Interaction Of Enrofloxacin With Model Membrane Systems. Implications In The Permeation Pathways, Revealed By Fluorescence And Conductance Studies

With the actual increasing menace of bacterial resistance, the antibiotic permeation mechanisms have been the centre of attention, in many research fields. OmpF is a pore forming protein, found in Gram-negative bacteria's outer membrane, known to have an important role in the uptake of nutrients and antibiotics (e.g. quinolones), towards the interior of the bacterial cell. Although several studies have been published of protein-drug interaction in the complex microbiological environment, an understanding of this mechanism at a molecular level is still lacking. Quinolones are one of the most prescribed classes of antibiotics, both in human and veterinary medicine, and this wide use seems to be the main cause for bacterial resistance. Fluoroquinolones were developed to increase quinolone antibacterial activity by changes in the quinolone structure. Enrofloxacin is a second generation fluoroquinolone being the first one to be introduced in Veterinary medicine. Its human analogue is the well known Ciprofloxacin. The main purpose of this work was to study Enrofloxacin permeation pathways. Permeation or interaction of antibiotics with lipid bilayers (liposomes) is of great importance in order to clarify the lipid component function's in membrane permeation. A first approach was the determination of partition coefficients Enrofloxacin/liposomes, which allowed the quantification of this interaction. After, OmpF was reconstituted in liposomes (proteoliposomes) by two different reconstitution methods (Gel exclusion chromatography and using detergent adsorbing beads) in order to compare size, homogeneity and protein orientation in proteoliposomes. Proteoliposomes quenching studies, using water soluble quencher KI and acrylamide, as well as ion conductance measurements were performed to establish the site of interaction/translocation of Enrofloxacin.