Kanamycin Uptake into Escherichia coli Is Facilitated by OmpF and OmpC Porin Channels Located in the Outer Membrane

Abstract

‡ Equally contributedThe complex cell envelope of Gram-negative bacteria comprises two membranes: the outer membrane (OM) and the cytoplasmic membrane. The two membranes delimit the periplasmic space of the bacterial cell and prevent the accumulation of toxic agents in the cytosol while regulating the access of nutrients vital for growth and cell function. The OM is the first barrier during compound uptake. It is composed of an asymmetric bilayer: an inner leaflet of phospholipids and an outer leaflet of lipopolysaccharides (LPS). Both OM leaflets combined prevent the efficient diffusion of hydrophilic as well as hydrophobic molecules. Porins, waterfilled channels spanning across the OM, enable passive diffusion of small, hydrophilic molecules into the periplasm. Substrate specificity is mainly defined by the constriction zone within the barrel structure of these porins, determining entry of molecules by factors such as size, shape, electric multipoles, and rigidity. 1,2,3 E. coli encodes multiple porins. The major porins OmpF and OmpC are highly abundant and both cation-selective, and it has been thought that they restrict the passage to compounds with a size-exclusion limit of about 600 Da. 4 However, it has recently been suggested that this limit should be redefined using other parameters. 5,6 The translocation of several classes of antibiotics, e.g. β-lactams and fluoroquinolones, through porins has been investigated extensively. Also, porin modification emerged as antibiotic resistance mechanism in clinical isolates, 7 based on specific changes in amino acid residues or decreased expression of wild-type porins. 8 Aminoglycosides (AGs) target the ribosome in the cytoplasm, thus they have to overcome both membranes in Gram-negative bacteria. Despite their frequent use as therapeutic agents, the mechanisms of their OM translocation remain incompletely understood. The self-promoted pathway is a proposed uptake mechanism. Here, divalent cations between LPS molecules are displaced by AGs, which leads to brief OM destabilization, thereby enabling OM translocation. 9

Dynamic interaction of fluoroquinolones with magnesium ions monitored using bacterial outer membrane nanopores

“…4,5 Methods to investigate the accumulation of antimicrobial agents inside a cell can be in vivo viability assay, 6 liposome swelling assay, 7 mass spectrometry and uorometry related approaches, [8][9][10] and electrophysiology. 5,11 Nanopore based single molecule tools could already investigate a single nucleotide segment, 12,13 and nucleotide base lesion analogues, 14,15 peptides [16][17][18][19] etc. Here we introduce outer membrane nanopores, which are pore forming proteins in the OM and especially general diffusion channels also called "porins", e.g., OmpF and OmpC from E. coli, facilitate the diffusion of hydrophilic nutrients and are slightly cation selective as found in both in situ as well as in silico characterization.…”

Permeation of β-Lactamase Inhibitors through the General Porins of Gram-Negative Bacteria

“…All the selected inhibitors are avibactam derivatives, belonging to the class of diazobicyclooctans. Thus, they are not β-lactams, and as it has been recently shown by using other scaffolds, [49,50] they will contribute to understand to a more general level the process of small-molecule permeation through Gram-negative general porins.…”

Large‐Peptide Permeation Through a Membrane Channel: Understanding Protamine Translocation Through CymA from Klebsiella Oxytoca**

“…Fort he latter molecules,a n extrapolation to similar concentration gradients reveals about 10 molecules per second with slightly shorter translocation times in the ms range.N ote that the slightly larger bcyclodextrin (M W = 1135 Da) is unable to permeate the membrane.A nother comparison can be made with the antibiotic molecule kanamycin (M W = 484 Da) for which very strong interactions with OmpF were observed during translocation. [32] Although the molecular size is an order of magnitude smaller than that of Ptm and the pore has half the diameter of that of DCymA, the event rate is about the same whereas the dwell time is in the ms range.Overall, this trend suggests that in the case of bacterial channels,t he molecular weight is only arough exclusion criterion whereas the molecular shape and pore-mediated interactions will finally control the translocation kinetics.M oreover, we conclude that even small concentration gradients are able to enforce the diffusion of large molecules like Ptm through narrow orifices.…”