Marco Lovelle Gomez

• The main goal of my PhD work was to try to elucidate the major pathways of antibiotics into the bacterial cell, and their interaction at the molecular level with bacterial membrane components. Bacterial membranes show high structural complexity. The outer membrane of Gram-negative bacteria, acting as a selectively permeable barrier, enables the bacterial cell to survive in various hostile environments. Restricted permeability is one important factor in determining antibiotic resistance as no antimicrobial agent will be effective unless it can penetrate the cell wall, so that knowledge of their composition and function becomes essential to the design and development of effective antibiotics. Channel forming proteins are a very important membrane component involved in the influx of several hydrophilic compounds, including different families of antibiotics. In the present work, fluorescence techniques are combined with high-resolution conductance measurements (black lipid bilayer technique) to characterize these biological structures in the presence and in the absence of antibiotics. Results obtained from fluorescence measurements can be successfully combined with bilayer measurements revealing details of the molecular interactions between the antibiotic and the the major porins of Escherichia coli. Cephalosporin studies proved the importance of the use of these two techniques and show that these interactions were higher for OmpF than for OmpC. Studies on single mutated OmpF porins demonstrated the drastic effect on channel permeation by substitution of a single amino acid. The replacement of a positively charged residue by a neutral one in the channel constriction zone results in a much lower main effective barrier for the translocation of antibiotics. Our studies with enrofloxacin not only corroborate this fact but also show that the replacement of an aminoacid residue away from the constriction zone also affects the antibiotic transport. The comprehensive information provided by the complementary and wide-ranging studies of drug-porin interactions can provide a thorough understanding of the mechanisms of drug intake and resistance, which can enable refinement of the current drug therapies and the design of new types of antibiotics to target resistant organisms.