Probing the biophysical properties of the Gram-negative cell envelope after treatment with low temperature plasma

Low temperature plasma (LTP) can produce reactive oxygen species (ROS), which are known antimicrobial agents. ROS can react with lipids and proteins within the bacterial cell envelope. These bactericidal effects offer the potential to combat infections by antibiotic-resistant bacterial strains. Cell viability assays were used to show that the cell envelope is damaged by LTP treatment,which produced transient pores across both membranes. By varying Lipopolysaccharide (LPS) oligosaccharide head group lengths in Escherichia coli, we demonstrated that LPS provides protection against LTP-induced poration. We found that fluorescent nanoparticles could be internalised after LTP-treatment and determined the minimum size of these pores to be 9 nm. This observation is consistent with the nanoparticles diffusing through the pores of the previously intact membranes. The propensity for pore formation increased as the length of the oligosaccharide head group in LPS decreased. Using fluorescence recovery after photobleaching (FRAP) microscopy, we measured the changes in membrane fluidity and the lateral mobility of specific targets in the cell envelope. The effect of LTP-treatment on membrane fluidity was difficult to quantify because the FRAP data were dependent on the styryl dye probe utilised. The mobility of both styryl dyes employed here increased in LTP-treated cells. Using a GFP-TolA fusion protein, we measured a decrease in lateral mobility in the inner membrane (IM) after LTP treatment. This decrease was also observed in the outer membrane (OM) for the OmpA protein bio-orthogonally labelled with Alexa fluor 568 (AF568). In contrast, no change in AF568-labelled LPS lateral mobility was detected in LTP-treated bacterial cells. Overall, LTP-treatment has been shown in this work to induce pronounced changes in the bacterial cell envelope. It is possible that some of these changes, like pore formation, may be exploited to deliver drug molecules which could render LTP a more effective antimicrobial agent.