Host binding proteins and bacterial adhesion: ecology and binding model

Defining the involvement of specific recognition and (or) adhesion molecules in the precise association formed between cells of an organism during development or between bacteria and specific host tissues has become a focus of extensive research. The possibility that the same molecules responsible for cellular adhesion in the host may also play a major role in determining host-bacterial interactions is now becoming more evident. The following review looks at the interaction of a group of host binding proteins, including lectins, fibronectin, and laminin, with respect to their specific association with bacteria. This information is dealt with both from the perspective of the ecology of the host and its autochthonous and pathogenic bacterial populations, as well as in terms of the difficulties in defining the nature of ligand associations even in the more simplified bacterial-host interaction.     

Nanoscale mapping and functional analysis of individual adhesins on living bacteria

Although much progress has been made in the identification and characterization of adhesins borne by pathogenic bacteria, the molecular details underlying their interaction with host receptors remain largely unknown owing to the lack of appropriate probing techniques. Here we report a method, based on atomic force microscopy (AFM) with tips bearing biologically active molecules, for measuring the specific binding forces of individual adhesins and for mapping their distribution on the surface of living bacteria. First, we determined the adhesion forces between the heparin-binding haemagglutinin adhesin (HBHA) produced by Mycobacterium tuberculosis and heparin, used as a model sulphated glycoconjugate receptor. Both the adhesion frequency and adhesion force increased with contact time, indicating that the HBHA-heparin complex is formed via multiple intermolecular bridges. We then mapped the distribution of single HBHA molecules on the surface of living mycobacteria and found that the adhesin is not randomly distributed over the mycobacterial surface, but concentrated into nanodomains.     

Sorption of lysine by molecularly imprinted carboxyl sorbents

Tuned (molecularly imprinted) and nontuned, with respect to lysine amino acid, carboxylic heteroreticular sorbents based on methacrylic acid and ethylene glycol dimethacrylate, were synthesized. Study of sorption of lysine within wide pH range and ionic strength indicated significant dissimilarities in amino acid sorption by tuned sorbents, which were expressed as an increase in the contribution of nonionic interaction, and resulted in a decrease in the ionic strength effect on the sorption capacity, as well as an increase in amino acid sorption selectivity.     

Subtle differences in dissociation rates of interactions between destabilized human carbonic anhydrase II mutants and immobilized benzenesulfonamide inhibitors probed by a surface plasmon resonance biosensor

The development of commercial biosensors based on surface plasmon resonance has made possible careful characterization of biomolecular interactions. Here, a set of destabilized human carbonic anhydrase II (HCA II) mutants was investigated with respect to their interaction kinetics with two different immobilized benzenesulfonamide inhibitors. Point mutations were located distantly from the active site, and the destabilization energies were up to 23 kJ/mol. The dissociation rate of wild-type HCA II, as determined from the binding to the inhibitor with higher affinity, was 0.019 s(-1). For the mutants, dissociation rates were faster (0.022-0.025 s(-1)), and a correlation between faster dissociation and a high degree of destabilization was observed. We interpreted these results in terms of increased dynamics of the tertiary structures of the mutants. This interpretation was supported by entropy determinations, showing that the entropy of the native structure significantly increased upon destabilization of the protein molecule. Our findings demonstrate the applicability of modern biosensor technology in the study of subtle details in molecular interaction mechanisms, such as the long-range effect of point mutations on interaction kinetics.     

Human diseases as representation of interspecies competition

Different aspects of competition between human and pathogenic microorganisms as well as their role in evolution of the human as biological species and development of its polymorphism were reviewed. Biological consequences of interaction between human and pathogenic microorganisms and also main factors of genetic selection such as tuberculosis, malaria, etc. were characterized. Numerous forms of polymorphism, which determine level of susceptibility and features of clinical course of many infections in humans, were described. It has been concluded that selective pressure of microorganisms results from direct selection due to excessive mortality of infected persons before integration in human genome as well as that immunologic pressure of mass immunization results in changes of pathogenic microorganisms populations leading to emergence of their antigenically distinct variants. New knowledge about competition between human and pathogenic microorganisms requires development of fundamentally new approaches in fight with infectious diseases.     

Atomic Force Microscopy: A Promising Tool for Deciphering the Pathogenic Mechanisms of Fungi in Cystic Fibrosis

During the past decades, atomic force microscopy (AFM) has emerged as a powerful tool in microbiology. Although most of the works concerned bacteria, AFM also permitted major breakthroughs in the understanding of physiology and pathogenic mechanisms of some fungal species associated with cystic fibrosis. Complementary to electron microscopies, AFM offers unprecedented insights to visualize the cell wall architecture and components through three-dimensional imaging with nanometer resolution and to follow their dynamic changes during cell growth and division or following the exposure to drugs and chemicals. Besides imaging, force spectroscopy with piconewton sensitivity provides a direct means to decipher the forces governing cell–cell and cell–substrate interactions, but also to quantify specific and non-specific interactions between cell surface components at the single-molecule level. This nanotool explores new ways for a better understanding of the structures and functions of the cell surface components and therefore may be useful to elucidate the role of these components in the host–pathogen interactions as well as in the complex interplay between bacteria and fungi in the lung microbiome.

     

Interaction forces and morphology of a protein-resistant poly(ethylene glycol) layer

The molecular interactions on a protein-resistant surface coated with low-molecular-weight poly(ethylene glycol) (PEG) copolymer brushes are investigated using the extended surface forces apparatus. The observed interaction force is predominantly repulsive and nearly elastic. The chains are extended with respect to the Flory radius, which is in agreement with qualitative predictions of scaling theory. Comparison with theory allows the determination of relevant quantities such as brush length and adsorbed mass. Based on these results, we propose a molecular model for the adsorbed copolymer morphology. Surface-force isotherms measured at high resolution allow distinctive structural forces to be detected, suggesting the existence of a weak equilibrium network between poly(ethylene glycol) and water--a finding in accordance with the remarkable solution properties of PEG. The occurrence of a fine structure is interpreted as a water-induced restriction of the polymer's conformational space. This restriction is highly relevant for the phenomenon of PEG protein resistance. Protein adsorption requires conformational transitions, both in the protein as well as in the PEG layer, which are energetically and kinetically unfavorable.     

A number of sorbents useful for the sorption of cephalosporin C

The physicochemical and sorption properties of a number of sorbents made in the USSR and abroad were studied with respect to the recovery of cephalosporin C from a model solution. It was shown that the sorption capacity of a sorbent depended on its specific surface. The macroporous styrene divinylbenzene sorbents with a specific surface of more than 500 m2/g had the best sorption properties.     

Spectroscopic characterization of furosemide binding to human carbonic anhydrase II

This study reports the interaction between furosemide and human carbonic anhydrase II (hCA II) using fluorescence, UV-vis and circular dichroism (CD) spectroscopy. Fluorescence data indicated that furosemide quenches the intrinsic fluorescence of the enzyme via a static mechanism and hydrogen bonding and van der Walls interactions play the major role in the drug binding. The binding average distance between furosemide and hCA II was estimated on the basis of the theory of F?rster energy transfer. Decrease of protein surface hydrophobicity was also documented upon furosemide binding. Chemical modification of hCA II using N-bromosuccinimide indicated decrease of the number of accessible tryptophans in the presence of furosemide. CD results suggested the occurance of some alterations in α-helical content as well as tertiary structure of hCA II upon drug binding.     

Mechanism of polyethylene glycol interaction with the molten globule folding intermediate of bovine carbonic anhydrase B.

Polyethylene glycol has been shown to bind to the molten globule intermediate on the bovine carbonic anhydrase B folding pathway. The mechanism of this interaction has been extensively probed. Polyethylene glycol (PEG) binds weakly to the molten globule first intermediate as measured by hydrophobic interaction chromatography, but PEG does not bind to either the native state or the second intermediate. The binding of PEG to the molten globule has been confirmed with both intrinsic fluorescence and fluorescence quenching experiments which indicate a single PEG-binding site on the molten globule. Electron paramagnetic resonance spectroscopic studies with nitroxide-labeled PEG also indicate a single binding site. Additional electron paramagnetic resonance studies with spin-labeled carbonic anhydrase B suggest that a conformational change occurs in the molten globule intermediate after PEG binds to the surface. The formation of a PEG-molten globule complex results in a reduction in self-association of this compact hydrophobic structure. PEG-molten globule complex formation is analogous to the observed interaction between chaperonins and a molten globule intermediate (Martin, J., Langer, T., Boteva, R., Schramel, A., Horwich, A.L., and Hartl, F.U. (1991) Nature 352, 36-42).     

The Major Surface-Associated Saccharides of Klebsiella pneumoniae Contribute to Host Cell Association

Analysing the pathogenic mechanisms of a bacterium requires an understanding of the composition of the bacterial cell surface. The bacterial surface provides the first barrier against innate immune mechanisms as well as mediating attachment to cells/surfaces to resist clearance. We utilised a series of Klebsiella pneumoniae mutants in which the two major polysaccharide layers, capsule and lipopolysaccharide (LPS), were absent or truncated, to investigate the ability of these layers to protect against innate immune mechanisms and to associate with eukaryotic cells. The capsule alone was found to be essential for resistance to complement mediated killing while both capsule and LPS were involved in cell-association, albeit through different mechanisms. The capsule impeded cell-association while the LPS saccharides increased cell-association in a non-specific manner. The electrohydrodynamic characteristics of the strains suggested the differing interaction of each bacterial strain with eukaryotic cells could be partly explained by the charge density displayed by the outermost polysaccharide layer. This highlights the importance of considering not only specific adhesin:ligand interactions commonly studied in adherence assays but also the initial non-specific interactions governed largely by the electrostatic interaction forces.     

A molecular dynamics simulation study of the effect of water–graphene interaction on the properties of confined water

The role of water in determining the structure and stability of biomacromolecules has been well studied. In this work, molecular dynamics simulations have been applied to investigate the effect of surface hydrophobicity on the structure and dynamics of water confined between graphene surfaces. In order to evaluate this effect, we apply various attractive/repulsive water–graphene interaction potentials (hydrophobicity). The properties of confined water are studied by applying a purely repulsive interaction potential between water–graphene (modelled as a repulsive r^?12 potential) and repulsive–attractive forces (modelled as an LJ(12-6) potential). Compared to the case of a purely repulsive graphene–water potential, the inclusion of repulsive–attractive forces leads to formation of sharp peaks for density and the number of hydrogen bonds. Also, it was found that repulsive–attractive graphene–water potential caused slower hydrogen bonds dynamics and restricted the diffusion coefficient of water. Consequently, it was found that hydrogen bond breakage and formation rate with the repulsive r^?12 potential model, will increase compared to the corresponding water confined with the LJ(12-6) potential.     

Preparative protein purification on underivatized silica

This article describes the use of underivatized silica gel as a preparative stationary phase for process purification of proteins. Although silica has been frequently used as a stationary phase backbone matrix, direct adsorption of proteins on underivatized silica has not been widely exploited for industrial applications. In this study an effort was made to fundamentally understand the interaction mechanisms between a protein and silica surface by using several proteins with a wide range of isoelectric points (pIs) and surface hydrophobicity. Interactions in silica were found to be largely dominated by a combination of ionic and hydrophobic forces. Accordingly, a predictive model was derived for describing linear retention of proteins on silica. Finally, a case study is described investigating the role of silica in an industrial purification process. It was found that the integration of the two modes of interaction confers silica with a unique selectivity that can be very effectively utilized in downstream bioprocessing.     

Equilibria for the adsorption of antibiotics onto neutral polymeric sorbents: Experimental and modeling studies

The objective of this work was to study the equilibria for adsorption of three antibiotics (penicillin V, tetracycline, and cephalosporin C) from water onto commercially available neutral polymeric sorbents. The pH was observed to be an important factor in adsorption as our results suggest that the neutral forms of penicillin V and cephalosporin C are preferentially adsorbed onto the neutral sorbents. Also, sorbent surface chemistry was observed to be important for adsorption, as the antibiotics adsorbed more favorably (both in terms of affinities and enthalpies) onto the aromatic sorbent as compared to the aliphatic ester sorbent. In addition to these thermodynamic measurements, molecular modeling studies and Monte Carlo simulations suggest that adsorption onto aromatic sorbents may involve specific interactions between the planar regions of the antibiotic molecules and the phenyl rings of the aromatic sorbent. The interaction energies predicted from Monte Carlo simulations were observed to provide qualitative agreement with experimentally determined adsorption affinities. (c) 1995 John Wiley & Sons, Inc.     

In vitro modeling of cell-scaffold interaction

A simple method of controlling the efficiency of surface ligand-cell receptor interaction has been developed in the course of modeling the specific adhesion of cells on a support with their subsequent proliferation and bone tissue formation, using affinity chromatography on macroporous monolithic sorbents. The biospecific peptide GRGDSP played the role of an active ligand on the support, whereas cells were simulated by polymeric (polystyrene) microparticles with the peptide EDYPVDIYYLMDLSYSMKDD immobilized on their surface. The latter peptide is part of the active site of the integrin molecule responsible for binding the RGD sequence. Thus, the monolithic ultrashort column (CIM® disk) represented a simplified model of the support (structural scaffold) possessing biospecific properties. The parameters of the interaction of affinity partners were quantitatively estimated by frontal analysis involving the construction of adsorption isotherms, followed by their linearization and mathematical processing. The data obtained indicate a high specificity of biological pairing, which is supported by the results of cell culture experiments.     

Carbonic anhydrase activity in Madin Darby canine kidney cells. Evidence for intercalated cell properties

Madin Darby canine kidney (MDCK) renal epithelial cell cultures have been investigated with respect to their potency to express carbonic anhydrase activity using histochemical methods. Acetazolamide inhibitable carbonic anhydrase activity could be detected in the cytoplasmic compartment as well as in the apical membrane of cells when grown on solid culture supports. Cells forming domes in MDCK monolayers exhibit the highest histochemically detectable enzyme activity. The attempt to subculture clonal cell lines from MDCK monolayer cultures resulted in the establishment of 5 clones, slightly different with respect to size and shape of cells and their potency to form domes. Scanning electron microscopy ensured the identification of one clone (1A4), which distinctly differed from the others with respect to the apical membrane architecture. Co-localization of peanut agglutinin and carbonic anhydrase activity at the plasma membrane always revealed a combined occurrence of enzyme reactivity and lectin binding in the apical membrane domain. Both, lectin binding and carbonic anhydrase activity were distinctly more intense in plasma membrane regions equipped with microvilli. From the results it is concluded that MDCK cells in tissue culture retained properties of intercalated cells of the nephron collecting duct segment.     

Helicobacter pylori and autoimmune pancreatitis: role of carbonic anhydrase via molecular mimicry?

Autoimmune pancreatitis is a recently defined nosological entity, which accounts for 4.6-6% of all forms of chronic pancreatitis and is often associated with other autoimmune diseases, particularly Sjogren's syndrome. Possession of the HLA DRB1*0405-DQB1*0401 genotype confers a risk for the development of autoimmune pancreatitis. Autoantibodies against carbonic anhydrase II and lactoferrin are frequently present in affected subjects and are suspected to have a pathogenic role. A link between gastric infection by Helicobacter pylori and autoimmune pancreatitis has been hypothesized. We used in silico protein analysis and search for HLA binding motifs to verify this hypothesis. We found a significant homology between human carbonic anhydrase II and alpha-carbonic anhydrase of Helicobacter pylori, an enzyme which is fundamental for the survival and proliferation of the bacterium in the gastric environment. Moreover, the homologous segments contain the binding motif of the HLA molecule DRB1*0405. Our data strengthen the hypothesis that gastric Helicobacter pylori infection can trigger autoimmune pancreatitis in genetically predisposed subjects.     

Controlled porous glass (CPG) with reactive epoxy groups as support for affinity chromatography I. Optimization of CPG modification and the binding of glucose with modified surface

The selective and complementary interaction between the ligand bound to support surface and isolated substance is the key of the affinity chromatography. This is the reason of the research in the field of certain sorbents. They are obtained by chemical reaction of matrix with proper ligands. It is carried out mainly by using bifunctional substances which make the reaction with the ligand in question possible. This paper deals with the preparation of controlled porosity glass with reactive epoxy groups. In order to obtain such support and avoid application of a proper epoxy silane compound, the double-step reaction using simple agents was employed. The sorbent syntheses were optimized. The prepared sorbents were applied for coupling some carbohydrates and amino acids.     

Effect of hemosorption on the ultrastructure of hepatocytes in toxic liver damage

Extracorporeal perfusion of toxic blood via carbonic sorbents is an effective method for correcting severe disturbances of hemostasis. Ultrastructural alterations in hepatic cells were studied in experimental toxic liver injury before and after hemosorption. It was established that after hemosorption the processes of intracellular regeneration were significantly activated in the liver parenchyma. The number of crysts in the mitochondria increased as did the electronic density of the matrix. At the same time the number of lysosomes rose as well. However, in persistent unresolved cholestasis, destructive alterations in the hepatic tissue progressed despite the performance of hemosorption.