Investigation of the antibacterial mechanisms of arginine-containing hemin derivatives <i>in vivo</i> against <i>S. aureus</i>

Continuing research in the field of synthetic amino acid and peptide derivatives of hemin (HD), in this work, we have identified specific aspects of the mechanism behind their pronounced antibacterial action, including action against resistant bacteria. Using gel electrophoresis, we demonstrated the ability of HD to interact with isolated plasmid DNA and genomic DNA inside living bacteria. No expected biological effect was observed on the electroporation of E. coli bacteria with a complex of plasmid DNA with HD. Electroporation of plasmid DNA isolated from this complex showed that its structure and function were preserved. Using laser scanning confocal microscopy with a fluorescent dye and labeled dextrans, we observed the rapid formation of defects smaller than 2–3 nm in the membrane of the antibiotic-sensitive S. aureus 209P strain. These damages, as was shown by the highly sensitive fluorescent ATP measurement method, may lead to the secretion of ATP from the intracellular space of the bacteria into the extracellular environment. At the same time, there is no secretion of proteins in these conditions. The formation of defects in bacterial membranes may be accompanied by the generation of alkyl peroxides inside the membrane due to the action of HD or as a result of electrostatic interactions between HD and lipid membranes. It can be concluded that the basic mechanism of HD’s antibacterial action is the formation of defects in bacterial membranes, followed by the secretion of ATP and possibly other low-molecular components of the cell into the external environment, along with interaction with genomic DNA.