Investigation of porin-dependent translocation of methylene blue and gentamicin through the outer membrane of gram-negative bacteria using molecular dynamics methods

The outer membrane of Gram-negative bacteria serves as a critical barrier to the permeability of antimicrobial compounds. Porin channels are believed to be the primary route for the penetration of hydrophilic molecules with a molecular weight up to 600 Da across this membrane. In this study, free energy profiles for the translocation of two different antimicrobial compounds through bacterial lipopolysaccharide membranes with and without integrated OmpF porin channels were generated using the method of nonequilibrium molecular dynamics. The compounds examined were gentamicin, an aminoglycoside antibiotic, and methylene blue, a phenothiazine antiseptic. For both compounds, similar free energy profiles were observed during translocation through the porin channel. However, gentamicin exhibited a higher energy barrier at the center of the channel, while methylene blue required less energy to exit the channel. The use of the umbrella sampling method allowed us to analyze in detail the molecular interactions between methylene blue and gentamicin and specific amino acid residues that line the OmpF porin channel, as well as identify those that contribute to the formation of barrier properties. It has been shown that, for methylene blue molecules, the penetration through the lipopolysaccharide membrane with embedded porin is energetically equivalent to that without it. At the same time, the porin-dependent transport route through the lipopolysaccharide membrane is preferable for gentamicin, as in the case of direct transfer through the membrane, this highly charged cationic compound would need to overcome a significant energy barrier of 50 kT. Molecular dynamic calculations can be very useful in studying porin-dependent mechanisms of resistance development in Gram-negative pathogens. They allow us to consider various modifications of porin structure and analyze their impact on the conductivity of channels for antimicrobial compounds.