Mechanisms of envelope permeability and antibiotic influx and efflux in Gram-negative bacteria (original) (raw)
Related papers
Breaching the Barrier: Quantifying Antibiotic Permeability across Gram-negative Bacterial Membranes
Journal of Molecular Biology, 2019
The double membrane cell envelope of Gram negative bacteria is a sophisticated barrier that facilitates the uptake of nutrients and protects the organism from toxic compounds. An antibiotic molecule must find its way through the negatively charged lipopolysaccharide layer on the outer surface, pass through either a porin or the hydrophobic layer of the outer membrane, then traverse the hydrophilic peptidoglycan layer only to find another hydrophobic lipid bilayer before it finally enters the cytoplasm, where it typically finds its target. This complex uptake pathway with very different physico-chemical properties is one reason that Gram-negatives are intrinsically protected against multiple classes of antibiotic-like molecules, and is likely the main reason that in vitro target based screening programmes have failed to deliver novel antibiotics for these organisms. Due to the lack of general methods available for quantifying the flux of drugs into the cell, little is known about permeation rates, transport pathways and accumulation at the target sites for particular molecules. Here we summarise the current tools available for measuring antibiotic uptake across the different compartments of Gram-negative bacteria.
mBio
Gram-negative bacteria are notoriously resistant to antibiotics, but the extent of the resistance varies broadly between species. We report that in significant human pathogens Acinetobacter baumannii , Pseudomonas aeruginosa , and Burkholderia spp., the differences in antibiotic resistance are largely defined by their penetration into the cell. For all tested antibiotics, the intracellular penetration was determined by a synergistic relationship between active efflux and the permeability barrier. We found that the outer membrane (OM) and efflux pumps select compounds on the basis of distinct properties and together universally protect bacteria from structurally diverse antibiotics. On the basis of their interactions with the permeability barriers, antibiotics can be divided into four clusters that occupy defined physicochemical spaces. Our results suggest that rules of intracellular penetration are intrinsic to these clusters. The identified specificities in the permeability barrier...
Journal of Antimicrobial Chemotherapy, 2000
Pseudomonas aeruginosa is intrinsically resistant to many antibiotics. Part of this resistance can be attributed to the relatively low permeability of the P. aeruginosa outer membrane to a variety of antibiotics. 1-3 Another part of the resistance appears to be caused by two recently discovered multidrug efflux systems. 4,5 In addition, in some cases, enzymes that specifically inactivate antibiotics lead to antibiotic resistance, e.g. the inducible-lactamase of P. aeruginosa. 6-8 A great deal of effort has gone into attempts to make the Gram-negative outer membrane permeable to antibiotics in the hope that this may prove useful clinically. Several polycations have been shown to make the outer membrane permeable, presumably by binding to lipopolysaccharide (LPS). These include polymixin B and its derivatives, 9 including deacylpolymixin B 10 and polymixin B nonapeptide. 10 Other polycationic permeabilizers include bactericidal/permeability-increasing protein, 11 protamine 9 and various polycationic peptides, 12-16 including lysine polymers, defensins, cecropins, magainins and mellitin. Chelators of divalent cations, such as ethylenediaminetetraacetate (EDTA), nitrilotriacetate and sodium hexametaphosphate, are all effective in making outer membranes permeable to antibiotics (reviewed in references 17-19). Chelators presumably render the membrane permeable by removing Ca 2ϩ and Mg 2ϩ from LPS, resulting in release of much of the LPS from the outer membrane and consequent outer membrane destabilization (reviewed in references 17-19). Phospholipids are also known to bind Ca 2ϩ and Mg 2ϩ effectively, 20 suggesting that certain phospholipids might make the outer membrane of P. aeruginosa permeable to-lactam antibiotics. This hypothesis is examined in the present study.
Research in Microbiology, 2018
Antibiotic resistance is a serious threat to public health. Significant efforts are currently directed toward containment of the spread of resistance, finding new therapeutic options concerning resistant human and animal pathogens, and addressing the gaps in the fundamental understanding of mechanisms of resistance. Experimental data and kinetic modeling revealed a major factor in resistance, the synergy between active efflux and the low permeability barrier of the outer membrane, which dramatically reduces the intracellular accumulation of many antibiotics. The structural and mechanistic particularities of trans-envelope efflux pumps amplify the effectiveness of cell envelopes as permeability barriers. An important feature of this synergism is that efflux pumps and the outer membrane barriers are mechanistically independent and select antibiotics based on different physicochemical properties. The synergism amplifies even weak polyspecificity of multidrug efflux pumps and creates a major hurdle in the discovery and development of new therapeutics against Gram-negative pathogens.
PLoS ONE, 2013
Active efflux of antimicrobial agents is a primary mechanism by which bacterial pathogens can become multidrug resistant. The combined use of efflux pump inhibitors (EPIs) with pump substrates is under exploration to overcome efflux-mediated multidrug resistance. Phenylalanine-arginine b-naphthylamide (PAbN) is a well-studied EPI that is routinely combined with fluoroquinolone antibiotics, but few studies have assessed its utility in combination with b-lactam antibiotics. The initial goal of this study was to assess the efficacy of b-lactams in combination with PAbN against the opportunistic pathogen, Pseudomonas aeruginosa. PAbN reduced the minimal inhibitory concentrations (MICs) of several b-lactam antibiotics against P. aeruginosa; however, the susceptibility changes were not due entirely to efflux inhibition. Upon PAbN treatment, intracellular levels of the chromosomally-encoded AmpC b-lactamase that inactivates b-lactam antibiotics were significantly reduced and AmpC levels in supernatants correspondingly increased, potentially due to permeabilization of the outer membrane. PAbN treatment caused a significant increase in uptake of 8-anilino-1-naphthylenesulfonic acid, a fluorescent hydrophobic probe, and sensitized P. aeruginosa to bulky antibiotics (e.g. vancomycin) that are normally incapable of crossing the outer membrane, as well as to detergent-like bile salts. Supplementation of growth media with magnesium to stabilize the outer membrane increased MICs in the presence of PAbN and restored resistance to vancomycin. Thus, PAbN permeabilizes bacterial membranes in a concentration-dependent manner at levels below those typically used in combination studies, and this additional mode of action should be considered when using PAbN as a control for efflux studies.
Method for Estimation of Low Outer Membrane Permeability to -Lactam Antibiotics
Antimicrobial Agents and Chemotherapy, 2002
The outer membrane of gram-negative bacteria plays a major role in -lactam resistance as it slows down antibiotic entry into the periplasm and therefore acts in synergy with -lactamases and efflux systems. Up to now, the quantitative estimation of low outer membrane permeability by the method of Zimmermann and Rosselet was difficult because of the secreted and cell surface-associated -lactamases. The method presented here uses the acylation of a highly sensitive periplasmic penicillin-binding protein (PBP) (BlaR-CTD) to assess the rate of -lactam penetration into the periplasm. The method is dedicated to measurement of low permeability and is only valid when the diffusion rate through the outer membrane is rate limiting. Cytoplasmic membrane associated PBPs do not interfere since they are acylated after the very sensitive BlaR-CTD. This method was used to measure the permeability of -lactamase-deficient strains of Enterobacter cloacae and Enterobacter aerogenes to benzylpenicillin, ampicillin, carbenicillin, cefotaxime, aztreonam, and cephacetrile. Except for that of cephacetrile, the permeability coefficients were equal to or below 10 ؊7 cm/s. For cephacetrile, carbenicillin, and benzylpenicillin, the outer membrane of E. cloacae was 20 to 60 times less permeable than that of Escherichia coli, whereas for cefotaxime, aztreonam, and ampicillin it was, respectively, 400, 1,000, and 700 times less permeable. The permeability coefficient for aztreonam is the lowest ever measured (P ؍ 3.2 ؋ 10 ؊9 cm/s). Using these values, the MICs for a -lactamase-overproducing strain of E. cloacae were successfully predicted, demonstrating the validity of the method.
European journal of medicinal chemistry, 2022
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