Interaction of aminoglycosides with the outer membranes and purified lipopolysaccharide and OmpF porin of Escherichia coli

Abstract

The mechanism of uptake of aminoglycosides across the outer membrane of Escherichia coli was reevaluated. Porin-deficient mutants showed no alteration in gentamicin or kanamycin susceptibility. Furthermore, the influence of kanamycin on intrinsic tryptophan fluorescence of porin OmpF (Y. Kobayashi, and T. Nakae, Eur. J. Biochem. 151:231-236, 1985) was shown to be strongly influenced by protein concentration and EDTA. This led to the hypothesis that aminoglycoside-mediated increases and decreases in intrinsic tryptophan fluorescence were due to aggregation-disaggregation of OmpF mediated by interaction at a divalent cation binding site on OmpF. Gentamicin, kanamycin, and polymyxin B increased E. coli outer membrane permeability to the hydrophobic fluorescent compound 1-N-phenyl-naphthylamine (NPN) and the peptidoglycan-degrading enzyme lysozyme. Addition of Mg2+ blocked these permeabilizing activities. Furthermore, gentamicin and polymyxin B bound to Mg2+-binding sites on E. coli lipopolysaccharide, as determined in dansyl polymyxin displacement experiments. A polymyxin-resistant, lipopolysaccharide-altered pmr mutant of E. coli had a fourfold-lower MIC of gentamicin and kanamycin and was more poorly permeabilized to 1-N-phenylnaphthylamine than was its parent strain. These data were consistent with uptake of aminoglycosides across the E. coli outer membrane by the self-promoted uptake mechanism.The outer membrane of gram-negative bacteria is a semipermeable barrier which restricts the access of all antibiotics to their targets (8,9,23). Hydrophilic molecules of sizes below a given exclusion limit can pass through the waterfilled channels of proteins called porins. Included among these molecules are the ,B-lactam antibiotics. The most convincing data suggesting that r-lactams are taken up through porin channels are the increases in MICs of P-lactams in mutants with deficiencies in porins (8,13,15,16,23), although this conclusion is supported by liposome swelling data showing the uptake of P-lactams into liposomes reconstituted with porins (32).In contrast, polycations, some of which are larger than the exclusion limit of the outer membrane, can be taken up by another mechanism, which we have termed the self-promoted uptake pathway (7,11,22). In this pathway, the polycations act to competitively displace divalent cations which cross-bridge adjacent lipopolysaccharide (LPS) molecules, thus disrupting these important outer membrane stabilizing sites. This, then, permeabilizes the outer membrane and is proposed to promote uptake of other molecules of the permeabilizing polycation. Mutants which interact more poorly with polymyxin B have been shown to resist killing by that polycation (11,22,25,29,30), while a mutant (totA) with LPS which better interacted with polycations was supersusceptible (26). These data show that self-promoted uptake is the first stage of uptake leading to cell killing by certain polycationic antibiotics.In the case of aminoglycosides, the mechanism of uptake across the outer membrane has been...

Photomanipulation of antibiotic susceptibility and biofilm formation of Escherichia coli heterologously expressing photoactivated adenylyl cyclase

“…Foulds and Chai reported that E. coli cells defi cient in ompF were more resistant to kanamycin and gentamicin (Foulds and Chai, 1978). On the other hand, Hancock et al reported that porin-defi cient mutants show no alteration in susceptibility to these antibiotics (Hancock et al, 1991). In addition to these observations, Kashiwagi et al reported that aminoglycosides are transported into cells by the oligopeptide transport system (Kashiwagi et al, 1998).…”

Viability states of bacteria—Specific mechanisms of selected probes

“…Contrarily, hydrophobic substances are supposed to diffuse through the cell wall and the IM but to a minor degree through the OM that seems to be an efficient barrier for hydrophobic substrates in Mycobacteria (8). Another process is described for the uptake of polycationic compounds (which comprise many antibiotics or diethylaminoethyl-or polylysinmoieties) which are thought to disorganize the OM, thereby mediating their own uptake in a process termed 'self-promoted uptake' (13). Moreover, many molecules can be specifically channeled into the cell by transporter proteins using energy-dependent processes or facilitated diffusion systems (for overview see (8)).…”

Polyamines decrease Escherichia coli outer membrane permeability

“…Our finding that polyamines can drastically affect porinmediated fluxes is important because it shows that (i) porins are functionally regulated channels and (ii) polyamines and related compounds could potentially serve as therapeutic agents specifically targeted at the outer membrane. Although the outer membrane is an effective barrier against hydrophilic and many hydrophobic compounds, some types of antibiotics, such as polymyxin B and aminoglycosides, have been shown to enter the cells by increasing outer membrane permeability (10,11,35). In their studies of polycation-induced sensitization of bacteria to antibiotics, Vaara and Vaara (36) reported that, as opposed to cations bearing a large number of positive charges (5 to 50), cadaverine, spermine, and spermidine were neither bactericidal nor active as outer membrane permeability-increasing agents.…”