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

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

Determination of thermodynamic parameters of Xerocomus chrysenteron lectin interactions with N-acetylgalactosamine and Thomsen-Friedenreich antigen by isothermal titration calorimetry

BACKGROUND: Lectins are carbohydrate-binding proteins which potentially bind to cell surface glycoconjugates. They are found in various organisms including fungi. A lectin from the mushroom Xerocomus chrysenteron (XCL) has been isolated recently. It shows insecticidal activity and has antiproliferative properties. RESULTS: As the monosaccharide binding specificity is an important determinant of lectin function, we determined the affinity of XCL for the galactose moiety. Isothermal titration calorimetry studies revealed a dissociation constant K(d )of 5.2 μM for the XCL:N-acetylgalactosamine interaction at 27degreesC. Higher affinities were observed at lower temperatures and higher osmotic pressures. The dissociation constant was five hundred times higher for the disaccharide beta-D-Gal(1–3)-D-GalNAc, Thomsen-Friedenreich (TF) antigen (Kd of 0.94 μM). By using fetuin and asialofetuin in interaction with the XCL, we revealed its ability to recognize the Thomsen-Friedenreich motif on glycoproteins. CONCLUSION: The XCL antiproliferative effect and the TF antigen specificity presented in this work suggest that XCL and ABL may have similar binding mechanisms. The recent structure determination of these two proteins lead us to analyse these interactions in the light of our thermodynamic data. The understanding of this type of interaction may be a useful tool for the regulation of cell proliferation

Protein encapsulation in liposomes: efficiency depends on interactions between protein and phospholipid bilayer.

BACKGROUND: We investigated the encapsulation mechanism of enzymes into liposomes. The existing protocols to achieve high encapsulation efficiencies are basically optimized for chemically stable molecules. Enzymes, however, are fragile and encapsulation requires in addition the preservation of their functionality. Using acetylcholinesterase as a model, we found that most protocols lead to a rapid denaturation of the enzyme with loss in the functionality and therefore inappropriate for such an application. The most appropriate method is based on lipid film hydration but had a very low efficiency. RESULTS: To improve it and to propose a standard procedure for enzyme encapsulation, we separate each step and we studied the effect of each parameter on encapsulation: lipid and buffer composition and effect of the different physical treatment as freeze-thaw cycle or liposomes extrusion. We found that by increasing the lipid concentration, increasing the number of freeze-thaw cycles and enhancing the interactions of the enzyme with the liposome lipid surface more than 40% of the initial total activity can be encapsulated. CONCLUSION: We propose here an optimized procedure to encapsulate fragile enzymes into liposomes. Optimal encapsulation is achieved by induction of a specific interaction between the enzyme and the lipid surface

Spontaneous lipid vesicle fusion with electropermeabilized cells

AbstractFusion is obtained between electropermeabilized mammalian cells and intact large unilamellar lipid vesicles. This is monitored by a fluorescence assay. Prepulse contact is obtained by Ca2+ when negatively charged lipids are present in the liposomes. The mixing of the liposome content in the cell cytoplasm is observed under conditions preserving cell viability. Electric conditions are such that free liposomes are not affected by the external field. Therefore destabilization of only one of the two membranes of the partners is sufficient for fusion. The comparison between the efficiency of dye delivery for different liposome preparations (multilamellar vesicles, large unilamellar vesicles, small unilamellar vesicles) is indicative that more metastable liposomes are more fusable with electropulsated cells. This observation is discussed within the framework of the recent hypothesis that occurrence of a contact induced electrostatic destabilization of the plasma membrane is a key step in the exocytosis process

Draft genome sequence of Dietzia maris DSM 43672, a gram-positivebacterium of the mycolata group

Here, we report the draft genome sequence of Dietzia maris, known previously as Rhodococcus maris. It is 3,505,372 bp in size with a G+C content of 73%. The draft genome sequence will improve our understanding of Dietzia maris related to other mycolata species and constitutes a basic tool for exploring the cell wall proteins

Liposome retention in size exclusion chromatography

BACKGROUND: Size exclusion chromatography is the method of choice for separating free from liposome-encapsulated molecules. However, if the column is not presaturated with lipids this type of chromatography causes a significant loss of lipid material. To date, the mechanism of lipid retention is poorly understood. It has been speculated that lipid binds to the column material or the entire liposome is entrapped inside the void. RESULTS: Here we show that intact liposomes and their contents are retained in the exclusion gel. Retention depends on the pore size, the smaller the pores, the higher the retention. Retained liposomes are not tightly fixed to the beads and are slowly released from the gels upon direct or inverted eluent flow, long washing steps or column repacking. Further addition of free liposomes leads to the elution of part of the gel-trapped liposomes, showing that the retention is transitory. Trapping reversibility should be related to a mechanism of partitioning of the liposomes between the stationary phase, water-swelled polymeric gel, and the mobile aqueous phase. CONCLUSION: Retention of liposomes by size exclusion gels is a dynamic and reversible process, which should be accounted for to control lipid loss and sample contamination during chromatography

Quantification of Fluoroquinolone Uptake through the Outer Membrane Channel OmpF of Escherichia coli.

Decreased drug accumulation is a common cause of antibiotic resistance in microorganisms. However, there are few reliable general techniques capable of quantifying drug uptake through bacterial membranes. We present a semiquantitative optofluidic assay for studying the uptake of autofluorescent drug molecules in single liposomes. We studied the effect of the Escherichia coli outer membrane channel OmpF on the accumulation of the fluoroquinolone antibiotic, norfloxacin, in proteoliposomes. Measurements were performed at pH 5 and pH 7, corresponding to two different charge states of norfloxacin that bacteria are likely to encounter in the human gastrointestinal tract. At both pH values, the porins significantly enhance drug permeation across the proteoliposome membranes. At pH 5, where norfloxacin permeability across pure phospholipid membranes is low, the porins increase drug permeability by 50-fold on average. We estimate a flux of about 10 norfloxacin molecules per second per OmpF trimer in the presence of a 1 mM concentration gradient of norfloxacin. We also performed single channel electrophysiology measurements and found that the application of transmembrane voltages causes an electric field driven uptake in addition to concentration driven diffusion. We use our results to propose a physical mechanism for the pH mediated change in bacterial susceptibility to fluoroquinolone antibiotics.This work was supported by a European Research Council (ERC) Grant (261101 Passmembrane) to UFK. JC acknowledges support from an Internal Graduate Studentship, Trinity College, Cambridge, and a Research Studentship from the Cambridge Philosophical Society. SP was supported by the Leverhulme Trust through an Early Career Fellowship. TM acknowledges support from the Konrad-Adenauer Foundation and the German National Merit Foundation. HB, YB and MW are part of the TRANSLOCATION consortium and have received support from the Innovative Medicines Joint Undertaking under grant agreement 115525, the European Union’s seventh framework program (FP7/2007-2013), and European Federation of Pharmaceutical Industries and Associates companies in-kind contribution. We thank Avelino Javer for help with the MATLAB scripts and Catalin Chimerel for helpful discussions.This is the author accepted manuscript. The final version is available from ACS via http://dx.doi.org/10.1021/jacs.5b0896

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

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 Escherichia coli 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 IC50 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

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

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 Escherichia coli 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 IC50 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