Evaluation of the carbohydrate recognition domain of the bacterial adhesin FimH: Design, synthesis and binding properties of mannoside ligands

Fimbriae are proteinogeneous appendages on the surface of bacteria, which mediate bacterial adhesion to the host cell glycocalyx. The so-called type 1 fimbriae exhibit specificity for alpha-d-mannosides and, therefore, they are assumed to mediate bacterial adhesion via the interaction of a fimbrial lectin and alpha-d-mannosyl residues exposed on the host cell surface. This carbohydrate-specific adhesive protein subunit of type 1 fimbriae has been identified as a protein called FimH. The crystal structure of this lectin is known and, based on this information, the molecular details of the interaction of mannoside ligands and FimH are addressed in this paper. Computer-based docking methods were used to evaluate known ligands as well as to design new ones. Then, a series of new mannosides with extended aglycon was synthesized and tested as inhibitors of type 1 fimbriae-mediated bacterial adhesion in an ELISA. The results obtained were compared to the predictions and findings as delivered by molecular modeling. This study led to an improved understanding of the ligand-receptor interactions under investigation.     

A new approach to decoupling of bacterial adhesion energies measured by AFM into specific and nonspecific components

A new method to decoupling of bacterial interactions measured by atomic force microscopy (AFM) into specific and nonspecific components is proposed. The new method is based on computing the areas under the approach and retraction curves. To test the efficacy of the new method, AFM was used to probe the repulsion and adhesion energies present between Listeria monocytogenes cells cultured at five pH values (5, 6, 7, 8, and 9) and silicon nitride (Si₃N₄). Overall adhesion energy was then decoupled into its specific and nonspecific components using the new method as well as using Poisson statistical approach. Poisson statistical method represents the most commonly used approach to decouple bacterial interactions into their components. For all pH conditions investigated, specific energies dominated the adhesion, and a transition in adhesion and repulsion energies for cells cultured at pH 7 was observed. When compared, the differences in the specific and nonspecific energies obtained using Poisson analysis and the new method were on average 2.2 % and 6.7 %, respectively. The relatively close energies obtained using the two approaches demonstrate the efficacy of the new method as an alternative way to decouple adhesion energies into their specific and nonspecific components.     

Surface thermodynamics and adhesion forces governing bacterial transmission in contact lens related microbial keratitis

Contact lens induced microbial keratitis results from bacterial transmission from one surface to another. We investigated the adhesion forces of Pseudomonas aeruginosa, Staphylococci and Serratia to different contact lenses, lens cases and corneal surfaces using AFM, and applied a Weibull analysis on these adhesion forces to calculate bacterial transmission probabilities from lens case to corneas with a contact lens as an intermediate. Also a new surface thermodynamic parameter was introduced, the interfacial free energy of transmission, which in essence compares the interfacial free energies of bacterial adhesion, calculated from measured contact angles with liquids on the donating and receiving surfaces in the transmission process. Bacterial adhesion forces were generally strongest among all eight strains for the lens case (-6.5 to -12.0 nN) and corneas (-3.5 to -11.5 nN), while contact lenses (-0.6 to -13.1 nN) exerted slightly smaller adhesion forces. Consequently, bacterial transmission from lens case to contact lens yielded a smaller contribution in the final transmission than from contact lens to cornea. Bacterial transmission probabilities as derived from force analyses were higher when the interfacial free energies of transmission were more negative, which is in line with surface thermodynamic principles. Therewith this parameter could provide useful in analyzing other bacterial transmission phenomena between donating and receiving surfaces as well.     

Influence of surface-energy components of Ni-P-TiO2-PTFE nanocomposite coatings on bacterial adhesion

The influence of total surface energy on bacterial adhesion has been investigated intensively with the frequent conclusion that bacterial adhesion is less on low-energy surfaces. However, there are also a number of contrary findings that high-energy surfaces have a smaller biofouling tendency. Recently, it was found that the CQ ratio, which is defined as the ratio of Lifshitz-van der Waals (LW) apolar to electron donor surface-energy components of substrates, has a strong correlation to bacterial adhesion. However, the electron donor surface-energy components of substrates varied over only a very limited range. In this article, a series of Ni-P-TiO(2)-PTFE nanocomposite coatings with wide range of surface-energy components were prepared using an electroless plating technique. The bacterial adhesion and removal on the coatings were evaluated with different bacteria under both static and flow conditions. The experimental results demonstrated that there was a strong correlation between bacterial attachment (or removal) and the CQ ratio. The coatings with the lowest CQ ratio had the lowest bacterial adhesion or the highest bacterial removal, which was explained using the extented DLVO theory.     

Bacterial adhesion to silica sand as related to Gibbs energy variations

Bacterial adhesion to silica sand was related to variations in system Gibbs energy DeltaG(adh). Two typical Gram-positive bacterial strains of Streptococcus mitis and Lactobacillus casei were used as the model bacteria in this research. Impacts of solution chemistry and goethite coating of silica sand on bacterial adhesion were also explored. S. mitis and L. casei had negative DeltaG(adh) with both uncoated and goethite-coated silica sand, demonstrating their adhesion potentials to these substrate. After goethite coating, DeltaG(adh) decreased (negatively increased) for both S. mitis and L. casei. In the presence of rhamnolipid biosurfactant, DeltaG(adh) increased (negatively decreased) in answer to the increase of the rhamnolipid biosurfactant concentration. Bacterial percentage adhesion to silica sand corresponded to DeltaG(adh). This study demonstrated that bacterial adhesion to substrate could be explained in terms of bacterial, substratum and intervening medium physicochemical surface properties, which can be independently determined based on contact angle measurements.     

One‐Pot Multicomponent Synthesis of Glycopolymers through a Combination of Host–Guest Interaction,Thiol‐ene,and Copper‐Catalyzed Click Reaction in Water

There is a common phenomenon that the heterogeneity of natural oligosaccharides contains various sugar units, which can be used to enhance affinity and selectivity toward a specific receptor, so the synthesis of heterogeneous glycopolymers is always an important issue in the glycopolymer field. Herein, this study conducts a one‐pot method to prepare polyrotaxane‐based heteroglycopolymers anchored with different sugar units and fluorescent moieties via the combination of host–guest interaction, thiol‐ene, and copper‐catalyzed click chemistry in water. Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, gel permeation chromatography, X‐ray diffraction, and Ellman's assay test are used in the paper to characterize the compounds. Quartz crystal microbalance‐dissipation (QCD‐D) experiments and bacterial adhesion assay are utilized to study the interactions of polyrotaxane‐based heteroglycopolymers with Con A and Escherichia coli . The results reveal that polyrotaxanes (PRs) with mannose and glucose present better specificity toward Con A and E. coli than PRs with glucose due to synergistic effects.     

Non-carbohydrate inhibitors of the lectin DC-SIGN

The C-type lectin dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is found on the surface of dendritic cells. It can mediate adhesion between dendritic cells and T lymphocytes and facilitate antigen capture and presentation. Many pathogens can exploit DC-SIGN binding for nefarious purposes. For example, DC-SIGN can facilitate the dissemination of viruses, like HIV-1. Alternatively, some microbes (e.g., Mycobacterium tuberculosis) use their ability to interact with DC-SIGN to evade immune detection. The diverse roles attributed to DC-SIGN provide impetus to identify ligands that can be used to explore its different functions. Such compounds also could serve as therapeutic leads. Most of the DC-SIGN ligands studied previously are mannose- or fucose-derived monosaccharides or oligosaccharides with inhibitory constants in the range of 0.1-10 mM. To identify monovalent ligands with more powerful DC-SIGN blocking properties, we devised a high-throughput fluorescence-based competition assay. This assay afforded potent non-carbohydrate, small molecule inhibitors (IC50 values of 1.6-10 microM). These compounds block not only DC-SIGN-carbohydrate interactions but also DC-SIGN-mediated cell adhesion. Thus, we anticipate that these non-carbohydrate inhibitors can be used to illuminate the role of DC-SIGN in pathogenesis and immune function.     

Multiscale dynamics of the cell envelope of Shewanella putrefaciens as a response to pH change

The bacterial surface properties of gram-negative Shewanella putrefaciens were characterized by microbial adhesion to hydrocarbons (MATH), adhesion to polystyrene dishes, and electrophoresis at different values of pH and ionic strength. The bacterial adhesion to these two apolar substrates shows significant variations according to pH and ionic strength. Such behavior could be partly explained by electrostatic repulsions between bacteria and the solid or liquid interface. However, a similar trend was also observed at rather high ionic strength where electrostatic interactions are supposed to be screened. The nanomechanical properties at pH 4 and 10 and at high ionic strength were investigated by using atomic force microscopy (AFM). The indentation curves revealed the presence of a polymeric external layer that swells and softens up with increasing pH. This suggests a concomitant increase of the water permeability and so did of the hydrophilicity of the bacterial surface. Such evolution of the bacterial envelope in response to changes in pH brings new insight to the pH dependence in the bacterial adhesion tests. It especially demonstrates the necessity to consider the hydrophobic/hydrophilic surface properties of bacteria as not univocal for the various experimental conditions investigated.     

Multiscale dynamics of the cell envelope of Shewanella putrefaciens as a response to pH change

The bacterial surface properties of gram-negative Shewanella putrefaciens were characterized by microbial adhesion to hydrocarbons (MATH), adhesion to polystyrene dishes, and electrophoresis at different values of pH and ionic strength. The bacterial adhesion to these two apolar substrates shows significant variations according to pH and ionic strength. Such behavior could be partly explained by electrostatic repulsions between bacteria and the solid or liquid interface. However, a similar trend was also observed at rather high ionic strength where electrostatic interactions are supposed to be screened. The nanomechanical properties at pH 4 and 10 and at high ionic strength were investigated by using atomic force microscopy (AFM). The indentation curves revealed the presence of a polymeric external layer that swells and softens up with increasing pH. This suggests a concomitant increase of the water permeability and so did of the hydrophilicity of the bacterial surface. Such evolution of the bacterial envelope in response to changes in pH brings new insight to the pH dependence in the bacterial adhesion tests. It especially demonstrates the necessity to consider the hydrophobic/hydrophilic surface properties of bacteria as not univocal for the various experimental conditions investigated.     

Microchip electrophoresis of bacteria using lipid-based liquid crystalline nanoparticles

The aggregation and adhesion of bacterial cells is a serious disadvantage for electrophoretic separations of bacteria. In this study, lipid-based liquid crystalline nanoparticles were used as a pseudostationary phase to minimise the bacterial aggregation and adsorption to the inner walls of microchannels. Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and Lactobacillus rhamnosus were selected as analytes and were separated by microchip electrophoresis (MCE) with laser-induced fluorescence (LIF) detection using 4.5 mM tris(hydroxymethyl) aminomethane (TRIS)-4.5 mM boric acid-0.1 mM ethylenediaminetetraacetate (EDTA) (TBE) containing poly(ethylene oxide) (PEO) and lipid-based nanoparticles as the running buffer. The mechanism of lipid-based nanoparticles affecting bacterial adhesion and aggregation was discussed and supported by zeta potential experiments. Under the optimal conditions, the three species of bacteria were identified with patterned peaks. This proposed MCE method using lipid-based nanoparticles as running buffer additives was also used to analyse a real yogurt sample, and valuable bacterial information was obtained by the electropherograms.     

Importance of molecular details in predicting bacterial adhesion to hydrophobic surfaces

Electrostatic and hydrophobic forces are generally recognized as important in bacterial adhesion. Current continuum models for these forces often wrongly predict measurements of bacterial adhesion forces. The hypothesis tested here is that even qualitative guides to bacterial adhesion often require more than continuum information about hydrophobic forces; they require knowledge about molecular details of the bacteria and substrate surface. In this study, four different strains of bacteria were adsorbed to silica surfaces hydrophobized with alkylsilanes. The thickness of the lipopolysaccharide layers varied on the different bacteria, and the lengths of the alkylsilane molecules were varied from experiment to experiment. Bacterial adhesion was assessed using column experiments and atomic force microscopy (AFM) experiments. Results show that hydrophobized surfaces have higher bacterial sticking coefficients and stronger adhesion forces than bare silica surfaces, as expected. However, adhesion decreased as the solution Debye length became longer than the alkylsilane, perhaps since the silane molecules could not "reach" the bacterial surface. Similarly, those bacteria with a long o-antigen layer had decreased adhesion, perhaps since the silane molecules could not reach surface-bound proteins on the bacteria. This study reveals that macroscopic measurements such as contact angle are not able to fully describe bacterial adhesion; rather, additional details such as the molecular length are required to predict adhesion.     

Bacterial interactions with proteins and cells relevant to the development of life-threatening endocarditis studied by use of a quartz-crystal microbalance

Implant-related infections are a major challenge in clinical routine because of severe complications, for example infective endocarditis (IE). The purpose of this study was to investigate the real-time interaction of S. gordonii with proteins and cells important in the development of IE, in a flow system, by use of a quartz-crystal microbalance (QCM). Acoustic sensors were biologically modified by preconditioning with sterile saliva, platelet-poor plasma (PPP), or platelet-rich plasma (PRP), followed then by perfusion of a bacterial suspension. After perfusion, additional fluorescence and scanning electron microscopic (SEM) studies were performed. The surface structure of S. gordonii was analyzed by atomic force microscopy (AFM). Compared with S. gordonii adhesion on the abiotic sensor surface following normal mass loading indicated by a frequency decrease, adhesion on saliva, PPP, or PRP-conditioned sensors resulted in an increase in frequency. Furthermore, adhesion induced slightly increased damping signals for saliva and PPP-coated sensors but a decrease upon bacterial adhesion to PRP, indicating the formation of a more rigid biofilm. Microscopic analysis confirmed the formation of dense and vital bacterial layers and the aggregation of platelets and bacteria. In conclusion, our study shows that the complex patterns of QCM output data observed are strongly dependent on the biological substrate and adhesion mechanisms of S. gordonii. Overall, QCM sheds new light on the pathways of such severe infections as IE.     

Force measurements of bacterial adhesion on metals using a cell probe atomic force microscope

The adhesion of microbial cells to metal surfaces in aqueous media is an important phenomenon in both the natural environment and engineering systems. The adhesion of two anaerobic sulfate-reducing bacteria (Desulfovibrio desulfuricans and a local marine isolate) and an aerobe (Pseudomonas sp.) to four polished metal surfaces (i.e., stainless steel 316, mild steel, aluminum, and copper) was examined using a force spectroscopy technique with an atomic force microscope (AFM). Using a modified bacterial tip, the attraction and repulsion forces (in the nano-Newton range) between the bacterial cell and the metal surface in aqueous media were quantified. Results show that the bacterial adhesion force to aluminum is the highest among the metals investigated, whereas the one to copper is the lowest. The bacterial adhesion forces to metals are influenced by both the electrostatic force and metal surface hydrophobicity. It is also found that the physiological properties of the bacterium, namely the bacterial surface charges and hydrophobicity, also have influence on the bacteria-metal interaction. The adhesion to the metals by Pseudomonas sp. and D. desulfuricans was greater than by the marine SRB isolate. The cell-cell interactions show that there are strong electrostatic repulsion forces between bacterial cells. Cell probe atomic force microscopy has provided some useful insight into the interactions of bacterial cells with the metal surfaces.     

Non-invasive vibrational SFG spectroscopy reveals that bacterial adhesion can alter the conformation of grafted "brush" chains on SAM

Understanding bacterial adhesion on a surface is a crucial step to design new materials with improved properties or to control biofilm formation and eradication. Sum Frequency Generation (SFG) vibrational spectroscopy has been employed to study in situ the conformational response of a self-assembled monolayer (SAM) of octadecanethiol (ODT) on a gold film to the adhesion of hydrophilic and hydrophobic ovococcoid model bacteria. The present work highlights vibrational SFG spectroscopy as a powerful and unique non-invasive biophysical technique to probe and control bacteria interaction with ordered surfaces. Indeed, the SFG vibrational spectral changes reveal different ODT SAM conformations in air and upon exposure to aqueous solution or bacterial adhesion. Furthermore, this effect depends on the bacterial cell surface properties. The SFG spectral modeling demonstrates that hydrophobic bacteria flatten the ODT SAM alkyl chain terminal part, whereas the hydrophilic ones raise this ODT SAM terminal part. Microorganism-induced alteration of grafted chains can thus affect the desired interfacial functionality, a result that should be considered for the design of new reactive materials.     

O‐Mannosylation Affords a Glycopeptide Hydrogel with Inherent Antibacterial Activities against E. coli via Multivalent Interactions between Lectins and Supramolecular Assemblies

Multivalent carbohydrate–lectin interactions play a crucial role in bacterial infection. Biomimicry of multivalent glycosystems represents a major strategy in the repression of bacterial growth. In this study, a new kind of glycopeptide (Naphthyl‐Phe‐Phe‐Ser‐Tyr, NMY) scaffold with mannose modification is designed and synthesized, which is able to perform supramolecular self‐assembly with the assistance of catalytic enzyme, and present multiple mannose ligands on its self‐assembled structure to target mannose‐binding proteins. Relying on multivalent carbohydrate–lectin interactions, the glycopeptide hydrogel is able to bind Escherichia coli (E. coli) in high specificity, and result in bacterial adhesion, membrane disruption and subsequent cell death. In vivo wound healing assays reveal that this glycopeptide hydrogel exhibits considerable potentials for promoting wound healing and preventing E. coli infection in a full‐thickness skin defect mouse model. Therefore, through a specific mannose–lectin interaction, a biocompatible hydrogel with inherent antibacterial activity against E. coli is achieved without the need to resort to antibiotic or antimicrobial agent treatment, highlighting the potential role of sugar‐coated nanomaterials in wound healing and control of bacterial pathogenesis.     

Recombinant single chain antibodies in bioelectrochemical sensors

Recombinant antibodies provide an emerging strategy in the development of new immunosensors. In particular, single chain antibodies (scFvs) can be isolated and expressed in bacterial systems that also allow their in vitro manipulation at the gene level. In this work, we present for the first time results of single-chain phage displayed antibodies combined with amperometric detection and its application as an immunosensor. The scFv is immobilized on a carbon electrode and used to capture and quantify its specific target antigen. We describe the detection of the sugar milk lactose, the bacteria Listeria monocytogenes, and the enzyme MtKatG, which is expressed by Mycobacteriumtuberculosis.     

Novel synthesis of oligosaccharides linked with carbamate and urea bonds utilizing modified Curtius rearrangement

We describe a novel synthesis of various carbamate- and urea-linked disaccharides stereospecifically using sugar carboxylic acids and sugar alcohols or sugar amines by the modified Curtius rearrangement. In this reaction, the reactivity of each hydroxyl group in glucose as an acceptor has been disclosed. Furthermore, we applied this method to the synthesis of carbamate-linked oligosaccharides including a dendritic molecule.     

Role of solution chemistry and ion valence on the adhesion kinetics of groundwater and marine bacteria

The role of solution chemistry on bacterial adhesion has been investigated using a radial stagnation point flow (RSPF) system. This experimental system utilized an optical microscope and an image-capturing device to directly observe the deposition kinetics of a groundwater bacterium, Burkholderia cepacia G4g, and a marine bacterium, Halomonas pacifica g. Experiments were carried out under well-controlled hydrodynamic and solution chemistry conditions, allowing for the sensitivity of bacterial adhesion behavior to be examined under a range of ionic strength and valence (KCl vs CaCl2) simulating groundwater and marine environments. Complimentary cell characterization techniques were conducted to evaluate the electrophoretic mobility, hydrophobicity, surface charge density, and viability of the bacteria under the same range of conditions. Solution chemistry was found to have a marked effect on the electrokinetic and surface properties of bacteria and the quartz collector, as well as on the resulting rate of bacterial deposition. Comparable adhesion trends were observed for B. cepacia G4g and H. pacifica g. Specifically, the deposition rates of the two bacteria species in both KCl and CaCl2 solutions increased with ionic strength, a trend consistent with traditional Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, which considers the combination of van der Waals and electrostatic double-layer interaction forces. However, in some cases, experimental results showed bacterial deposition behavior to deviate from DLVO predictions. On the basis of the systematic investigation of bacterial cell characteristics, it was found that Ca2+ ions play a distinct role on bacterial surface charge, hydrophobicity, and deposition behaviors. It is further suggested that bacterial adhesion is determined by the combined influence of DLVO interactions, electrosteric interactions associated with solution chemistry, and the hydrodynamics of the deposition system.     

Kinetic adhesion of bacterial cells to sand: cell surface properties and adhesion rate

Correlation between microbial surface thermodynamics using the extended DLVO (XDLVO) theory and kinetic adhesion of various bacterial cells to sand was investigated. Two experimental setups were utilized. Adhesion tests were conducted in batch reactors with slow agitation. Also, bacteria were circulated through small sand columns in a closed loop and the results were analyzed with a simple model which accounted for the rate of the adhesion phenomena (omega in h(-1)) and adhesion percentage. Cells surface properties were derived from contact angle measurements. The wicking method was utilized to characterize the sand. Zeta potentials were measured for the sand and the cells. Kinetic of bacterial retention by the porous media was largely influenced by the electrostatic interactions which are correlated with omega from the model (R(2)=0.71). Negative zeta potentials resulted in electrostatic repulsions occurring between the sand and the bacterial cells which in result delayed bacterial adhesion. While no correlation was found between the adhesion percentage and the total interaction energy calculated with the XDLVO theory the respective behavior of hydrophobic and hydrophilic bacteria as well as the importance of electrostatic interactions was evidenced. All the bacterial strains studied adhered more in the column experiments than in the adhesion tests, presumably due to enhanced collision efficiency and wedging in porous media, while filtration could be ignored except for the larger Bacillus strains. Approximate XDLVO calculations due to solid surface nanoscale roughness, retention in a secondary minimum and population heterogeneity are discussed. Our results obtained with a large variety of different physicochemical bacterial strains highlights the influence of both surface thermodynamics and porous media related effects as well as the limits of using the XDLVO theory for evaluating bacterial retention through porous media.     

Synthesis and reactivity profiles of phosphinated poly(alkyl aryl ether) dendrimers

Poly(alkyl aryl ether) dendrimers were utilized to synthesize a series of new triphenylphosphine functionalized dendrimers. Zero, first, second and third generation dendrimers, carrying 3, 6, 12 and 24 triphenylphosphine units, were prepared and characterized. The new triphenylphosphine containing dendrimers were assessed for their reactivity profiles and in this instance, the dendrimers were used as reagents to mediate Mitsunobu etherification reaction between phenol and various primary, secondary and benzylic alcohols. In addition, dendritic poly-phenols were also tested in an O-benzylation reaction. A monomeric methoxy group attached triphenylphosphine acted as a control for comparison of reactivity profiles of dendrimers. It was observed that the etherification reaction was mediated efficiently by the dendritic reagent, and in addition, the dendritic phosphine oxide reagents could be recovered quantitatively by precipitation methods. The recovered dendritic phosphine oxides were reduced subsequently to the corresponding phosphines and used as reagents for the Mitsunobu reaction, repetitively.