Microcalorimetric study on the influence of temperature on bacterial coaggregation

Binding isotherms and heats of interaction have been determined at 15, 25, and 40 degrees C for a coaggregating and a non-coaggregating oral bacterial pair. Heats of interaction were measured upon three consecutive injections of streptococci into an actinomyces suspension using isothermal titration calorimetry. After each injection, the number of streptococci injected remaining free in suspension was quantified microscopically and the degree of binding between the two bacterial strains was established. The coaggregating pair shows positive cooperative binding. The highest cooperativity, at 25 degrees C, correlates with a strong, macroscopically visible coaggregation. The non-coaggregating pair shows low cooperativity and lacks macroscopically visible coaggregation. Interactions between the coaggregating partners seem to be mainly due to specific, enthalpically saturable and favorable binding sites. Even though the enthalpic part of the interaction is saturated, cooperativity increases with consecutive injections, implying that the coaggregation phenomenon is driven by entropy gain. The change in heat capacity (DeltaC(p)) is positive for the non-coaggregating pair from 15-40 degrees C as well as for the coaggregating pair beyond 25 degrees C. At lower temperatures the coaggregating pair causes a negative DeltaC(p). The decrease in heat capacity together with an increase in entropy is considered to be indicative of hydrophobic interactions playing an important role in the formation of large coaggregates as observed for the coaggregating pair at 25 degrees C.     

Structure, Stability, and Activity of Adsorbed Enzymes

A proteolytic enzyme, α-chymotrypsin, and a lipolytic enzyme, cutinase, were adsorbed from aqueous solution onto a hydrophobic Teflon surface and a hydrophilic silica surface. We investigated the influence of adsorption on the structure, the structure thermal stability and the activity of these enzymes. Probing the protein structure by circular dichroism spectroscopy indicates that Teflon promotes the formation of helical structure in α-chymotrypsin, but the reverse effect is found with cutinase. The perturbed protein structures on Teflon are remarkably stable, showing no heat-induced structural transitions up to 100°C, as monitored by differential scanning calorimetry. Contact with the hydrophilic silica surface leads to a loss in the helix content of both proteins. Differential scanning calorimetry points to a heterogeneous population of adsorbed protein molecules with respect to their conformational states. The fraction of the native-like conformation in the adsorbed layer increases with increasing coverage of the silica surface by the proteins. The specific enzymatic activity in the adsorbed state qualitatively correlates with the fraction of proteins in the native-like conformation.     

A modified Poisson-Boltzmann model including charge regulation for the adsorption of ionizable polyelectrolytes to charged interfaces, applied to lysozyme adsorption on silica

The equilibrium adsorption of polyelectrolytes with multiple types of ionizable groups is described using a modified Poisson-Boltzmann equation including charge regulation of both the polymer and the interface. A one-dimensional mean-field model is used in which the electrostatic potential is assumed constant in the lateral direction parallel to the surface. The electrostatic potential and ionization degrees of the different ionizable groups are calculated as function of the distance from the surface after which the electric and chemical contributions to the free energy are obtained. The various interactions between small ions, surface and polyelectrolyte are self-consistently considered in the model, such as the increase in charge of polyelectrolyte and surface upon adsorption as well as the displacement of small ions and the decrease of permittivity. These interactions may lead to complex dependencies of the adsorbed amount of polyelectrolyte on pH, ionic strength, and properties of the polymer (volume, permittivity, number, and type of ionizable groups) and of the surface (number of ionizable groups, pK, Stern capacity). For the adsorption of lysozyme on silica, the model qualitatively describes the gradual increase of adsorbed amount with pH up to a maximum value at pHc, which is below the iso-electric point, as well as the sharp decrease of adsorbed amount beyond pHc. With increasing ionic strength the adsorbed amount decreases (for pH > pHc), and pHc shifts to lower values.     

Path-dependency of the interaction between coaggregating and between non-coaggregating oral bacterial pairs--a thermodynamic approach

Coaggregation, i.e. specific recognition between bacteria from different species, is a well-described phenomenon in the human oral cavity but remains physically poorly understood. With our study we aimed at elucidating some aspects of the mechanism of the coaggregation between the oral bacteria Streptococcus oralis J22 and Actinomyces naeslundii 147, in particular with respect to the driving force for coaggregation and its pathway-dependency. To that end, the macroscopic turbidity of the bacterial suspension, the morphology of the coaggregates, binding isotherms and heats of interaction were compared between the above-mentioned coaggregating bacterial pair and a non-coaggregating pair, Streptococcus sanguis PK1889 and A. naeslundii 147. The coaggregating pair forms large aggregates, which rapidly sediment from the suspension while the non-coaggregating pair forms only very small coaggregates that remain homogeneously suspended. Coaggregation is further characterized by a high affinity between the partner cells that bind to each other in a strong cooperative mode. The interactions between both pairs occur under the release of heat and are thus enthalpically favorable. More heat is released for the coaggregating than for the non-coaggregating pair. Adding the coaggregating bacteria in steps to each other leads to saturation of enthalpically favorable binding sites. This is observed when the streptococcus is added to the actinomyces as well as when the addition is done the other way around. It is concluded that the cooperativity of the coaggregation process is based on an increase of entropy. It is furthermore shown that the density of the coaggregates as well as the heat effect of formation of these coaggregates depend on the number of steps in which the partner cells are added to each other. Adding S. oralis J22 in three steps to A. naeslundii 147 results in the formation of denser coaggregates under the release of less heat, as compared to that of addition in one step. These differences point to a larger entropy increase when in a step-wise mixing the coaggregating bacteria are allowed to form more densely-packed coaggregates.     

Softness of the bacterial cell wall of Streptococcus mitis as probed by microelectrophoresis

Chemical and structural complexity of bacterial cell surfaces complicate accurate quantification of cell surfaces properties. The presence of fibrils, fimbriae or other surface appendages on bacterial cell surfaces largely influence those properties and would therefore play a major function in interfacial phenomena as aggregation and adhesion. The electrophoretic softness and fixed charge density in the polyelectrolyte layer of nine Streptococcus mitis strains, usually carrying long sparsely distributed fibrils, were determined by the soft particle analysis using measured electrophoretic mobilities as a function of the ionic strength. In general, S. mitis cell surfaces are electrophoretically soft (1.0-2.5 nm) with a fixed negative charge density of -1.2 to -4.3 x 10(6) Cm(-3). Further, a comparison with surfaces of other bacterial strains that are reported to be soft indicates that the Ohshima soft layer model does not provide information on the surface morphology causing the softness. The most likely reason is that the electroosmotic flow occurs only in the very outer region of thick extracellular surface layers. Nevertheless, determining the surface softness is essential for proper characterization of the cell surface electrostatics.     

Interaction of bovine serum albumin and human blood plasma with PEO-tethered surfaces: influence of PEO chain length, grafting density, and temperature

Solid surfaces are modified by grafting poly(ethylene oxide), PEO, to influence their interaction with indwelling particles, in particular molecules of bovine serum albumin and human plasma proteins. As a rule, the grafted PEO layers suppress protein adsorption. The suppression is most effective when the PEO layer is in a molecular brush conformation having a reciprocal grafting density (area per grafted PEO chain) less than the dimensions of the protein molecules. Nevertheless, the protein molecules may penetrate the PEO brush to some extent. For a given grafting density, the penetration is facilitated by increasing thickness of the brush. Tenuous brushes of reciprocal grafting densities exceeding the protein molecular dimensions enhance protein adsorption. The results point to a weak attractive interaction between PEO and protein. The protein repellency of a densely PEO-brushed surface is ascribed to a high activation energy for the protein molecules to enter the brush. Varying the temperature between 22 and 38 degrees C does not significantly affect the range of grafting density over which the brush changes from protein-attractive to protein-repellent.     

Adsorption of an endoglucanase from the hyperthermophilic Pyrococcus furiosus on hydrophobic (polystyrene) and hydrophilic (silica) surfaces increases protein heat stability

The interaction of an endoglucanase from the hyperthermophilic microorganism Pyrococcus furiosus with two types of surfaces, that is, hydrophobic polystyrene and hydrophilic silica, was investigated, and the adsorption isotherms were determined. The adsorbed hyperthermostable enzyme did not undergo loss of biological activity. A model was proposed for the mechanism of interaction of the enzyme with the surface based on the shape of the adsorption isotherm, the morphological characteristics of the enzyme, and the thermodynamic parameters of the system. The enzyme was irreversibly immobilized at the solid/liquid interface even at high temperatures, and most interestingly, it acquired further heat stabilization upon adsorption. The denaturation temperature increased from 108 degrees C in solution to 116 degrees C upon adsorption on hydrophilic silica particles. Adsorption on the hydrophobic polystyrene surface even shifted the denaturation temperature to 135 degrees C, the most extreme experimentally determined protein denaturation temperature ever reported. Maintenance of the biological function particularly at high temperatures is important for the development of solid substrate immobilized enzymes for applications in biocatalysis and biotechnology. This also presents an additional stabilization mechanism employed by nature where the extracellular hyperthermostable enzyme remains folded and active at the extreme temperatures of its natural environment by adsorption on the surface of rocks and other materials appearing in the surroundings of the microorganism.     

Complex formation in mixtures of lysozyme-stabilized emulsions and human saliva

In this paper, we studied the interaction between human unstimulated saliva and lysozyme-stabilized oil-in-water emulsions (10 wt/wt% oil phase, 10 mM NaCl, pH 6.7), to reveal the driving force for flocculation of these emulsions. Confocal scanning laser microscopy (CSLM) showed formation of complexes between salivary proteins and lysozyme adsorbed at the oil-water interface and lysozyme in solution as well. To assess the electrostatic nature of the interaction in emulsion/saliva mixtures, laser-diffraction and rheological measurements were conducted in function of the ionic strength by adding NaCl to the mixture in the range between 0 and 168 mM. Increasing the ionic strength reduced the ability of saliva to induce emulsion flocculation as shown by the decreased floc size and the effect on the viscosity. Turbidity experiments with varying pH (3-7) and ionic strength also showed decreased complex formation in mixtures between saliva and lysozyme in solution upon NaCl addition up to 200 mM. Decreasing the pH increased the turbidity, in line with the increase of the positive net charge on the lysozyme molecule. We conclude that electrostatic attraction is the main driving force for complex formation between saliva components and lysozyme adsorbed at the oil droplets and in solution.     

DLVO and steric contributions to bacterial deposition in media of different ionic strengths

The deposition of eight bacterial strains on Teflon and glass in aqueous media with ionic strengths varying between 0.0001 and 1 M was measured and interpreted. Two types of interactions were considered: (1) those described by the DLVO theory, which comprise van der Waals attraction and electrostatic repulsion (bacteria and surfaces are both negatively charged); and (2) steric interactions between the outer cell surface macromolecules and the substrata. As a trend, at low ionic strength (<0.001 M), deposition is inhibited by DLVO-type electrostatic repulsion, but at high ionic strength (≥0.1 M) it is dominated by steric interactions. The ionic strength at which the transition from the DLVO-controlled to the sterically controlled deposition occurs, is determined by the extension of the macromolecules into the surrounding medium, which varied between 5 and 100 nm among the bacterial strains studied. The steric interactions either promote deposition by bridging or inhibit it by steric repulsion. Between Teflon and hydrophobic bacteria, bridging is generally observed. The surface of one bacterial strain contains amphiphilic macromolecules that form bridges with Teflon but induce steric repulsion on glass. The presence of highly polar anionic polysaccharide coatings on the cell impedes attachment on both glass and Teflon. For practice, the general conclusion is that the deposition of most bacteria is: (1) strongly inhibited by DLVO-type electrostatic repulsion in aqueous environments of low ionic strength such as rain water, streams and lakes; (2) controlled by DLVO and/or steric interactions in systems as domestic waste waters and saliva; and (3) determined by steric interactions only in more saline environments as milk, urine, blood and sea water.     

Adsorption of proteins from solution at the solid-liquid interface

The purpose of this article is to present some general principles and rules for the adsorption of proteins from aqueous solution on solid surfaces, emphasizing conformational and reversibility aspects. Special attention is paid to the relation between structural properties of the protein molecule and its adsorption behavior and to the role of small ions in the overall adsorption process. Thermodynamic analysis reveals that, under many conditions, the adsorption is driven by an entropy increase that is (partly) related to changes in the structure of the protein molecules.