How β-Lactam Antibiotics Enter Bacteria: A Dialogue with the Porins (original) (raw)
Related papers
The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria
Nature Reviews Microbiology, 2008
Multidrug resistance (MDR) is frequ-ently reported in clinical Gram-negative bacteria. This limits which therapeutic options are available and is a major cause of mortality when acquired as a nosocomial infection 1,2 . Moreover, no truly novel active antibacterial compound is currently in clinical trials. Thus, it is important to decipher the molecular basis of the MDR mechanisms 3-5 . MDR is prevalent in key Gram-negative clinical pathogens, such as Escherichia coli, Salmonella spp., Klebsiella spp., Enterobacter spp., Campylobacter spp., Acinetobacter spp. and Pseudomonas spp. Three major bacterial strategies have emerged for the development of drug resistance: the membrane barrier limits the intracellular access of an antibiotic; the enzymatic barrier produces detoxifying enzymes that degrade or modify the antibiotic; and the target protection barrier impairs target recognition and thus antimicrobial activity 6 . These mechanisms can act simultaneously in clinical isolates, generating a high level of resistance. There are two different aspects to transport systems across the bacterial membrane -influx and efflux. Here, we focus on the influx of antibiotics, as the efflux has been extensively discussed in recent reviews 5-8 .
Toward Screening for Antibiotics with Enhanced Permeation Properties through Bacterial Porins
Biochemistry, 2010
Gram-negative bacteria are protected by an outer membrane barrier, and to reach their periplasmic target, penicillins have to diffuse through outer membrane porins such as OmpF. Here we propose a structuredynamics-based strategy for improving such antibiotic uptake. Using a variety of experiments (high-resolution single channel recording, Minimum Inhibitory Concentration (MIC), liposome swelling assay) and accelerated molecular simulations, we decipher the subtle balance of interactions governing ampicillin diffusion through the porin OmpF. This suggests mutagenesis of a hot spot residue of OmpF for which additional simulations reveal drastic changes in the molecular and energetic pathway of ampicillin's diffusion. Inverting the problem, we predict and describe how benzylpenicillin diffuses with a lower effective energy barrier by interacting differently with OmpF. The thorough comparison between the theoretical predictions and the three independent experiments, which were set up to measure the kinetics of transport and biological activity, gives insights on how to combine such different investigation techniques with the aim of providing complementary validation. Our study illustrates the importance of microscopic interactions at the constriction region of the biological channel to control the antibiotic flux through it. We conclude by providing a complete inventory of the channel and antibiotic hot spots and discuss the implications in terms of antibacterial screening and design.
Journal of Biomolecular Screening, 2010
1 School of engineering and Science, Jacobs university bremen, bremen, germany. 2 nanion technologies gmbh, munich, germany. a chip-based automated patch-clamp technique provides an attractive biophysical tool to quantify solute permeation through membrane channels. Proteo-giant unilamellar vesicles (proteo-guvs) were used to form a stable lipid bilayer across a micrometer-sized hole. because of the small size and hence low capacitance of the bilayer, single-channel recordings were achieved with very low background noise. the latter allowed the characterization of the influx of 2 major classes of antibioticscephalosporins and fluoroquinolones-through the major Escherichia coli porins ompf and ompc. analyzing the ion current fluctuations in the presence of antibiotics revealed transport properties that allowed the authors to determine the mode of permeation. the chip-based setup allows rapid solution exchange and efficient quantification of antibiotic permeation through bacterial porins on a single-molecule level. (Journal of Biomolecular Screening 2010:302-307)
Microscopic mechanism of antibiotics translocation through a porin
OmpF from the outer membrane of Escherichia coli is a general porin considered to be the main pathway for b-lactam antibiotics. The availability of a high-resolution crystal structure of OmpF and new experimental techniques at the singlemolecule level have opened the way to the investigation of the microscopic mechanisms that allow the passage of antibiotics through bacterial pores. We applied molecular dynamics simulations to investigate the translocation process of ampicillin (Amp) through OmpF. Using a recent algorithm capable of accelerating molecular dynamics simulations we have been able to obtain a reaction path for the translocation of Amp through OmpF. The mechanism of passage depends both on the internal degrees of freedom of Amp and on interactions of Amp with OmpF. Understanding this mechanism would help us design more efficient antibiotics and shed light on nature's way of devising channels able to enhance the transport of molecules through membranes.
2021
Gram-negative bacteria pose a serious public health concern, primarily due to a higher frequency of antibiotic resistance conferred to them as a result of low permeability of their outer membrane (OM). Antibiotics capable of traversing the OM typically permeate through OM porins; thus, understanding the permeation properties of these porins is instrumental to the development of new antibiotics. A common macroscopic feature of many OM porins is their ability to transition between functionally distinct open and closed states that regulate transport properties and rate. To obtain a molecular basis for these processes, we performed tens of microseconds of molecular dynamics simulations of E. coli OM porin, OmpF. We observed that large-scale motion of the internal loop, L3, leads to widening and narrowing of the pore, suggesting its potential role in gating. Furthermore, Markov state analysis revealed multiple energetically stable conformations of L3 corresponding to open and closed stat...
PLoS ONE, 2011
Antibiotic-resistant bacteria, particularly Gram negative species, present significant health care challenges. The permeation of antibiotics through the outer membrane is largely effected by the porin superfamily, changes in which contribute to antibiotic resistance. A series of antibiotic resistant E. coli isolates were obtained from a patient during serial treatment with various antibiotics. The sequence of OmpC changed at three positions during treatment giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and OmpC33, in which OmpC20 was derived from the first clinical isolate). We demonstrate that expression of the OmpC K12 porin in the clinical isolates lowers the MIC, consistent with modified porin function contributing to drug resistance. By a range of assays we have established that the three mutations that occur between OmpC20 and OmpC33 modify transport of both small molecules and antibiotics across the outer membrane. This results in the modulation of resistance to antibiotics, particularly cefotaxime. Small ion unitary conductance measurements of the isolated porins do not show significant differences between isolates. Thus, resistance does not appear to arise from major changes in pore size. Crystal structures of all four OmpC clinical mutants and molecular dynamics simulations also show that the pore size is essentially unchanged. Molecular dynamics simulations suggest that perturbation of the transverse electrostatic field at the constriction zone reduces cefotaxime passage through the pore, consistent with laboratory and clinical data. This subtle modification of the transverse electric field is a very different source of resistance than occlusion of the pore or wholesale destruction of the transverse field and points to a new mechanism by which porins may modulate antibiotic passage through the outer membrane.
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...
Biophysical journal, 2010
Our aim in this study was to provide an atomic description of ampicillin translocation through OmpF, the major outer membrane channel in Escherichia coli and main entry point for b-lactam antibiotics. By applying metadynamics simulations, we also obtained the energy barriers along the diffusion pathway. We then studied the effect of mutations that affect the charge and size at the channel constriction zone, and found that in comparison to the wild-type, much lower energy barriers are required for translocation. The expected higher translocation rates were confirmed on the macroscopic scale by liposome-swelling assays. A microscopic view on the millisecond timescale was obtained by analysis of temperature-dependent ion current fluctuations in the presence of ampicillin and provide the enthalpic part of the energy barrier. By studying antibiotic translocation over various timescales and length scales, we were able to discern its molecular mechanism and rate-limiting interactions, and draw biologically relevant conclusions that may help in the design of drugs with enhanced permeation rates.