Molecular Bases Basis of Antibiotic Translocation Across Outer Membrane Porins of Enterobacter Aerogenes

Abstract

Multi-drug resistant (MDR) bacteria are an increasingly serious threat to global public health. In the last decades, many studies showed that general outer membrane porins (OMPs) from Gram-negative bacteria represent the major route for antibiotics to translocate through the outer membrane.To explore the molecular rules for efficient drug transport and therefore cell killing, molecule-protein interaction is analyzed at single molecule level via electrophysiology. We incorporated OMPs into planar bilayer and single-channel currents were measured by an automated planar lipid bilayer station (Orbit 16, Nanion). The role of OMPs in the molecule-protein interactions can be characterized by the interaction kinetics. Here, we show by using 11 clinical relevant antibiotics and Omp35, Omp36 porins from Enterobacter aerogenes, that Omp35 has generally higher antibiotic-porin interaction than Omp36. Interestingly, E. aerogenes preferentially expresses Omp36 in human hosts. These in vitro results also correlate well with in vivo data collected from bacterial viability assays.On the other side, we studied the effect of magnesium on the antibiotic-porin interactions in order to approach physiological conditions. Since fluoroquinolones antibiotics have relatively heavy dipole, they can be used as probes to detect the electrostatic properties at the constriction zone of the porin channels. We found that the addition of magnesium strengthened the interaction between norfloxacin antibiotics and Omp36. The results indicate that the electrostatic properties of the constriction zone are altered and show a more efficient route to transport the norfloxacin.In conclusion, the porin properties can be better understood when considering the host environment. Improved understanding of antibiotic-porin interaction kinetics and the correlated antibiotic translocation based cell killing would facilitate the understanding on porin specificity thus the design of new antibiotics that can more efficiently reach high intracellular concentration.