lipid aldehydes and carboxylic acids were able to induce membrane permeabilization but could possess different mechanisms. The molecular conformation of carboxylic acids in membranes depended on the pH of the suspension, while aldehydes remained probably unaffected. At a pH of 7.4, the sn-2 tail of truncated carboxylic acids pointed to the aqueous phase, resulting in intrinsic positive molecular curvature, inducing hence the formation of highly curved isotropic structures. Nevertheless, lipid aldehydes were likely to permeabilize membrane towards larger molecules while lipid carboxylic acids tended to permeabilize membrane to smaller molecules or possibly induce lipid flip-flop. At lower pH, the sn-2 tail of truncated aldehydes and carboxylic acids stayed inside the membrane bilayer which induced stronger permeabilization for the carboxylic acid. Membrane packing was largely affected by both lipid oxides at the membrane-aqueous phase interface. Further, the formed oxidized lipid species were partly released from membrane bilayers. In this work, we performed a systematic study to explore the mechanisms of how CAP kills bacteria depending on various conditions and we investigated how membrane damage is involved in these changes. We used both bacteria and liposomes as research models and carried out a series of experiments to compare and correlate alterations in biochemical and biophysical parameters. The results show that CAP has strong and very diverse effects on membranes, depending on environmental conditions and membrane properties. This study hence provides a better understanding of antibacterial mechanisms by CAP, contributing a step forward in the plasma medicine research field.