Biophysical characterization of E. coli TolC interaction with the known blocker hexaamminecobalt.

BACKGROUND: The tripartite efflux pump AcrAB-TolC in E. coli is involved in drug resistance by transporting antibiotics out of the cell. The outer membrane protein TolC can be blocked by various cations, including hexaamminecobalt, thereby TolC represents a potential target for reducing antimicrobial resistance as its blockage may improve efficacy of antibiotics. METHODS: We utilized single channel electrophysiology measurements for studying TolC conductance in the absence and presence of the known TolC blocker hexaamminecobalt. Association and dissociation constants of hexaamminecobalt were determined using surface plasmon resonance measurements. Minimum inhibitory concentration (MIC) assays in the absence and presence of antibiotics were carried out for investigating the antibacterial effect of hexaamminecobalt and its potential to reduce MICs. RESULTS: TolC gating in the absence of any ligand is voltage dependent and asymmetric at high applied voltages. Hexaamminecobalt binds to TolC with high affinity and kinetic data revealed fast association and dissociation rates. Despite potent binding to TolC, hexaamminecobalt does not possess an intrinsic antimicrobial activity against E. coli nor does it reduce MIC values of antibiotics erythromycin and fusidic acid. CONCLUSIONS: TolC opening can be effectively blocked by small molecules. More potent channel blockers are needed in order to investigate the eligibility of TolC as drug target. GENERAL SIGNIFICANCE: TolC, a potentially interesting pharmaceutical target can be addressed by small molecules, blocking the channel. Biophysical characterization of the binding processes will support future identification and optimisation of more potent TolC blockers in order to validate TolC as a pharmaceutical target.

Antifungal compounds redirect metabolic pathways in yeasts: metabolites as indicators of modes of action.

AIMS: Metabolic pathways, e.g. biosynthesis of ergosterol or carbohydrate metabolism including respiration, are well-known targets of several fungicides. With our study we wanted to prove that metabolite profiles can be used to classify fungicides according to their mode of action and that concentrations of key metabolites are changed even without detectable reduced growth rates. METHODS AND RESULTS: We exposed the yeasts Candida albicans and Saccharomyces cerevisiae to inhibitors of the electron transport chain and to compounds known to interact with osmotic stress defence pathways. Glycerol and ethanol were chosen as key metabolites of branches of glucose catabolism. Increased glycerol concentrations were observed not only when the osmotic stress response was activated, but also as response to the inhibition of the electron transfer chain, whereas elevated ethanol levels were observed only when the respiratory pathways were blocked. CONCLUSIONS: The treatment of the yeasts Candida albicans and Saccharomyces cerevisiae with antimycotic compounds led to a redirection of metabolic pathways, which could be followed by the quantification of both the metabolites ethanol and glycerol. Only the combination of both concentration profiles allowed the clear distinction between inhibitors of the respiratory chain and effects on the osmotic stress response pathway. IMPACT OF STUDY: The extension of the number of metabolites to a comprehensive quantitative metabolic profile of compound-treated test organisms can be an additional tool in fungicide research allowing the detection of compounds which act on fungi and, moreover, the elucidation of modes of action.

Development of a rapid, quantitative glucosyltransferase assay based on a screen-printed fructose enzyme electrode and application to optimization studies on gtfD expression in recombinant Escherichia coli.

A biosensor for fructose determination was used as basis of an assay for the determination of glucosyltransferase (GTF) activities and applied to monitoring recombinant enzyme production. GTFs catalyze the synthesis of glucans from sucrose leading to the release of fructose. Specific fructose determinations in the microM concentration range were achieved with a fructose electrode based on fructose dehydrogenase, which was immobilized on a screen-printed platinum electrode. This electrode was used as basis of the new assay for GTF activity determinations. Depending on the amount of enzyme, the assay was completed within 15-30 min compared to 1-2 h for the traditional photometric assay. From the amount of fructose released in a given reaction time, GTF activities were determined down to approx. 20 U/L. Even unpurified samples from a recombinant GTF-S production process could be analyzed without any problems, and a good correlation was obtained to data obtained from the photometric assay. Analysis of samples from cultures of various rGTF-S-producing recombinant E. coli strains grown on different media with SDS-PAGE and with the new assay identified the same strain and culture medium as optimum for recombinant GTF-S production.

Optimisation of the composition of a screen-printed acrylate polymer enzyme layer with respect to an improved selectivity and stability of enzyme electrodes.

Glucose oxidase (GOD) was immobilized on screen-printed platinum electrodes by entrapment in a screen printable paste polymerized by irradiation with UV-light. The influences of different additives, in particular polymers and graphite, on the sensitivity and stability of the sensor and the permeability of the enzyme layer for a possible electrochemical interferent were investigated. The chosen additives were Gafquat 755N, poly-L-lysine, bovine serum albumin (BSA), sodium dodecylsulfate (SDS), polyethylene glycol (PEG), Nafion and graphite. All additives led to increases of glucose signals, i.e. improved the sensitivity of glucose detection with Gafquat 755N, poly-L-lysine, SDS and graphite showing the strongest influences with increases by a factor 4, 6.5, 5 and 10, respectively. Ascorbic acid was used as a model interferent showing the influence of the enzyme layer composition on the selectivity of the sensor. The addition of Gafquat 755N or poly-L-lysine led to higher signals not only for glucose, but also for ascorbic acid. SDS addition already reduced the influence of ascorbic acid, which was almost completely eliminated when Nafion (5%) and PEG (10%) were added. A comparable beneficial effect on the selectivity of the sensors was also observed for the addition of 0.5% graphite. Thus, the enzyme electrodes with PEG, Nafion or graphite as additives in the enzyme layer were applied to glucose determinations in food samples and samples obtained from E. coli cultivations.

Optimisation of glass surfaces for optical immunosensors.

The surfaces of glass sensor chips were modified with dextran to generate a layer protecting the sensor surface from unspecific protein binding and also serving as a matrix for covalent protein immobilisation. Dextran was coupled to the glass surface in different concentrations either covalently on amino-functionalised glass chips or via biotin-avidin binding. Unspecific binding of BSA was monitored with the grating coupler system, and was increasingly suppressed with increasing dextran concentrations. Using a solution with 100 mg/ml carboxymethylated dextran decreased the signals to approximately 2% of those obtained at an untreated glass chip. Antibodies were successfully immobilised in the dextran and binding to the corresponding Cy5-labelled antigen was repeatedly monitored using a fluorescence sensor system (total internal reflection fluorescence (TIRF)).

Development of a multichannel fluorescence affinity sensor system

A multichannel fluorometer is proposed for analysis of biochemical reactions. The sensor is based on the luminescence generation in the evanescent field of a totally reflected laser beam. For transduction, multiple reflection elements are used. Multichannel operation is realized, including the possibility of applying different solutions to each channel at the same time. First experimental results, obtained with fluorescein or Cy5 as labels in a model hybridization assay, demonstrate the applicability and allow the detection of 3-10 fmol injected fluorescently labeled oligonucleotide.

Enrichment of hydrophobic proteins via Triton X-114 phase partitioning and hydroxyapatite column chromatography for mass spectrometry.

Membrane proteins are the starting point of several signal transduction pathways. Therefore, the separation and identification of these proteins are of great interest in proteome analysis. However, the specific properties of membrane proteins seriously impede their analysis. We present an effective and highly reproducible method for the two-dimensional separation of extremely hydrophobic proteins and demonstrate the advantages of special preseparation procedures for the identification of proteins which have very similar Mr and p/. Using the example of the integral membrane protein very low density lipoprotein (VLDL) receptor (NCBI Acc. # 1730111) and the soluble heat shock protein (HSP) 90 (NCBI Acc. # 386786) we present the applicability of a phase-separation system with Triton X-114. Using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) of the protein spots after 2-D separation of the hydrophilic and the strongly hydrophobic protein fraction of human endothelial cells (ECV cell line), we were able to distinguish both proteins.

Production and structure elucidation of glycoglycerolipids from a marine sponge-associated microbacterium species.

The bacterium Microbacterium sp., isolated from the sponge Halichondria panicea, produced four unusual cell-associated glycoglycerolipids and one diphosphatidylglycerol when grown on marine broth and on artificial seawater media. The lipids were isolated by chromatography on silica columns and their structures elucidated using a combination of multidimensional NMR and MS techniques. The main compound was 1-O-acyl-3-[alpha-glucopyranosyl-(1-3)-(6-O-acyl-alpha-mannopyranosyl )]glycerol (GGL.2) with 14-methyl-hexadecanoic acid and 12-methyl-tetradecanoic acid positioned at C-6 of the mannose unit and at the glycerol moiety. Glycolipid production was correlated with growth and reached a maximum value of 200 mg/L when grown on artificial seawater medium with 20 g/L glucose. The main compound decreased the surface tension of water down to 33 mN/m and the interfacial tension of the water/n-hexadecane system down to 5 mN/m. In addition to this good surface-active behavior, the main glycoglycerolipid showed antitumor activities.

Electrochemical reduction of flavocytochromes 2B4 and 1A2 and their catalytic activity.

The present study shows that cytochromes P450 2B4 and 1A2 with a covalently attached riboflavin (semisynthetic flavocytochromes RfP450 2B4 and RfP450 1A2) can be reduced electrochemically on rhodium-graphite electrodes at a potential of -500 mV (vs Ag/AgCl). In the presence of substrates such as aminopyrine, aniline, 7-ethoxyresorufin, and 7-pentoxyresorufin, N-demethylation, p-hydroxylation, and O-dealkylation reactions proceeded, as was confirmed by product analysis. Rates of electrocatalytically driven reactions are comparable to those obtained using NAD(P)H as the source of reducing equivalents. These results suggest the practicality of developing flavocytochrome P450s as catalysts for oxidation reactions with different classes of organic substrates.

Development of an automated microbial sensor system.

An automated whole cell biosensor system was developed by integration of immobilized microbial cells in a flow-through system with screen-printed flow-through electrodes as detectors. The detectors used were thick-film Pt-electrodes in a 3-electrode configuration constructed as sandwich flow-through cells with a volume of about 36 microliters polarized at -900 mV. The measuring principle was the determination of oxygen consumption due to the microbial metabolism. Fructose was used as model analyte. The microorganisms were immobilized on cellulose-acetate membranes and integrated into a newly created reaction chamber (membrane reactor). The microbial cells used were Rhodococcus erythropolis and Issatchenkia orientalis known to be suitable for the determination of biological oxygen demand.

Application of screen-printed electrodes as transducers in affinity flow-through sensor systems.

An affinity flow-through sensor system based on a heterogeneous competitive affinity assay for the determination of low molecular weight compounds is described using the examples of biotin and atrazine determination. The binding proteins, either streptavidin or a biotinylated monoclonal antibody, were immobilized on a biotinylated screen-printed electrode, where the competition between the analyte and an analyte-enzyme-conjugate took place. Determination of the bound enzyme was done through the supply of suitable enzyme substrates and electrochemical determination of an enzyme reaction product. In the assays described here, peroxidase was used as enzyme label. As hydrogen peroxide and hydroquinone were used as enzyme substrates, the amount of enzyme retained at the screen-printed graphite electrode was determined amperometrically at a reducing potential of -600 mV vs a screen-printed platinum electrode. The activation of the electrode by biotinylation was done in a batch procedure outside the system, before the electrode was inserted. All following steps of the assay were performed automatically in an unsegmented flow-through system through an appropriate delivery of required reagents. The system was optimized mainly through the determination of biotin. This assay was based on the competition between biotin and biotinylated peroxidase for the binding sites of streptavidin. The method showed a linear range from 0.045 to 2 micrograms/l (r2 = 0.9997, n = 7) with RSD lower than 3.8%. The system was modified further by using a biotinylated monoclonal antibody against atrazine for analyte recognition and performing a competitive assay between atrazine and a triazine-peroxidase-conjugate. The linear range was from 0.01 to 10 micrograms/l, with IC50 = 0.4 microgram/l and RSD lower than 4.6%. The method was also applied to atrazine spiked water samples. Regeneration of the sensor surface was based on removal of streptavidin in both assays.

A solid-phase enzyme linked immunosorbent assay using monoclonal antibodies, for the detection of African swine fever virus antigens and antibodies.

An improved solid-phase enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies was developed to detect an African swine fever virus protein (VP73) in pig samples. The use of monoclonal antibodies against VP73 allowed a sensitive and specific sandwich ELISA. This assay detected a limiting antigen concentration of 0.05 microgram/ml of VP73, lower than the detection limit of 0.6 microgram/ml obtained by using polyclonal antibodies by the same ELISA. The whole virus particle was detected with this method to a limit of 2.3 x 10(2) PFU/ml. At the same time, an indirect ELISA was developed to detect ASFV antibodies. The results also indicate that this method may be a useful technique for epidemiological surveys.

Real-time observation of affinity reactions using grating couplers: determination of the detection limit and calculation of kinetic rate constants.

The use of integrated optical grating couplers for the analysis of bioaffinity reactions in order to calculate kinetic rate constants was investigated. The specificity of the sensor surface was determined by adsorptive or covalent attachment of the specific ligands. As an evanescent field sensor, the specific interaction of the corresponding ligand could be observed in real time and without labels. The detection limit in terms of the molecular weight of the analyte was studied by the specific binding of biotinylated proteins of different molecular weights to avidin-loaded sensors. It was shown that grating coupler sensors allowed detection of compounds of at least 2000 daltons using high-affinity receptors, while the direct sensing of low molecular analytes, such as biotin, could not be significantly achieved. Association rate constants were calculated for the interaction of the different biotinylated proteins to avidin-covered sensors from single binding curves. Due to the strong binding between avidin and biotin, the dissociation of the formed complex could not be observed. Kinetic rate constants and equilibrium constants were determined by studying the interaction of human immunoglobulin with the immobilized receptor, protein G. For the four human immunoglobulin subclasses a high affinity to protein G was determined with affinity constants ranging from 3.3 to 8.4 x 10(8) M-1.

Synthesis of mono- and bifunctional peptide-dextran conjugates for the immobilization of peptide antigens on ELISA plates: properties and application.

Dextran has been used as a carrier molecule for the synthesis of monofunctional peptide-dextran conjugates. The immunodetection of such carrier immobilized peptides on ELISA plates was compared to that of peptides adsorbed directly to immunoplates. The main features observed with peptide-dextran conjugates were as follows: only small amounts of peptide (1-2 mg) were necessary for coupling via alpha- or epsilon-amino groups to NaIO4-activated dextran (4 mg); the coupling yield was up to 68%; an amino acid analysis of the conjugate enabled the amount of carrier immobilized peptide to be calculated; an estimated 15-17 peptides were bound per dextran molecule (MW 73,500); using a carbohydrate as carrier reduces the possibility of non-specific interactions because no hydrophobic or ionic sites and no protein-like epitopes exist on the carrier apart from the peptide ligand. It can be assumed that some peptide ligands provide the forces for an interaction with the plate surface whereas other remain free for the interaction with the antibody. Thus, the detection with monoclonal anti-peptide antibodies allowed peptide-dextran conjugates to be used at coating concentrations of 1-3 nM peptide, corresponding to 0.6-2.6 ng peptide-dextran per well. In contrast, concentrations of 150-500 nM were required for coating with peptides. The applicability of monofunctional peptide-dextran conjugates was demonstrated by investigating the titer and specificity of a polyclonal anti-peptide serum developed against human gastrointestinal glutathione peroxidase. The introduction of biotin as a second ligand of the dextran conjugate permitted its capture on streptavidin coated plates. This synthesis of bifunctional peptide-biotin-dextran conjugates opens up additional possibilities for applications.

Analysis of the biotin-binding protein actinavidin using affinity capillary electrophoresis.

Affinity capillary electrophoresis (ACE) was applied to study the bioaffinity of ligand-receptor interaction between the microbial biotin-binding protein actinavidin and biotin. The ACE method is based on short time incubation of a mixture of actinavidin and increasing concentrations of biotinylated oligonucleotide (bio-ON), which was found to be an effective affinity ligand. Separation of intermediate loading forms of actinavidin from unbound ligand in the presence of micellar phase and by capillary zone electrophoresis enabled the quantitation of free bio-ON, permitting the evaluation of the biotin-binding capacity of actinavidin in absence and presence of sodium dodecyl sulfate (SDS). Although in the latter case actinavidin lost a part of its binding capacity (not more than 12%), it was still possible to develop an indirect, noncompetitive assay for the determination of actinavidin in culture liquid, utilizing the combination of micellar electrokinetic capillary chromatography (MEKC) and ACE. Due to the affinity interaction, actinavidin in the sample decreases the amount of bio-ON added, enabling quantitation of the protein. SDS, which is required in this assay to prevent protein adsorption to the capillary wall, greatly enhances the reproducibility and peak shape. Actinavidin levels determined are in agreement with those obtained by commonly used solid-phase analysis. The limit of detection was about 500 ng/mL. Thus the proposed method was found to be well suited for the evaluation of actinavidin affinity and monitoring of its levels in cultivation process.

Development and evaluation of a dipstick immunoassay format for the determination of atrazine residues on-site.

On the basis of a semi-quantitative dipstick immunoassay (IA) for atrazine with visual detection (Giersch, T., J. Agric. Food Chem., 1993, 41, 1006), a quantitative format suitable as a field assay for the analysis of pesticide residues in water and liquid food samples on-site is described. For antibody immobilization, different membranes and immobilization techniques were investigated. The measuring range for atrazine was 0.3-10 micrograms l-1 using reflectance detection. The total assay time was about 25 min with dipsticks previously coated with antibody. Atrazine-spiked water and liquid food samples were selected for assay evaluation. The samples could be measured directly without the need for any prior enrichment or clean-up steps. A satisfactory agreement was found between the results of the dipstick IA and HPLC or GC measurements of both the original and spiked samples.

Gas phase detection of cocaine by means of immunoanalysis.

Immunoanalytical techniques based on an indirect competitive ELISA to determine cocaine in the gas phase are described. A test-gas generator was developed and evaluated by determining saturation vapour pressures and the sublimation enthalpy of cocaine base. To achieve quantitative recovery of the drug, the saturated air stream was sucked through a retention fluid. For validation of the test gas generator, samples were analysed with a microtitre plate cocaine-ELISA based on monoclonal antibodies. To simplify the sampling and analysis procedures by analysing the retention fluid directly in the sampling vessel, particle-based immunoassay formats were developed. According to the ELISA format, avidin was immobilized on the particles. The application of various solid particles consisting of different materials with a wide range of diameters (0.5-550 microns) to the analysis of cocaine standards and gas samples showed good correlation with the results obtained with the microtitre plate ELISA with an average IC50 value (end-point of the test; concentration at 50% binding) of 9.7 ng ml-1. The particle-based assays showed IC50 values in the range of 5-54 ng ml-1 and signal background ratios ranging from 2 to 11. The application of particle-based assays for direct analysis of cocaine in the sample fluid was successfully performed with glass beads, precoated with avidin and a biotin-cocaine conjugate. Recovery of cocaine from the gas phase depended on the volume of sample fluid and on the geometry of the sample tube.

Microbial biosensor for free fatty acids using an oxygen electrode based on thick film technology.

A microbial biosensor based on thick film technology was developed. The microorganisms, Arthrobacter nicotianae, were immobilized in Ca-alginate directly on the electrode surface. For the stability of the calcium alginate gel the addition of 0.5 mM CaCl2 to the assay buffer was necessary. The respiratory activity of the microorganisms was monitored by oxygen consumption at -600 mV vs. Ag/AgCl reference electrode. The sensor was used in a batch system and was applied to the determination of free fatty acids in milk. Short-chain fatty acids (C4:0-C12:0) were the preferential substrates, with butyric acid being the main substrate. Consequently, the concentration of free short-chain fatty acids was represented as the butyric acid equivalent. The sensor showed linearity over the concentration range 9.5-165.5 microM (correlation coefficient, r = 0.99920). The response time of the sensor was approximately 3 min. No additional dialysis membrane was necessary, which led to a high sensitivity of the sensor and fast response times. Recovery rates of 98-113% were found for butyric acid in milk samples using the sensor without any additional membrane and a sample dilution of 200 by the assay. Two widespread disadvantages of microbial sensors, long response times and long times to return to the baseline signal after use, could be overcome.

Optimization of biosensing using grating couplers: immobilization on tantalum oxide waveguides.

This paper presents a comparative study of immobilization strategies for integrated optical grating couplers using tantalum oxide waveguides. As a model system the affinity reaction between protein G and human IgG was investigated. The receptors were coupled to the waveguide by adsorption, covalent attachment and avidin-biotin bridges after modifying the sensor surface by silanization introducing epoxy and amino groups. In addition, the results obtained by a monolayer coverage of the sensors with the receptors were compared with those using a carboxymethyl-dextran matrix allowing the immobilization of an increased amount of protein. The different coupling procedures were assessed by the system response due to the specific binding of human IgG to the sensing layer and are discussed with respect to their effectiveness, stability and the absence of non-specific binding.