Publications by authors named "Mathias Winterhalter"

161 Publications

How to Enter a Bacterium: Bacterial Porins and the Permeation of Antibiotics.

Chem Rev 2021 Mar 16. Epub 2021 Mar 16.

Department of Life Sciences and Chemistry, Jacobs University Bremen, Bremen 28759, Germany.

Despite tremendous successes in the field of antibiotic discovery seen in the previous century, infectious diseases have remained a leading cause of death. More specifically, pathogenic Gram-negative bacteria have become a global threat due to their extraordinary ability to acquire resistance against any clinically available antibiotic, thus urging for the discovery of novel antibacterial agents. One major challenge is to design new antibiotics molecules able to rapidly penetrate Gram-negative bacteria in order to achieve a lethal intracellular drug accumulation. Protein channels in the outer membrane are known to form an entry route for many antibiotics into bacterial cells. Up until today, there has been a lack of simple experimental techniques to measure the antibiotic uptake and the local concentration in subcellular compartments. Hence, rules for translocation directly into the various Gram-negative bacteria via the outer membrane or via channels have remained elusive, hindering the design of new or the improvement of existing antibiotics. In this review, we will discuss the recent progress, both experimentally as well as computationally, in understanding the structure-function relationship of outer-membrane channels of Gram-negative pathogens, mainly focusing on the transport of antibiotics.
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http://dx.doi.org/10.1021/acs.chemrev.0c01213DOI Listing
March 2021

Rapid fabrication of teflon apertures by controlled high voltage pulses for formation of free standing planar lipid bilayer membrane.

Biomed Microdevices 2021 Feb 27;23(1):12. Epub 2021 Feb 27.

Department of Biomedical Engineering, Ankara Yıldırım Beyazıt University, Ankara, Turkey.

Free standing artificial lipid bilayers are widely used in the study of biological pores. In these types of studies, the free standing planar lipid bilayer is formed over a micron-sized aperture consisting of either polymer such as Polytetrafluoroethylene (PTFE, Teflon) or glass. Teflon is chemically inert, has a low dielectric constant, and has a high electrical resistance which combined allow for obtaining low noise recordings. This study investigates the reproducible generation of micropores in the range of 50-100 microns in diameter in a Teflon film using a high energy discharge set-up. The discharger set-up consists of a microprocessor, a transformer, a voltage regulator, and is controlled by a computer. We compared two approaches for pore creation: single and multi-pulse methods. The results showed that the multi-pulse method produced narrower aperture size distributions and is more convenient for lipid bilayer formation, and thus would have a higher success rate than the single-pulse method. The bilayer stability experiments showed that the lipid bilayer lasts for more than 33 h. Finally, as a proof-of-concept, we show that the single and multi-channel electrophysiology experiments were successfully performed with the apertures created by using the mentioned discharger. In conclusion, the described discharger provides reproducible Teflon-pores in a cheap and easy-to-operate manner.
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http://dx.doi.org/10.1007/s10544-021-00553-4DOI Listing
February 2021

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

Angew Chem Int Ed Engl 2021 04 3;60(15):8089-8094. Epub 2021 Mar 3.

Department of Life Sciences and Chemistry, Jacobs University, 28759, Bremen, Germany.

Quantifying the passage of the large peptide protamine (Ptm) across CymA, a passive channel for cyclodextrin uptake, is in the focus of this study. Using a reporter-pair-based fluorescence membrane assay we detected the entry of Ptm into liposomes containing CymA. The kinetics of the Ptm entry was independent of its concentration suggesting that the permeation through CymA is the rate-limiting factor. Furthermore, we reconstituted single CymA channels into planar lipid bilayers and recorded the ion current fluctuations in the presence of Ptm. To this end, we were able to resolve the voltage-dependent entry of single Ptm peptide molecules into the channel. Extrapolation to zero voltage revealed about 1-2 events per second and long dwell times, in agreement with the liposome study. Applied-field and steered molecular dynamics simulations added an atomistic view of the permeation events. It can be concluded that a concentration gradient of 1 μm Ptm leads to a translocation rate of about one molecule per second and per channel.
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http://dx.doi.org/10.1002/anie.202016943DOI Listing
April 2021

Towards understanding single-channel characteristics of OccK8 purified from Pseudomonas aeruginosa.

Eur Biophys J 2021 Jan 22;50(1):87-98. Epub 2021 Jan 22.

School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759, Bremen, Germany.

Antibiotic resistance in Gram-negative bacteria causes serious health issues worldwide. Bacteria employ several resistance mechanisms to cope with antimicrobials. One of their strategies is to reduce the permeability of antibiotics either through general diffusion porins or substrate-specific channels. In this study, one of the substrate-specific channels from Pseudomonas aeruginosa, OccK8 (also known as OprE), was investigated using single-channel electrophysiology. The study also includes the investigation of permeability properties of several amino acids with different charged groups (i.e. arginine, glycine and glutamic acid) through OccK8. We observed four different conformations of the same OccK8 channel when inserted in lipid bilayers. This is in contrast to previous studies where heterologous expressed OccK8 in E. coli showed only one conformation. We hypothesized that the difference in our study was due to the expression and purification of the native channel from P. aeruginosa. The single-channel uptake characteristics of the porin showed that negatively charged glutamic acid preferentially interacted with the channel while the positively charged arginine molecule showed infrequent interaction with OccK8. The neutral amino acid glycine did not show any interaction at the physiological conditions.
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http://dx.doi.org/10.1007/s00249-021-01498-5DOI Listing
January 2021

Antibiotic uptake through porins located in the outer membrane of Gram-negative bacteria.

Expert Opin Drug Deliv 2020 Nov 13:1-9. Epub 2020 Nov 13.

Department of Life Sciences and Chemistry, Jacobs University , Bremen, Germany.

: Making selective inhibitors of novel Gram-negative targets is not a substantial challenge - getting them into Gram-negative bacteria to reach their lethal target is the bottleneck. Poor permeability of the antibiotic requires high concentration causing off target activity. The lack of simple experimental techniques to measure antibiotic uptake as well as the local concentration at the target site creates a particular bottleneck in understanding and in improving the antibiotic activity. : Here we recall current approaches to quantify the uptake. For a few antibiotics with known evidence for channel-limited permeation, the flux across a single OmpF or OmpC channel has been measured. For a typical concentration gradient of 1 µM of antibiotics the uptake varies between one up to few hundred molecules per second and per channel. : The current research effort is on quantifying the flux for a larger list of compounds on a cellular (mass spectra, fluorescence) or at single channel level (electrophysiology). A larger dataset of single channel permeabilities under various condition will be a powerful tool for understanding and improving the activity of antibiotics.
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http://dx.doi.org/10.1080/17425247.2021.1847080DOI Listing
November 2020

The Beauty of Asymmetric Membranes: Reconstitution of the Outer Membrane of Gram-Negative Bacteria.

Front Cell Dev Biol 2020 14;8:586. Epub 2020 Jul 14.

Division of Biophysics, Priority Research Area Infection, Research Center Borstel Leibniz Lung Center, Borstel, Germany.

The architecture of the lipid matrix of the outer membrane of Gram-negative bacteria is extremely asymmetric: Whereas the inner leaflet is composed of a phospholipid mixture, the outer leaflet is built up by glycolipids. For most Gram-negative species, these glycolipids are lipopolysaccharides (LPS), for a few species, however, glycosphingolipids. We demonstrate experimental approaches for the reconstitution of these asymmetric membranes as (i) solid supported membranes prepared by the Langmuir-Blodgett technique, (ii) planar lipid bilayers prepared by the Montal-Mueller technique, and (iii) giant unilamellar vesicles (GUVs) prepared by the phase transfer method. The asymmetric GUVs (aGUVs) composed of LPS on one leaflet are shown for the first time. They are characterized with respect to their phase behavior, flip-flop of lipids and their usability to investigate the interaction with membrane active peptides or proteins. For the antimicrobial peptide LL-32 and for the bacterial porin OmpF the specificity of the interaction with asymmetric membranes is shown. The three reconstitution systems are compared with respect to their usability to investigate domain formation and interactions with peptides and proteins.
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http://dx.doi.org/10.3389/fcell.2020.00586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7381204PMC
July 2020

Computational Modeling of Ion Transport in Bulk and through a Nanopore Using the Drude Polarizable Force Field.

J Chem Inf Model 2020 06 15;60(6):3188-3203. Epub 2020 Jun 15.

Department of Physics and Earth Sciences, Jacobs University Bremen, 28759 Bremen, Germany.

In the past two decades, molecular dynamics simulations have become the method of choice for elucidating the transport mechanisms of ions through various membrane channels. Often, these simulations heavily rely on classical nonpolarizable force fields (FFs), which lack electronic polarizability in the treatment of the electrostatics. The recent advancements in the Drude polarizable FF lead to a complete set of parameters for water, ions, protein, and lipids, allowing for a more realistic modeling of membrane proteins. However, the quality of these Drude FFs remains untested for such systems. Here, we examine the quality of this FF set in two ways, i.e., (i) in simple ionic aqueous solution simulations and (ii) in more complex membrane channel simulations. First, the aqueous solutions of KCl, NaCl, MgCl, and CaCl salts are simulated using the polarizable Drude and the nonpolarizable CHARMM36 FFs. The bulk conductivity has been estimated for both FF sets using applied-field simulations for several concentrations and temperatures in the case of all investigated salts and compared to experimental findings. An excellent improvement in the ability of the Drude FF to reproduce the experimental bulk conductivities for KCl, NaCl, and MgCl solutions can be observed but not in the case of CaCl. Moreover, the outer membrane channel OmpC from the bacterium has been employed to examine the ability of the polarizable and nonpolarizable FFs to reproduce ion transport-related quantities known from experiment. Unbiased and applied-field simulations have been performed in the presence of KCl using both FF sets. Unlike for the bulk systems of aqueous salt solutions, it has been found that the Drude FF is not accurate in modeling KCl transport properties across the OmpC porin.
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http://dx.doi.org/10.1021/acs.jcim.0c00389DOI Listing
June 2020

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

ACS Infect Dis 2020 07 20;6(7):1855-1865. Epub 2020 May 20.

IOM/CNR, Sezione di Cagliari, Cittadella Universitaria di Monserrato, 09042 Monserrato, Italy.

Despite decades of therapeutic application of aminoglycosides, it is still a matter of debate if porins contribute to the translocation of the antibiotics across the bacterial outer membrane. Here, we quantified the uptake of kanamycin across the major porin channels OmpF and OmpC present in the outer membrane of . Our analysis revealed that, despite its relatively large size, about 10-20 kanamycin molecules per second permeate through OmpF and OmpC under a 10 μM concentration gradient, whereas OmpN does not allow the passage. Molecular simulations elucidate the uptake mechanism of kanamycin through these porins. Whole-cell studies with a defined set of porin mutants provide evidence that translocation of kanamycin via porins is relevant for antibiotic potency. The values are discussed with respect to other antibiotics.
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http://dx.doi.org/10.1021/acsinfecdis.0c00102DOI Listing
July 2020

Rapid lipid bilayer membrane formation on Parylene coated apertures to perform ion channel analyses.

Biomed Microdevices 2020 04 30;22(2):32. Epub 2020 Apr 30.

Institute for Microsensors, -actuators and -systems (IMSAS), University of Bremen, Microsystems Center Bremen (MCB), Bremen, Germany.

We present a chip design allowing rapid and robust lipid bilayer (LBL) membrane formation using a Parylene coated thin silicon nitride aperture. After bilayer formation, single membrane channels can be reconstituted and characterized by electrophysiology. The ability for robust reconstitution will allow parallelization and enhanced screening of small molecule drugs acting on or permeating across the membrane channel. The aperture was realized on a microfabricated silicon nitride membrane by using standard clean-room fabrication processes. To ensure the lipid bilayer formation, the nitride membrane was coated with a hydrophobic and biocompatible Parylene layer. We tested both Parylene-C and Parylene-AF4. The contact angle measurements on both Parylene types showed very good hydrophobic properties and affinity to lipids. No precoating of the Parylene with an organic solvent is needed to make the aperture lipophilic, in contradiction to Teflon membranes. The chips can be easily placed in an array utilizing a 3D printed platform. Experiments show repetitive LBL formation and destruction (more than 6 times) within a very short time (few seconds). Through measurements we have established that the LBL layers are very thin. This allows the investigation of the fusion process of membrane proteins i.e. outer membrane protein (OmpF) in the LBL within a few minutes.
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http://dx.doi.org/10.1007/s10544-020-0473-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192868PMC
April 2020

Electroosmosis Dominates Electrophoresis of Antibiotic Transport Across the Outer Membrane Porin F.

Biophys J 2020 06 19;118(11):2844-2852. Epub 2020 Apr 19.

Department of Life Sciences and Chemistry, Jacobs University, Bremen, Germany. Electronic address:

We report that the dynamics of antibiotic capture and transport across a voltage-biased OmpF nanopore is dominated by the electroosmotic flow rather than the electrophoretic force. By reconstituting an OmpF porin in an artificial lipid bilayer and applying an electric field across it, we are able to elucidate the permeation of molecules and their mechanism of transport. This field gives rise to an electrophoretic force acting directly on a charged substrate but also indirectly via coupling to all other mobile ions, causing an electroosmotic flow. The directionality and magnitude of this flow depends on the selectivity of the channel. Modifying the charge state of three different substrates (norfloxacin, ciprofloxacin, and enoxacin) by varying the pH between 6 and 9 while the charge and selectivity of OmpF is conserved allows us to work under conditions in which electroosmotic flow and electrophoretic forces add or oppose. This configuration allows us to identify and distinguish the contributions of the electroosmotic flow and the electrophoretic force on translocation. Statistical analysis of the resolvable dwell times reveals rich kinetic details regarding the direction and the stochastic movement of antibiotics inside the nanopore. We quantitatively describe the electroosmotic velocity component experienced by the substrates and their diffusion coefficients inside the porin with an estimate of the energy barrier experienced by the molecules caused by the interaction with the channel wall, which slows down the permeation by several orders of magnitude.
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http://dx.doi.org/10.1016/j.bpj.2020.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264812PMC
June 2020

The challenge of intracellular antibiotic accumulation, a function of fluoroquinolone influx versus bacterial efflux.

Commun Biol 2020 04 28;3(1):198. Epub 2020 Apr 28.

UMR_MD1, U-1261, Aix-Marseille University, INSERM, IRBA, MCT, Marseille, France.

With the spreading of antibiotic resistance, the translocation of antibiotics through bacterial envelopes is crucial for their antibacterial activity. In Gram-negative bacteria, the interplay between membrane permeability and drug efflux pumps must be investigated as a whole. Here, we quantified the intracellular accumulation of a series of fluoroquinolones in population and in individual cells of Escherichia coli according to the expression of the AcrB efflux transporter. Computational results supported the accumulation levels measured experimentally and highlighted how fluoroquinolones side chains interact with specific residues of the distal pocket of the AcrB tight monomer during recognition and binding steps.
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http://dx.doi.org/10.1038/s42003-020-0929-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189378PMC
April 2020

Serial femtosecond crystallography on in vivo-grown crystals drives elucidation of mosquitocidal Cyt1Aa bioactivation cascade.

Nat Commun 2020 03 2;11(1):1153. Epub 2020 Mar 2.

Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, Grenoble, F-38000, France.

Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfide-bridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution.
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http://dx.doi.org/10.1038/s41467-020-14894-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052140PMC
March 2020

Structural insights into the main S-layer unit of reveal a massive protein complex with porin-like features.

J Biol Chem 2020 03 18;295(13):4224-4236. Epub 2020 Feb 18.

Department of Plant Physiology, Warsaw University of Life Sciences-SGGW, Nowoursynowska Str. 159, 02776 Warsaw, Poland. Electronic address:

In the extremophile bacterium , the outermost surface layer is tightly connected with the rest of the cell wall. This integrated organization provides a compact structure that shields the bacterium against environmental stresses. The fundamental unit of this surface layer (S-layer) is the S-layer deinoxanthin-binding complex (SDBC), which binds the carotenoid deinoxanthin and provides both, thermostability and UV radiation resistance. However, the structural organization of the SDBC awaits elucidation. Here, we report the isolation of the SDBC with a gentle procedure consisting of lysozyme treatment and solubilization with the nonionic detergent -dodecyl-β-d-maltoside, which preserved both hydrophilic and hydrophobic components of the SDBC and allows the retention of several minor subunits. As observed by low-resolution single-particle analysis, we show that the complex possesses a porin-like structural organization, but is larger than other known porins. We also noted that the main SDBC component, the protein DR_2577, shares regions of similarity with known porins. Moreover, results from electrophysiological assays with membrane-reconstituted SDBC disclosed that it is a nonselective channel that has some peculiar gating properties, but also exhibits behavior typically observed in pore-forming proteins, such as porins and ionic transporters. The functional properties of this system and its porin-like organization provide information critical for understanding ion permeability through the outer cell surface of S-layer-carrying bacterial species.
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http://dx.doi.org/10.1074/jbc.RA119.012174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105295PMC
March 2020

Electrophysiological Characterization of Transport Across Outer-Membrane Channels from Gram-Negative Bacteria in Presence of Lipopolysaccharides.

Angew Chem Int Ed Engl 2020 05 24;59(22):8517-8521. Epub 2020 Mar 24.

Department of Life Sciences and Chemistry, Jacobs University Bremen, 28759, Bremen, Germany.

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we present an approach using fusion of native outer membrane vesicles (OMVs) into a planar lipid bilayer, allowing characterization of membrane protein channels in their native environment. Two major membrane channels from E. coli, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion surprisingly revealed only single or few channel activities. The asymmetry of the OMVs translates after fusion into the lipid membrane with the lipopolysaccharides (LPS) dominantly present at the side of OMV addition. Compared to the conventional reconstitution method, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution and significantly lower permeation. We suggest using outer membrane vesicles for functional and structural studies of membrane channels in the native membrane.
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http://dx.doi.org/10.1002/anie.201913618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317717PMC
May 2020

The Structural Basis for Low Conductance in the Membrane Protein VDAC upon β-NADH Binding and Voltage Gating.

Structure 2020 02 17;28(2):206-214.e4. Epub 2019 Dec 17.

Biozentrum, University of Basel, Basel 4056, Switzerland. Electronic address:

The voltage-dependent anion channel (VDAC) forms the primary diffusion pore of the outer mitochondrial membrane. In its apo form, VDAC adopts an open conformation with high conductance. States of lower conductance can be induced by ligand binding or the application of voltage. Here, we clarify at the atomic level how β-NADH binding leads to a low-conductance state and characterize the role of the VDAC N-terminal helix in voltage gating. A high-resolution NMR structure of human VDAC-1 with bound NADH, combined with molecular dynamics simulation show that β-NADH binding reduces the pore conductance sterically without triggering a structural change. Electrophysiology recordings of crosslinked protein variants and NMR relaxation experiments probing different time scales show that increased helix dynamics is present in the open state and that motions of the N-terminal helices are involved in the VDAC voltage gating mechanism.
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http://dx.doi.org/10.1016/j.str.2019.11.015DOI Listing
February 2020

Porins and small-molecule translocation across the outer membrane of Gram-negative bacteria.

Nat Rev Microbiol 2020 03 2;18(3):164-176. Epub 2019 Dec 2.

UMR-MD1, U-1261, Aix-Marseille Université, INSERM, SSA, IRBA, MCT, Faculté de Pharmacie, Marseille, France.

Gram-negative bacteria and their complex cell envelope, which comprises an outer membrane and an inner membrane, are an important and attractive system for studying the translocation of small molecules across biological membranes. In the outer membrane of Enterobacteriaceae, trimeric porins control the cellular uptake of small molecules, including nutrients and antibacterial agents. The relatively slow porin-mediated passive uptake across the outer membrane and active efflux via efflux pumps in the inner membrane creates a permeability barrier. The synergistic action of outer membrane permeability, efflux pump activities and enzymatic degradation efficiently reduces the intracellular concentrations of small molecules and contributes to the emergence of antibiotic resistance. In this Review, we discuss recent advances in our understanding of the molecular and functional roles of general porins in small-molecule translocation in Enterobacteriaceae and consider the crucial contribution of porins in antibiotic resistance.
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http://dx.doi.org/10.1038/s41579-019-0294-2DOI Listing
March 2020

Manipulation of charge distribution in the arginine and glutamate clusters of the OmpG pore alters sugar specificity and ion selectivity.

Biochim Biophys Acta Biomembr 2019 10 13;1861(10):183021. Epub 2019 Jul 13.

Division of Biochemistry and Applied Protein Center Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, D-91058 Erlangen, Germany; Department Biology, Division of Plant Physiology, Philipps-University Marburg, D-35043 Marburg, Germany. Electronic address:

OmpG is a general diffusion pore in the E. coli outer membrane with a molecular architecture comprising a 14-stranded β-barrel scaffold and unique structural features. In contrast to other non-specific porins, OmpG lacks a central constriction zone and has an exceptionally wide pore diameter of about 13 Å. The equatorial plane of OmpG harbors an annulus of four alternating basic and acidic patches whose function is only poorly characterized. We have investigated the role of charge distribution for ion selectivity and sugar transport with the help of OmpG variants mutated in the annulus. Substituting the glutamate residues of the annulus for histidines or alanines led to a strong reduction in cation selectivity. Replacement of the glutamates in the annulus by histidine residues also disfavored the passage of pentoses and hexoses relative to disaccharides. Our results demonstrate that despite the wide pore diameter, an annulus only consisting of two opposing basic patches confers reduced cation and monosaccharide transport compared to OmpG wild type. Furthermore, randomization of charged residues in the annulus had the potential to abolish pH-dependency of sugar transport. Our results indicate that E, E, R, R and R in the annulus form electrostatic interactions with R, E and D in loop L6 that influence pH-dependency of sugar transport.
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http://dx.doi.org/10.1016/j.bbamem.2019.07.009DOI Listing
October 2019

Outer Membrane Porins.

Subcell Biochem 2019 ;92:79-123

UMR_MD1, Inserm U1261, IRBA, Membranes et Cibles Thérapeutiques, Facultés de Médecine et de Pharmacie, Aix-Marseille Université, Marseille, France.

The transport of small molecules across membranes is essential for the import of nutrients and other energy sources into the cell and, for the export of waste and other potentially harmful byproducts out of the cell. While hydrophobic molecules are permeable to membranes, ions and other small polar molecules require transport via specialized membrane transport proteins . The two major classes of membrane transport proteins are transporters and channels. With our focus here on porins-major class of non-specific diffusion channel proteins , we will highlight some recent structural biology reports and functional assays that have substantially contributed to our understanding of the mechanism that mediates uptake of small molecules, including antibiotics, across the outer membrane of Enterobacteriaceae . We will also review advances in the regulation of porin expression and porin biogenesis and discuss these pathways as new therapeutic targets.
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http://dx.doi.org/10.1007/978-3-030-18768-2_4DOI Listing
July 2019

Identification and Characterization of Approved Drugs and Drug-Like Compounds as Covalent ClpP Inhibitors.

Int J Mol Sci 2019 May 31;20(11). Epub 2019 May 31.

Fraunhofer Institute for Molecular Biology and Applied Ecology IME, ScreeningPort, 22525 Hamburg, Germany.

The serine protease Caseinolytic protease subunit P (ClpP) plays an important role for protein homeostasis in bacteria and contributes to various developmental processes, as well as virulence. Therefore, ClpP is considered as a potential drug target in Gram-positive and Gram-negative bacteria. In this study, we utilized a biochemical assay to screen several small molecule libraries of approved and investigational drugs for ClpP inhibitors. The approved drugs bortezomib, cefmetazole, cisplatin, as well as the investigational drug cDPCP, and the protease inhibitor 3,4-dichloroisocoumarin (3,4-DIC) emerged as ClpP inhibitors with IC values ranging between 0.04 and 31 µM. Compound profiling of the inhibitors revealed cefmetazole and cisplatin not to inhibit the serine protease bovine α-chymotrypsin, and for cefmetazole no cytotoxicity against three human cell lines was detected. Surface plasmon resonance studies demonstrated all novel ClpP inhibitors to bind covalently to ClpP. Investigation of the potential binding mode for cefmetazole using molecular docking suggested a dual covalent binding to Ser97 and Thr168. While only the antibiotic cefmetazole demonstrated an intrinsic antibacterial effect, cDPCP clearly delayed the bacterial growth recovery time upon chemically induced nitric oxide stress in a ClpP-dependent manner.
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http://dx.doi.org/10.3390/ijms20112686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600665PMC
May 2019

Breaching the Barrier: Quantifying Antibiotic Permeability across Gram-negative Bacterial Membranes.

J Mol Biol 2019 08 5;431(18):3531-3546. Epub 2019 Apr 5.

Department of Life Sciences and Chemistry, Jacobs University Bremen, Campus Ring 1, D-28759 Bremen, Germany. Electronic address:

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-negative are intrinsically protected against multiple classes of antibiotic-like molecules, and is likely the main reason that in vitro target-based screening programs 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 summarize the current tools available for measuring antibiotic uptake across the different compartments of Gram-negative bacteria.
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http://dx.doi.org/10.1016/j.jmb.2019.03.031DOI Listing
August 2019

The 2018 correlative microscopy techniques roadmap.

J Phys D Appl Phys 2018 Nov 31;51(44):443001. Epub 2018 Aug 31.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.

Developments in microscopy have been instrumental to progress in the life sciences, and many new techniques have been introduced and led to new discoveries throughout the last century. A wide and diverse range of methodologies is now available, including electron microscopy, atomic force microscopy, magnetic resonance imaging, small-angle x-ray scattering and multiple super-resolution fluorescence techniques, and each of these methods provides valuable read-outs to meet the demands set by the samples under study. Yet, the investigation of cell development requires a multi-parametric approach to address both the structure and spatio-temporal organization of organelles, and also the transduction of chemical signals and forces involved in cell-cell interactions. Although the microscopy technologies for observing each of these characteristics are well developed, none of them can offer read-out of all characteristics simultaneously, which limits the information content of a measurement. For example, while electron microscopy is able to disclose the structural layout of cells and the macromolecular arrangement of proteins, it cannot directly follow dynamics in living cells. The latter can be achieved with fluorescence microscopy which, however, requires labelling and lacks spatial resolution. A remedy is to combine and correlate different readouts from the same specimen, which opens new avenues to understand structure-function relations in biomedical research. At the same time, such correlative approaches pose new challenges concerning sample preparation, instrument stability, region of interest retrieval, and data analysis. Because the field of correlative microscopy is relatively young, the capabilities of the various approaches have yet to be fully explored, and uncertainties remain when considering the best choice of strategy and workflow for the correlative experiment. With this in mind, the Journal of Physics D: Applied Physics presents a special roadmap on the correlative microscopy techniques, giving a comprehensive overview from various leading scientists in this field, via a collection of multiple short viewpoints.
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http://dx.doi.org/10.1088/1361-6463/aad055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372154PMC
November 2018

Small-Molecule Permeation across Membrane Channels: Chemical Modification to Quantify Transport across OmpF.

Angew Chem Int Ed Engl 2019 03 27;58(14):4737-4741. Epub 2019 Feb 27.

Department of Life Sciences and Chemistry, Jacobs University, Campus Ring 1, 28759, Bremen, Germany.

Biological channels facilitate the exchange of molecules across membranes, but general tools to quantify transport are missing. Electrophysiology is the method of choice to study the functional properties of channels. However, analyzing the current fluctuation of channels typically does not identify successful transport, that is, distinguishing translocation from binding. To distinguish both processes, we added an additional barrier at the channel exit acting as a molecular counter. To identify permeation, we compare the molecule residence time in the native channel with one that is chemically modified at the exit. We use the well-studied outer membrane channel from E. coli, OmpF. Position 181, which is below the constriction region, was subsequently mutated into cysteine (E181C) in an otherwise cysteine-free system, then functionalized by covalent binding with one of the two blockers MTSES or GLT. We measured the passage of model peptides, mono-, tri-, hepta-arginine and of norfloxacin, as an example for antibiotic permeation.
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http://dx.doi.org/10.1002/anie.201814489DOI Listing
March 2019

Mechanistic aspects of maltotriose-conjugate translocation to the Gram-negative bacteria cytoplasm.

Life Sci Alliance 2019 02 28;2(1):e201800242. Epub 2018 Dec 28.

Aix Marseille Univ, Institut National de la Santé et de la Recherche Médicale, Service de Santé des Armées, Institut de Recherche Biomédicale des Armées, Membranes et Cibles Thérapeutiques, Marseille, France.

Small molecule accumulation in Gram-negative bacteria is a key challenge to discover novel antibiotics, because of their two membranes and efflux pumps expelling toxic molecules. An approach to overcome this challenge is to hijack uptake pathways so that bacterial transporters shuttle the antibiotic to the cytoplasm. Here, we have characterized maltodextrin-fluorophore conjugates that can pass through both the outer and inner membranes mediated by components of the maltose regulon. Single-channel electrophysiology recording demonstrated that the compounds permeate across the LamB channel leading to accumulation in the periplasm. We have also demonstrated that a maltotriose conjugate distributes into both the periplasm and cytoplasm. In the cytoplasm, the molecule activates the maltose regulon and triggers the expression of maltose binding protein in the periplasmic space indicating that the complete maltose entry pathway is induced. This maltotriose conjugate can (i) reach the periplasmic and cytoplasmic compartments to significant internal concentrations and (ii) auto-induce its own entry pathway the activation of the maltose regulon, representing an interesting prototype to deliver molecules to the cytoplasm of Gram-negative bacteria.
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http://dx.doi.org/10.26508/lsa.201800242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311466PMC
February 2019

Fosfomycin Permeation through the Outer Membrane Porin OmpF.

Biophys J 2019 01 8;116(2):258-269. Epub 2018 Dec 8.

Department of Physics and Earth Sciences, Jacobs University Bremen, Bremen, Germany. Electronic address:

Fosfomycin is a frequently prescribed drug in the treatment of acute urinary tract infections. It enters the bacterial cytoplasm and inhibits the biosynthesis of peptidoglycans by targeting the MurA enzyme. Despite extensive pharmacological studies and clinical use, the permeability of fosfomycin across the bacterial outer membrane is largely unexplored. Here, we investigate the fosfomycin permeability across the outer membrane of Gram-negative bacteria by electrophysiology experiments as well as by all-atom molecular dynamics simulations including free-energy and applied-field techniques. Notably, in an electrophysiological zero-current assay as well as in the molecular simulations, we found that fosfomycin can rapidly permeate the abundant Escherichia coli porin OmpF. Furthermore, two triple mutants in the constriction region of the porin have been investigated. The permeation rates through these mutants are slightly lower than that of the wild type but fosfomycin can still permeate. Altogether, this work unravels molecular details of fosfomycin permeation through the outer membrane porin OmpF of E. coli and moreover provides hints for understanding the translocation of phosphonic acid antibiotics through other outer membrane pores.
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http://dx.doi.org/10.1016/j.bpj.2018.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6350074PMC
January 2019

Effects of H-bonds on sugar binding to chitoporin from Vibrio harveyi.

Biochim Biophys Acta Biomembr 2019 03 19;1861(3):610-618. Epub 2018 Dec 19.

Biochemistry-Electrochemistry Research Unit and School of Chemistry, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC), Payupnai, Wangchan District, Rayong, Thailand. Electronic address:

Background: VhChiP is a sugar-specific-porin present in the outer membrane of the marine bacterium Vibrio harveyi and responsible for chitin uptake, with a high selectivity for chitohexaose.

Methods: VhChiP and its mutants were expressed and purified from BL21 (DE3) Omp8 Rosetta strain. After reconstitution into planar lipid bilayers, the ion current fluctuations caused by chitohexaose entering the channel were measured in deuterium oxide and in water.

Results: The role of hydrogen-bonding in sugar binding was investigated by comparing channel occlusion by chitohexaose in buffers containing HO and DO. The BLM results revealed the significant contribution of hydrogen bonding to the binding of chitohexaose in the constriction zone of VhChiP. Replacing HO as solvent by DO significantly decreased the on- and off-rates of sugar penetration into the channel. The importance of hydrogen bonding inside the channel was more noticeable when the hydrophobicity of the constriction zone was diminished by replacing Trp with the charged residues Asp or Arg. The on- and off-rates decreased up to 2.5-fold and 4-fold when Trp was replaced by Arg, or 5-fold and 3-fold for Trp replacement by Asp, respectively. Measuring the on-rate at different temperatures and for different channel mutants revealed the activation energy for chitohexaose entrance into VhChiP channel.

Conclusions: Hydrogen-bonds contribute to sugar permeation.
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http://dx.doi.org/10.1016/j.bbamem.2018.12.012DOI Listing
March 2019

α-Amino Diphenyl Phosphonates as Novel Inhibitors of Escherichia coli ClpP Protease.

J Med Chem 2019 01 11;62(2):774-797. Epub 2019 Jan 11.

Laboratory of Medicinal Chemistry , University of Antwerp , Universiteitsplein 1 , B-2610 Antwerp , Belgium.

Increased Gram-negative bacteria resistance to antibiotics is becoming a global problem, and new classes of antibiotics with novel mechanisms of action are required. The caseinolytic protease subunit P (ClpP) is a serine protease conserved among bacteria that is considered as an interesting drug target. ClpP function is involved in protein turnover and homeostasis, stress response, and virulence among other processes. The focus of this study was to identify new inhibitors of Escherichia coli ClpP and to understand their mode of action. A focused library of serine protease inhibitors based on diaryl phosphonate warheads was tested for ClpP inhibition, and a chemical exploration around the hit compounds was conducted. Altogether, 14 new potent inhibitors of E. coli ClpP were identified. Compounds 85 and 92 emerged as most interesting compounds from this study due to their potency and, respectively, to its moderate but consistent antibacterial properties as well as the favorable cytotoxicity profile.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01466DOI Listing
January 2019

A Multidisciplinary Approach toward Identification of Antibiotic Scaffolds for Acinetobacter baumannii.

Structure 2019 02 13;27(2):268-280.e6. Epub 2018 Dec 13.

Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK. Electronic address:

Research efforts to discover potential new antibiotics for Gram-negative bacteria suffer from high attrition rates due to the synergistic action of efflux systems and the limited permeability of the outer membrane (OM). One strategy to overcome the OM permeability barrier is to identify small molecules that are natural substrates for abundant OM channels and use such compounds as scaffolds for the design of efficiently permeating antibacterials. Here we present a multidisciplinary approach to identify such potential small-molecule scaffolds. Focusing on the pathogenic bacterium Acinetobacter baumannii, we use OM proteomics to identify DcaP as the most abundant channel during infection in rodents. The X-ray crystal structure of DcaP reveals a trimeric, porin-like structure and suggests that dicarboxylic acids are potential transport substrates. Electrophysiological experiments and all-atom molecular dynamics simulations confirm this notion and provide atomistic information on likely permeation pathways and energy barriers for several small molecules, including a clinically relevant β-lactamase inhibitor.
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http://dx.doi.org/10.1016/j.str.2018.10.021DOI Listing
February 2019

Translocation of small molecules through engineered outer-membrane channels from Gram-negative bacteria.

Eur Phys J E Soft Matter 2018 Sep 24;41(9):111. Epub 2018 Sep 24.

Department of Life Sciences and Chemistry, Jacobs University Bremen, D-28719, Bremen, Germany.

Selective permeability is a key feature of biological membranes. It is controlled by the physico-chemical properties of the lipid bilayer and by channel-forming membrane proteins. Here we focus on the permeation of small molecules across channel-forming proteins in Gram-negative bacteria called porins and present a new approach based on artificial amino acids. We introduced Hco, a fluorescent amino acid with characteristic excitation and emission wavelengths, into OmpF and measured FRET from Hco to dissolved Bocillin FL using solubilized OmpF porins. We examined four variants of OmpF and by doing so, we were able to show that small molecules, like Bocillin FL, are remaining long enough in the porin in order to undergo FRET and produce a reproducible fluorescence signal. This finding opens the way to quantify translocation in the future by the simultaneous detection of resistive pulses and differential fluorescence with FRET as an example.
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http://dx.doi.org/10.1140/epje/i2018-11721-9DOI Listing
September 2018

Getting Drugs through Small Pores: Exploiting the Porins Pathway in Pseudomonas aeruginosa.

ACS Infect Dis 2018 10 8;4(10):1519-1528. Epub 2018 Aug 8.

Department of Physics , University of Cagliari , SP Monserrato-Sestu Km 0.8 , Monserrato , 09042 , Italy.

Understanding molecular properties of outer membrane channels of Gram-negative bacteria is of fundamental significance as they are the entry point of polar antibiotics into bacteria. Outer membrane proteomics revealed OccK8 (OprE) to be among the five most expressed substrate specific channels of the clinically important Pseudomonas aeruginosa. The high-resolution X-ray structure and electrophysiology highlighted a very narrow pore. However, experimental in vitro methods showed the transport of natural amino acids and antibiotics, among them ceftazidime. We used molecular dynamics simulations to reveal the importance of the physicochemical properties of ceftazidime in modulating the translocation through OccK8, proposing a structure-function relationship. As in general porins, the internal electric field favors the translocation of polar molecules by gainful energy compensation in the central constriction region. Importantly, the comparatively narrow OccK8 pore can undergo a substrate-induced expansion to accommodate relatively large-sized substrates.
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http://dx.doi.org/10.1021/acsinfecdis.8b00149DOI Listing
October 2018

Getting Drugs into Gram-Negative Bacteria: Rational Rules for Permeation through General Porins.

ACS Infect Dis 2018 10 17;4(10):1487-1498. Epub 2018 Aug 17.

Department of Physics , University of Cagliari, Cittadella Universitaria di Monserrato , SP Monserrato-Sestu Km 0.8 , Monserrato , 09042 , Italy.

Small, hydrophilic molecules, including most important antibiotics in clinical use, cross the Gram-negative outer membrane through the water-filled channels provided by porins. We have determined the X-ray crystal structures of the principal general porins from three species of Enterobacteriaceae, namely Enterobacter aerogenes, Enterobacter cloacae, and Klebsiella pneumoniae, and determined their antibiotic permeabilities as well as those of the orthologues from Escherichia coli. Starting from the structure of the porins and molecules, we propose a physical mechanism underlying transport and condense it in a computationally efficient scoring function. The scoring function shows good agreement with in vitro penetration data and will enable the screening of virtual databases to identify molecules with optimal permeability through porins and help to guide the optimization of antibiotics with poor permeation.
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http://dx.doi.org/10.1021/acsinfecdis.8b00108DOI Listing
October 2018