Measurements of conformational changes during adhesion of lipid and protein (polylysine and S-layer) surfaces

The adhesion forces between various surfaces were measured using the "surface forces apparatus" technique. This technique allows for the thickness of surface layers and the adhesion force between them to be directly measured in controlled vapor or liquid environments. Three types of biological surfaces were prepared by depositing various lipid-protein monolayers (with thicknesses ranging from 1 to 4 nm) on the inert, molecularly smooth mica surface: (i) hydrophobic lipid monolayers; (ii) amphiphilic polyelectrolyte surfaces of adsorbed polylysine; and (iii) deposited bacterial S-layer proteins. The adhesion, swelling, and wetting properties of these surfaces was measured as a function of relative humidity and time. Initial adhesion is due mainly to the van der Waals forces arising from nonpolar (hydrophobic) contacts. Following adhesive contact, significant molecular rearrangements can occur which alter their hydrophobic-hydrophilic balance and increase their adhesion with time. Increased adhesion is generally enhanced by (i) increased relative humidity (or degree of hydration); (ii) increased contact time; and (iii) increased rates of separation. The results are likely to be applicable to the adhesion of many other biosurfaces, and show that the hydrophobicity of a lipid or protein surface is not an intrinsic property of that surface but depends on its environment (e.g., on whether it is in aqueous solution or exposed to the atmosphere), and on the relative humidity of the atmosphere. It also depends on whether the surface is in adhesive contact with another surface and-when considering dynamic (nonequilibrium) conditions-on the time and previous history of its interaction with that surface. (c) 1993 John Wiley & Sons, Inc.     

Interactions between pathogenic fungi and human epithelial and endothelial surfaces

The adhesion of fungi to host cells is an important area of study. Knowledge of the molecular mechanisms involved in these interactions can be used to devise methods to interfere with them. Similar to many pathogens, loss of fungal adhesion to epithelial or endothelial cell surfaces results in a marked decrease in virulence when evaluated in both in vivo and in vitro disease models. This review emphasizes literature from the past year and focuses on the molecular mechanisms by which fungi in the genera Candida, Cryptococcus, Sporothrix, Pneumocystis, and Aspergillus adhere to epithelial and/or endothelial host surfaces. The methodologies used to conduct these studies are also discussed.     

Weak rolling adhesion enhances bacterial surface colonization

Bacterial adhesion to and subsequent colonization of surfaces are the first steps toward forming biofilms, which are a major concern for implanted medical devices and in many diseases. It has generally been assumed that strong irreversible adhesion is a necessary step for biofilm formation. However, some bacteria, such as Escherichia coli when binding to mannosylated surfaces via the adhesive protein FimH, adhere weakly in a mode that allows them to roll across the surface. Since single-point mutations or even increased shear stress can switch this FimH-mediated adhesion to a strong stationary mode, the FimH system offers a unique opportunity to investigate the role of the strength of adhesion independently from the many other factors that may affect surface colonization. Here we compare levels of surface colonization by E. coli strains that differ in the strength of adhesion as a result of flow conditions or point mutations in FimH. We show that the weak rolling mode of surface adhesion can allow a more rapid spreading during growth on a surface in the presence of fluid flow. Indeed, an attempt to inhibit the adhesion of strongly adherent bacteria by blocking mannose receptors with a soluble inhibitor actually increased the rate of surface colonization by allowing the bacteria to roll. This work suggests that (i) a physiological advantage to the weak adhesion demonstrated by commensal variants of FimH bacteria may be to allow rapid surface colonization and (ii) antiadhesive therapies intended to prevent biofilm formation can have the unintended effect of enhancing the rate of surface colonization.     

Dynamic study of the transition from hyaluronan- to integrin-mediated adhesion in chondrocytes

Membrane-bound hyaluronan mediates the initial adhesive interactions between many cell types and external surfaces. In RCJ-P chondrocytes, such early contacts are mediated through a thick hyaluronidase-sensitive coat. The early adhesion is followed by integrin-mediated interactions and the formation of stable focal adhesions. During this process, the distance between the cell membrane and the surface is reduced from micrometers to few tens of nanometers. The transition from hyaluronan- to integrin-mediated adhesion was studied on glass surfaces by total internal reflection fluorescence microscopy. Hyaluronan-mediated adhesion precedes focal adhesions formation by 2-10 min. After these initial interactions, the pericellular hyaluronan remains sequestered into discrete pockets between the cell and the surface, which are a few hundreds nanometers thick and a few micrometers wide, and are flanked by focal adhesions. The hyaluronan coat facilitates the nucleation of small paxillin-rich contacts, which later mature into focal adhesions. These dynamic studies demonstrate that pericellular hyaluronan mediates initial cell-surface adhesion, and regulates the formation of focal adhesions.     

N‐isopropylacrylamide‐based thermoresponsive polyelectrolyte multilayer films for human mesenchymal stem cell expansion

Human mesenchymal stem cells (hMSCs) are colony‐forming unit fibroblasts (CFU‐F) derived from adult bone marrow and have significant potential for many cell‐based tissue‐engineering applications. Their therapeutic potential, however, is restricted by their diminishing plasticity as they are expanded in culture. In this study, we used N‐isopropylacrylamide (NIPAM)‐based thermoresponsive polyelectrolyte multilayer (N‐PEMU) films as culture substrates to support hMSC expansion and evaluated their effects on cell properties. The N‐PEMU films were made via layer‐by‐layer adsorption of thermoresponsive monomers copolymerized with charged monomers, positively charged allylamine hydrochloride (PAH), or negatively charged styrene sulfonic acid (PSS) and compared to fetal bovine serum (FBS) coated surfaces. Surface charges were shown to alter the extracellular matrix (ECM) structure and subsequently regulate hMSC responses including adhesion, proliferation, integrin expression, detachment, and colony forming ability. The positively charged thermal responsive surfaces improved cell adhesion and growth in a range comparable to control surfaces while maintaining significantly higher CFU‐F forming ability. Immunostaining and Western blot results indicate that the improved cell adhesion and growth on the positively charged surfaces resulted from the elevated adhesion of ECM proteins such as fibronectin on the positively charge surfaces. These results demonstrate that the layer‐by‐layer approach is an efficient way to form PNIPAM‐based thermal responsive surfaces for hMSC growth and removal without enzymatic treatment. The results also show that surface charge regulates ECM adhesion, which in turn influences not only cell adhesion but also CFU‐forming ability and their multi‐lineage differentiation potential. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Processes of bioadhesion on stainless steel surfaces and cleanability: A review with special reference to the food industry

Biofouling of equipment surfaces in the food industry is due initially to physico-chemical adhesion processes, and subsequently to the proliferation of microbes within an extracellular polymer matrix. Two physico-chemical theories can be applied to predict simple cases of bacterial adhesion. However, these models are limited in their applicability owing to the complexity of bacterial surfaces and the surrounding medium. Various factors that can affect the bacterial adhesion process have been listed, all directly linked to the solid substratum, the suspension liquid or the microorganism. For stainless steel surfaces, it is important to take into account the grade of steel, the type of finish, surface roughness, the cleaning procedures used and the age of the steel. Regarding the suspension fluid within which adhesion takes place, pH, ionic composition and the presence of macromolecules are important variables. In addition, the adhering microorganisms have extremely complex surfaces and many factors must be taken into account when conducting adhesion tests, such as the presence of cell appendages, the method of culture, the contact time between the microorganism and the surface, and exopolymer synthesis. Research on biofilms growing on stainless steel has confirmed results obtained with other materials, regarding resistance to disinfectants, the role of the extracellular matrix and the process by which the biofilm forms. However, it appears that the bactericidal activity of disinfectants on biofilms differs according to the type of surface on which they are growing. The main cleaners and disinfectants used in the food industry are alkaline and acid detergents, peracetic acid, quaternary ammonium chlorides and iodophors. The cleanability and disinfectability of stainless steel surfaces have been compared with those of other materials. According to the published research findings, stainless steel is comparable in its biological cleanability to glass, and significantly better than polymers, aluminium or copper. Moreover, microorganisms in a biofilm developing on a stainless steel surface can be killed with lower concentrations of disinfectant than those on polymer surfaces.     

Surface Physicochemistry and Ionic Strength Affects eDNA’s Role in Bacterial Adhesion to Abiotic Surfaces

Extracellular DNA (eDNA) is an important structural component of biofilms formed by many bacteria, but few reports have focused on its role in initial cell adhesion. The aim of this study was to investigate the role of eDNA in bacterial adhesion to abiotic surfaces, and determine to which extent eDNA-mediated adhesion depends on the physicochemical properties of the surface and surrounding liquid. We investigated eDNA alteration of cell surface hydrophobicity and zeta potential, and subsequently quantified the effect of eDNA on the adhesion of Staphylococcus xylosus to glass surfaces functionalised with different chemistries resulting in variable hydrophobicity and charge. Cell adhesion experiments were carried out at three different ionic strengths. Removal of eDNA from S. xylosus cells by DNase treatment did not alter the zeta potential, but rendered the cells more hydrophilic. DNase treatment impaired adhesion of cells to glass surfaces, but the adhesive properties of S. xylosus were regained within 30 minutes if DNase was not continuously present, implying a continuous release of eDNA in the culture. Removal of eDNA lowered the adhesion of S. xylosus to all surfaces chemistries tested, but not at all ionic strengths. No effect was seen on glass surfaces and carboxyl-functionalised surfaces at high ionic strength, and a reverse effect occurred on amine-functionalised surfaces at low ionic strength. However, eDNA promoted adhesion of cells to hydrophobic surfaces irrespective of the ionic strength. The adhesive properties of eDNA in mediating initial adhesion of S. xylosus is thus highly versatile, but also dependent on the physicochemical properties of the surface and ionic strength of the surrounding medium.     

Activated platelets form protected zones of adhesion on fibrinogen and fibronectin-coated surfaces

Leukocytes form zones of close apposition when they adhere to ligand- coated surfaces. Because plasma proteins are excluded from these contact zones, we have termed them protected zones of adhesion. To determine whether platelets form similar protected zones of adhesion, gel-filtered platelets stimulated with thrombin or ADP were allowed to adhere to fibrinogen- or fibronectin-coated surfaces. The protein- coated surfaces with platelets attached were stained with either fluorochrome-conjugated goat anti-human fibrinogen or anti-human fibronectin antibodies, or with rhodamine-conjugated polyethylene glycol polymers. Fluorescence microscopy revealed that F(ab')2 anti- fibrinogen (100 kD) did not penetrate into the contact zones between stimulated platelets and the underlying fibrinogen-coated surface, while Fab antifibrinogen (50 kD) and 10 kD polyethylene glycol readily penetrated and stained the substrate beneath the platelets. Thrombin- or ADP-stimulated platelets also formed protected zones of adhesion on fibronectin-coated surfaces. F(ab')2 anti-fibronectin and 10 kD polyethylene glycol were excluded from these adhesion zones, indicating that they are much less permeable than those formed by platelets on fibrinogen-coated surfaces. The permeability properties of protected zones of adhesion formed by stimulated platelets on surfaces coated with both fibrinogen and fibronectin were similar to the zones of adhesion formed on fibronectin alone. mAb 7E3, directed against the alpha IIb beta 3 integrin blocked the formation of protected adhesion zones between thrombin-stimulated platelets and fibrinogen or fibronectin coated surfaces. mAb C13 is directed against the alpha 5 beta 1 integrin on platelets. Stimulated platelets treated with this mAb formed protected zones of adhesion on surfaces coated with fibronectin. These protected zones were impermeable to F(ab')2 antifibronectin but were permeable to 10 kD polyethylene glycol. These results show that activated platelets form protected zones of adhesion and that the size of molecules excluded from these zones depends upon the composition of the matrix proteins to which the platelets adhere. They also show that formation of protected zones of adhesion by platelets requires alpha IIb beta 3 integrins while the permeability properties of these zones of adhesion are regulated by both alpha IIb beta 3 and alpha 5 beta 1 integrins.     

Shear stress affects the kinetics of Staphylococcus aureus adhesion to collagen

Staphylococcus aureus is a major human pathogen that has been shown to bind collagen under static conditions. However, many staphylococcal infections are hematogenously acquired and adhesion events may be influenced by shear stress. In this study, we used a dynamic experimental system consisting of a parallel-plate perfusion chamber and phase-contrast video microscope to study the effects of shear stress on the adhesion kinetics of intact S. aureus to collagen surfaces in vitro. The adhesion of S. aureus Phillips to collagen types I, II, and IV was investigated over a physiologically relevant range of wall shear stresses at 37 degrees C. S. aureus PH100, a collagen adhesin-deficient mutant strain, was used as a control strain for the experiments. We found that S. aureus Phillips could adhere to collagens I, II, and IV at wall shear stresses less than 15 dyn/cm(2) and that the kinetics of the adhesion process were wall shear stress-dependent. Similar studies with PH100 demonstrated that these cells are unable to adhere firmly to collagen surfaces. Transient interactions between PH100 and the collagen surfaces were observed at low levels of shear stress suggesting that S. aureus may also interact with collagen by an alternative mechanism that does not lead to firm adhesion.     

Studies on cell adhesion and recognition. I. Extent and specificity of cell adhesion triggered by carbohydrate-reactive proteins (glycosidases and lectins) and by fibronectin

The extent and the specificity of the initial cell attachment induced by various proteins coated on plastic surfaces have been studied with the following results: (a) Cell adhesion on the surfaces coated with sialidase and beta-galactosidase was as strong as on concanavalin A and limulus lectin-coated surfaces and the reactions were strongly inhibited by glycosidase inhibitors or by competitive substrates. The adhesion on sialidase was inhibited by 2-deoxy-2,3-dehydro-N- acetylneuraminic acid and by polysialoganglioside (GT1b) at low concentration (0.05-0.1 mM). The cell adhesion on beta-galactosidase coat was inhibited by 1,4-D-galactonolactone and beta-methylgalactoside but not by alpha-methylgalactoside. Thus, the initiation of cell adhesion on glycosidase surfaces could be mediated through the interactions of the specific binding sites of the enzyme surface with the cell surface substrates under physiological conditions. (b) Cell adhesion on various lectins could be blocked by various competing monosaccharides at the concentrations similar to the inhibitory concentrations for binding of lectins from solution to the cells. (c) Cell adhesion on fibronectin surfaces as well as on gelatin-coated surfaces was equally inhibited by GT1b at relatively high concentrations (0.25-0.5 mM). Lower concentrations of GT1b (0.05-0.1 mM) inhibited the cell adhesion on surfaces of Limulus lectin and sialidase. It is suggested that the cell adhesion mediated by fibronectin is based on yet unknown interactions in contrast to a specific cell adhesion through glycosidases and lectins.     

Kinetics of specific and nonspecific adhesion of red blood cells on glass.

Fixed spherical human red blood cells suspended in 17% sucrose were allowed to adhere on either clean glass surfaces or glass surfaces preincubated with antibodies specific to a certain blood group antigen. The adhesion experiments were performed in an impinging jet apparatus, in which the cells are subjected to stagnation point flow. The objective of this study was to compare the efficiencies of nonspecific and specific (antigen-antibody mediated) adhesion of red blood cells on glass surfaces. The efficiency was defined as the ratio of the experimental adhesion rate to that calculated based on numerical solutions of the mass transfer equation, taking into account hydrodynamic interactions as well as colloidal forces. The efficiency for nonspecific adhesion was nearly unity at flow rates lower than 85 microliter/s (corresponding to a wall shear rate, Gw, of 30 s-1 at a radial distance of 110 microns from the stagnation point). The values of efficiency dropped at higher flow rates, due to an increase in the tangential force. The critical deposition concentration is found to occur at 120-150 mM NaCl, which is consistent with the theoretically predicted values. At low salt concentrations, the experimental values are higher than the theoretical ones. Similar discrepancies have been found in many colloidal systems. Introducing steric repulsion by adsorbing a layer of albumin molecules on the glass completely prevents nonspecific adhesion at flow rates below 60 microliter/s (Gw congruent to 15 s-1). The efficiency of specific adhesion depends both on the concentration of antibody molecules on the surface and the flow rate. Normal red cells adhere more readily through antigen-antibody bonds than fixed cells. Fixed spherical cells have a higher adhesion efficiency than fixed biconcave ones.     

The mucus binding of Bifidobacterium lactis Bb12 is enhanced in the presence of Lactobacillus GG and Lact. delbrueckii subsp. bulgaricus

The ability to adhere to mucosal surfaces is related to many probiotic health effects. In the presence of Lactobacillus GG or Lact. bulgaricus, the adhesion of Bifidobacterium lactis Bb12 to a mucus model was more than doubled. Other tested lactobacilli did not affect the adhesion, nor was the adhesion of the lactobacilli influenced by the bifidobacteria. Co-aggregation between Bif. lactis Bb12 and the tested lactobacilli was insignificant and does not explain the observed effect. The results suggest that combinations of probiotics strains may have synergistic adhesion effects. Such specific strain combinations should also be assessed in clinical studies.     

Friction between cellulose surfaces and effect of xyloglucan adsorption

The forces and friction between cellulose spheres have been measured in the absence and presence of xyloglucan using an atomic force microscope. The forces between cellulose are monotonically repulsive with negligible adhesion after contact is achieved. The friction coefficient is observed to be unusually high in comparison with other nanotribological systems. We have confirmed that xyloglucan adsorbs strongly to cellulose, which results in a much stronger adhesion, which is dependent on the time the surfaces are in contact. Xyloglucan also increases the repulsion on approach of the cellulose surfaces, and the friction is markedly reduced. The apparently incompatible observations of decreased friction in combination with increased adhesion fulfills many of the necessary criteria for a papermaking additive.     

Nanoscale Pulling of Type IV Pili Reveals Their Flexibility and Adhesion to Surfaces over Extended Lengths of the Pili

Type IV pili (T4P) are very thin protein filaments that extend from and retract into bacterial cells, allowing them to interact with and colonize a broad array of chemically diverse surfaces. The physical aspects that allow T4P to mediate adherence to many different surfaces remain unclear. Atomic force microscopy (AFM) nanoscale pulling experiments were used to measure the mechanical properties of T4P of a mutant strain of Pseudomonas aeruginosa PAO1 unable to retract its T4P. After adhering bacteria to the end of an AFM cantilever and approaching surfaces of mica, gold, or polystyrene, we observed adhesion of the T4P to all of the surfaces. Pulling of single and multiple T4P on retraction of the cantilever from the surfaces could be described using the worm-like chain (WLC) model. Distinct peaks in the measured distributions of the best-fit values of the persistence length L_p on two different surfaces provide strong evidence for close-packed bundling of very flexible T4P. In addition, we observed force plateaus indicating that adhesion of the T4P to both hydrophilic and hydrophobic surfaces occurs along extended lengths of the T4P. These data shed new light, to our knowledge, on T4P flexibility and support a low-affinity, high-avidity adhesion mechanism that mediates bacteria-surface interactions.     

Cell surface carbohydrates in cell adhesion.

Carbohydrates are ubiquitous constituents of cell surfaces, and possess many characteristics that make them ideal candidates for recognition molecules. In many systems where cell adhesion plays a critical role, carbohydrate binding proteins have been shown to bind to cell surface carbohydrates and participate in cell-cell interactions. Such systems include fertilization, development, pathogen-host recognition and inflammation. In particular the recent discovery of the LEC-CAMs and their importance in leukocyte biology has refocused attention on lectin-mediated cell adhesion. The LEC-CAMs offer good targets for the development of therapeutics based on carbohydrate structures.     

Effect of antimicrobial residues on early adhesion and biofilm formation by wild-type and benzalkonium chloride-adapted Pseudomonas aeruginosa

Antimicrobial residue deposition can change the physico-chemical properties of bacteria and surfaces and thus promote or impair bacterial adhesion. This study focuses on benzalkonium chloride (BC) deposition on polystyrene (PS) surfaces and the influence of this conditioning film on the physico-chemical properties of PS and on early adhesion and biofilm formation by Pseudomonas aeruginosa wild-type and its laboratory BC-adapted strain. The latter readily acquired the ability to grow in BC, and also exhibited physico-chemical surface changes. The existence of residues on PS surfaces altered their hydrophobicity and favoured adhesion as determined by the free energy and early adhesion characterization. Adapted bacteria revealed a higher ability to adhere to surfaces and to develop biofilms, especially on BC-conditioned surfaces, which thereby could enhance resistance to sanitation attempts. These findings highlight the importance of investigations concerning the antimicrobial deposition effect after cleaning procedures, which may encourage bacterial adhesion, especially of bacteria that have been previously exposed to chemical stresses.     

Bacterial strains isolated from different niches can exhibit different patterns of adhesion to substrata

Various mechanisms have been demonstrated to be operative in bacterial adhesion to surfaces, but whether bacterial adhesion to surfaces can ever be captured in one generally valid mechanism is open to question. Although many papers in the literature make an attempt to generalize their conclusions, the majority of studies of bacterial adhesion comprise only two or fewer strains. Here we demonstrate that three strains isolated from a medical environment have a decreasing affinity for substrata with increasing surface free energy, whereas three strains from a marine environment have an increasing affinity for substrata with increasing surface free energy. Furthermore, adhesion of the marine strains related positively with substratum elasticity, but such a relation was absent in the strains from the medical environment. This study makes it clear that strains isolated from a given niche, whether medical or marine, utilize different mechanisms in adherence, which hampers the development of a generalized theory for bacterial adhesion to surfaces.     

Bacterial Strains Isolated from Different Niches Can Exhibit Different Patterns of Adhesion to Substrata

Various mechanisms have been demonstrated to be operative in bacterial adhesion to surfaces, but whether bacterial adhesion to surfaces can ever be captured in one generally valid mechanism is open to question. Although many papers in the literature make an attempt to generalize their conclusions, the majority of studies of bacterial adhesion comprise only two or fewer strains. Here we demonstrate that three strains isolated from a medical environment have a decreasing affinity for substrata with increasing surface free energy, whereas three strains from a marine environment have an increasing affinity for substrata with increasing surface free energy. Furthermore, adhesion of the marine strains related positively with substratum elasticity, but such a relation was absent in the strains from the medical environment. This study makes it clear that strains isolated from a given niche, whether medical or marine, utilize different mechanisms in adherence, which hampers the development of a generalized theory for bacterial adhesion to surfaces.     

Influence of calcium and other cations on surface adhesion of bacteria and diatoms: A review

Association with a surface is an important aspect of survival for microorganisms in natural and manmade environments/Both bacteria and diatoms are involved in such associations. In many cases, this leads to surface fouling, which often results in surface deterioration and mechanical failure in industrial systems. We now know that microorganisms exploit many strategies to establish associations with surfaces. As in the case of other cellular processes, calcium ions seem to play an important role in adhesion of cells to surfaces. Calcium is involved in non-specific interactions such as neutralization of the electrical double layer between cell and substratum surface as well as specific adhesive interactions that cannot be replaced by other cations. The unique properties of calcium ions promote both specific and non-specific interactions with protein and polysaccha-ride adhesin molecules at the cell surface. As important, but less well understood, calcium ions also influence the way microbial cells interact with different substrata.