Interaction of secretory immunoglobulin A antibodies with Naegleria fowleri trophozoites and collagen type I

In this work, we analyzed the in vitro interaction of human secretory immunoglobulin A (sIgA) antibodies with Naegleria fowleri trophozoites and the capacity of these antibodies to inhibit amoeba adherence to collagen type I. We also studied N. fowleri antigens that are recognized by sIgA, using immunoblot assays. Immunocytochemical analysis of the interaction showed a redistribution of antigens on the surface of trophozoites by sIgA antibodies. Ultrastructural analysis of antibody-amoeba interaction showed that besides the patching and cap formation, parasites were capable of eliminating the antigen-antibody complex produced on the surface. sIgA antibodies were capable of inhibiting the in vitro adhesion of trophozoites to collagen type I. We suggest that nonsymptomatic infections by N. fowleri may stimulate a local specific immunity that prevents trophozoite adhesion and invasion of nasal mucosa.     

The measurement of Bacillus mycoides spore adhesion using atomic force microscopy,simple counting methods,and a spinning disk technique

An atomic force microscope has been used to study the adhesion of Bacillus mycoides spores to a hydrophilic glass surface and a hydrophobic-coated glass surface. AFM images of spores attached to the hydrophobic-coated mica surface allowed the measurement of spore dimensions in an aqueous environment without desiccation. The spore exosporium was observed to be flexible and to promote the adhesion of the spore by increasing the area of spore contact with the surface. Results from counting procedures using light microscopy matched the density of spores observed on the hydrophobic-coated glass surface with AFM. However, no spores were observed on the hydrophilic glass surface with AFM, a consequence of the weaker adhesion of the spores at this surface. AFM was also used to quantify directly the interactions of B. mycoides spores at the two surfaces in an aqueous environment. The measurements used "spore probes" constructed by immobilizing a single spore at the apex of a tipless AFM cantilever. The data showed that stretching and sequential bond breaking occurred as the spores were retracted from the hydrophilic glass surface. The greatest spore adhesion was measured at the hydrophobic-coated glass surface. An attractive force on the spores was measured as the spores approached the hydrophobic-coated surface. At the hydrophilic glass surface, only repulsive forces were measured during the approach of the spores. The AFM force measurements were in qualitative agreement with the results of a hydrodynamic shear adhesion assay that used a spinning disk technique. Quantitatively, AFM measurements of adhesive force were up to 4 x 10(3) times larger than the estimates made using the spinning disk data. This is a consequence of the different types of forces applied to the spore in the different adhesion assays. AFM has provided some unique insights into the interactions of spores with surfaces. No other instrument can make such direct measurements for single microbiological cells.     

The distribution of sugar chains on the vomeronasal epithelium observed with an atomic force microscope

The distribution of sugar chains on tissue sections of the rat vomeronasal epithelium, and the adhesive force between the sugar and its specific lectin were examined with an atomic force microscope (AFM). AFM tips were modified with a lectin, Vicia villosa agglutinin, which recognizes terminal N-acetyl-D-galactosamine (GalNAc). When a modified tip scanned the luminal surface of the sensory epithelium, adhesive interactions between the tip and the sample surface were observed. The final rupture force was calculated to be approximately 50 pN based on the spring constant of the AFM cantilever. Distribution patterns of sugar chains obtained from the force mapping image were very similar to those observed using fluorescence-labeled lectin staining. AFM also revealed distribution patterns of sugar chains at a higher resolution than those obtained with fluorescence microscopy. Most of the adhesive interactions disappeared when the scanning solution contained 1 mM GaINAc. The adhesive interactions were restored by removing the sugar from the solution. Findings suggest that the adhesion force observed are related to the binding force between the lectin and the sugars distributed across the vomeronasal epithelium.     

Nanoscale investigation on adhesion of E. coli to surface modified silicone using atomic force microscopy

Bacterial adhesion on biomaterial surfaces is the initial step in establishing infections and leads to the formation of biofilms. In this study, silicone was modified with different biopolymers and silanes, including: heparin, hyaluronan, and self-assembled octadecyltrichlorosilane (OTS), and fluoroalkylsilane (FAS). The aim was to provide a stable and bacteria-resistant surface by varying the degree of hydrophobicity and the surface structure. The adhesion of Escherichia coli (JM 109) on different modified silicone surfaces was investigated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Mica, an ideal hydrophilic and smooth surface, was employed as a control specimen to study the effect of hydrophobicity and surfaces roughness on bacterial adhesion. AFM probes were coated with E. coli and the force measurements between the bacteria-immobilized tip and various materials surfaces were obtained while approaching to and retracting from the surfaces. A short-range repulsive force was observed between the FAS coated silicone and bacteria. The pull-off force of bacteria to FAS was the smallest among coated surfaces. On the other hand, heparin exhibited a long-range attractive force during approach and required a higher pull-off force in retraction. Both AFM and SEM results indicated that FAS reduced bacterial adhesion whereas heparin enhanced the adhesion compared to pure silicone. The work demonstrates that hydrophobicity cannot be used as a criterion to predict bacterial adhesion. Rather, both the native properties of the individual strain of bacteria and the specific functional structure of the surfaces determine the strength of force interaction, and thus the extent of adhesion.     

Predicting the chemical composition and structure of Aspergillus nidulans hyphal wall surface by atomic force microscopy

In fungi, cell wall plays an important role in growth and development. Major macromolecular constituents of the aspergilli cell wall are glucan, chitin, and protein. We examined the chemical composition and structure of the Aspergillus nidulans hyphal wall surface by an atomic force microscope (AFM). To determine the composition of the cell wall surface, the adhesion forces of commercially available β-glucan, chitin, and various proteins were compared to those of corresponding fractions prepared from the hyphal wall. In both setups, the adhesion forces of β-glucan, chitin, and protein were 25–50, 1000–3000, and 125–300 nN, respectively. Adhesion force analysis demonstrated that the cell surface of the apical tip region might contain primarily chitin and β-glucan and relatively a little protein. This analysis also showed the chemical composition of the hyphal surface of the mid-region would be different from that of the apical region. Morphological images obtained by the tapping mode of AFM revealed that the hyphal tip surface has moderate roughness.     

The NTA-His6 bond is strong enough for AFM single-molecular recognition studies

There is a need in current atomic force microscopy (AFM) molecular recognition studies for generic methods for the stable, functional attachment of proteins on tips and solid supports. In the last few years, the site-directed nitrilotriacetic acid (NTA)-polyhistidine (Hisn) system has been increasingly used towards this goal. Yet, a crucial question in this context is whether the NTA-Hisn bond is sufficiently strong for ensuring stable protein immobilization during force spectroscopy measurements. Here, we measured the forces between AFM tips modified with NTA-terminated alkanethiols and solid supports functionalized with His6-Gly-Cys peptides in the presence of Ni2+. The force histogram obtained at a loading rate of 6600 pN s(-1) showed three maxima at rupture forces of 153 +/- 57 pN, 316 +/- 50 pN and 468 +/- 44 pN, that we attribute primarily to monovalent and multivalent interactions between a single His6 moiety and one, two and three NTA groups, respectively. The measured forces are well above the 50-100 pN unbinding forces typically observed by AFM for receptor-ligand pairs. The plot of adhesion force versus log (loading rate) revealed a linear regime, from which we deduced a kinetic off-rate constant of dissociation, k(off) approximately 0.07 s(-1). This value is in the range of that estimated for the multivalent interaction involving two NTA, using fluorescence measurements, and may account for an increased binding stability of the NTA-His6 bond. We conclude that the NTA-His6 system is a powerful, well-suited platform for the stable, oriented immobilization of proteins in AFM single-molecule studies.     

Morphology of modified regenerated model cellulose II surfaces studied by atomic force microscopy: effect of carboxymethylation and heat treatment

Model cellulose II surfaces with different surface charge have been prepared from carboxymethylated wood pulp. AFM tapping-mode imaging in air showed that the introduction of charged groups into the film does not appreciably change the surface morphology. However, after a mild heat treatment (heating at 105 degrees C for 6 h), an irreversible surface structure change, from near spherical-type aggregates to a fibrillar structure, was observed. This might be attributed to the formation of strong hydrogen bonds in the crystalline region of the films while the amorphous regions shrank upon drying. The suitability of these charged cellulose films for surface forces studies was also investigated. At pH below the pK(a) of the carboxyl groups present in the film, the interaction force could be fit by a van der Waals force interaction. At higher pH, the interaction was of a purely electrostatic nature with no van der Waals component observable due to the swelling of the surfaces.     

Simultaneous height and adhesion imaging of antibody-antigen interactions by atomic force microscopy.

Specific molecular recognition events, detected by atomic force microscopy (AFM), so far lack the detailed topographical information that is usually observed in AFM. We have modified our AFM such that, in combination with a recently developed method to measure antibody-antigen recognition on the single molecular level (Hinterdorfer, P., W. Baumgartner, H. J. Gruber, K. Schilcher, and H. Schindler, Proc. Natl. Acad. Sci. USA 93:3477-3481 (1996)), it allows imaging of a submonolayer of intercellular adhesion molecule-1 (ICAM-1) in adhesion mode. We demonstrate that for the first time the resolution of the topographical image in adhesion mode is only limited by tip convolution and thus comparable to tapping mode images. This is demonstrated by imaging of individual ICAM-1 antigens in both the tapping mode and the adhesion mode. The contrast in the adhesion image that was measured simultaneously with the topography is caused by recognition between individual antibody-antigen pairs. By comparing the high-resolution height image with the adhesion image, it is possible to show that specific molecular recognition is highly correlated with topography. The stability of the improved microscope enabled imaging with forces as low as 100 pN and ultrafast scan speed of 22 force curves per second. The analysis of force curves showed that reproducible unbinding events on subsequent scan lines could be measured.     

Kinetic and thermodynamic analyses of adhesion of a peptide,Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), and human formyl peptide receptor (hFPR)

The kinetic and thermodynamic properties of a peptide–receptor interaction was investigated by measuring the adhesion force in the reaction via atomic force microscopy (AFM). Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), considered as a model system in the present study, is a potent neutrophil chemo-attractant. Since being identified as an agonist for formyl peptide receptor (FPR), WKYMVm’s high affinity to FPR has been verified through investigation of its kinetic and physiological behaviors via conventional methods. However, there have been no reports on the adhesion force of WKYMVm-FPR. In this research, we measured the adhesion force of WKYMVm-FPR using AFM. Kinetic parameters obtained from the relationship between the adhesion force and loading rate were used to characterize the thermodynamic properties of WKYMVm-hFPR binding.     

Nanostructure and force spectroscopy analysis of human peripheral blood CD4+ T cells using atomic force microscopy

To date, nanoscale imaging of the morphological changes and adhesion force of CD4⁺ T cells during in vitro activation remains largely unreported. In this study, we used atomic force microscopy (AFM) to study the morphological changes and specific binding forces in resting and activated human peripheral blood CD4⁺ T cells. The AFM images revealed that the volume of activated CD4⁺ T cells increased and the ultrastructure of these cells also became complex. Using a functionalized AFM tip, the strength of the specific binding force of the CD4 antigen-antibody interaction was found to be approximately three times that of the unspecific force. The adhesion forces were not randomly distributed over the surface of a single activated CD4⁺ T cell, indicated that the CD4 molecules concentrated into nanodomains. The magnitude of the adhesion force of the CD4 antigen-antibody interaction did not change markedly with the activation time. Multiple bonds involved in the CD4 antigen-antibody interaction were measured at different activation times. These results suggest that the adhesion force involved in the CD4 antigen-antibody interaction is highly selective and of high affinity.     

Nanomechanical recognition of sphingomyelin-rich membrane domains by atomic force microscopy

Sphingomyelin (SM) is a reservoir of signaling lipids and forms specific lipid domains in biomembranes together with cholesterol. In this study, atomic force microscopy (AFM) and force measurement were applied to investigate the interaction of SM-binding protein toxin, lysenin, with N-palmitoyl-D-erythro-sphingosylphosphorylcholine (palmitoyl sphingomyelin, PSM) bilayer spread over a mica substrate, in an aqueous buffer solution. Lysenin molecules were grafted on a silicon nitride tip for AFM by siloxane-thiol-amide coupling. The bilayers were prepared by the Langmuir-Blodgett (LB)/Langmuir-Schaefer (LS) method. By repeating cycles of tip approach/retraction motion, single-molecular adhesion motions were observed on the force curve, characterized as "fishing curves". The addition of cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) did not alter the peak force but increased the peak extension. Mixtures of PSM/DOPC/cholesterol exhibited 2-dimensional two-phase domain separation. The characteristic fishing curves were observed exclusively in one of the phases, indicating the selective interaction of the lysenin tip to PSM-rich membrane domains. Our results indicate that the AFM tips conjugated with lysenin are useful to detect the surface distribution of SM-rich membrane domains as well as the nanomechanical properties of the domains.     

Interaction force of chitin-binding domains onto chitin surface

Interaction force of chitin-binding domains (ChBD1 and ChBD2) from a thermostable chitinase onto chitin surface was directly measured by atomic force microscopy (AFM) in a buffer solution. In the force curve measurement, multiple pull-off events were observed for the AFM tips functionalized with either ChBD1 or ChBD2, whereas the AFM tips terminated with nitrilotriacetic acid groups without ChBD showed no interaction peak, suggesting that the detected forces are derived from the binding functions of ChBDs onto the chitin surface. The force curve analyses indicate that the binding force of ChBD2 is stronger than that of ChBD1. This result suggests that ChBD1 and ChBD2 play different roles in adsorption onto chitin surface.     

Quantification of bacterial adhesion forces using atomic force microscopy (AFM)

This study demonstrated that atomic force microscopy (AFM) can be used to obtain high-resolution topographical images of bacteria, and to quantify the tip-cell interaction force and the surface elasticity. Results show that the adhesion force between the Si3N4 tip and the bacteria surface was in the range from -3.9 to -4.3 nN. On the other hand, the adhesion forces at the periphery of the cell-substratum contact surface ranged from -5.1 to -5.9 nN and those at the cell-cell interface ranged from -6.5 to -6.8 nN. The two latter forces were considerably greater than the former one, most likely due to the accumulation of extracellular polymer substance (EPS). Results also show that the elasticity varied on the cell surface.     

Observation of living cells using the atomic force microscope.

We used an atomic force microscope (AFM) to image samples immersed in a fluid in order to study the dynamic behavior of the membranes of living cells. AFM images of cultured cells immersed in a buffer were obtained without any preliminary preparation. We observed surface changes and displacements which suggest that the cells were still alive during the measurements. Some membrane details imaged with the AFM have also been observed using a scanning electron microscope and their dynamic behavior has been confirmed by microcinematography. We believe that the AFM will offer new insights into the exploration of dynamic changes affecting cell membranes.     

Heterogeneity in bacterial surface polysaccharides,probed on a single-molecule basis

The heterogeneity in bacterial surface macromolecules was probed by examining individual macromolecules on the surface of Pseudomonas putida KT2442 via single-molecule force spectroscopy (SMFS). Using an atomic force microscope (AFM), the silicon nitride tip was brought into contact with biopolymer molecules on bacterial cells and these macromolecules were stretched. Force-extension measurements on different bacterial cells showed a range of adhesion affinities and polymer lengths. However, substantial heterogeneity was also observed in the force-extension curves on a single bacterium. A given bacterium has biopolymers that range in size from tens to hundreds of nanometers, with adhesion affinities for the AFM tip from nearly zero to greater than 1 nN. A distribution of polymer sizes was confirmed by size-exclusion chromatography. The freely jointed chain (FJC) model for polymer elasticity was applied to individual force-extension curves in order to estimate the contour lengths and segment lengths of the polymer chains. A range of segment lengths was obtained using the FJC model, from 0.154-0.45 nm in water, 0.154-0.32 nm in 0.01 M KCl, and 0.154-0.65 nm in 0.1 M KCl. The modeling confirms that the heterogeneity in biopolymers is more than a matter of differences in molecular weights, since a range of stiffnesses (segment lengths) was also observed. The effect of salt concentration on biopolymer conformation and adhesion was also explored. While the biopolymers were flexible in all solvents, they were slightly more extended in water than in either of the salt solutions (0.01 and 0.1 M KCl). The adhesion of polysaccharides with the AFM tip was not dependent on salt concentration, because the polymers were not highly charged and heterogeneity overwhelmed any trends that could be observed in adhesion with respect to solution ionic strength. These experiments indicate that heterogeneity in biopolymer properties on an individual bacterium and within a population of bacterial cells may be much greater than previously believed and should be incorporated into models of bacterial adhesion.     

Mechanical force analysis of peptide interactions using atomic force microscopy

Some peptides have previously been reported to bind low molecular weight chemicals. One such peptide with the amino acid sequence His-Ala-Ser-Tyr-Ser was selectively screened from a phage library and bound to a cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphine (TMpyP), with a binding constant of 10(5) M(-1) (J. Kawakami, T. Kitano, and N. Sugimoto, Chemical Communications, 1999, pp. 1765-1766). The proposed binding was due to pi-electron stacking from two aromatic amino acids of histidine and tyrosine. In this study, the weak interactions between TMpyP and the peptide were further investigated by force curve analysis using atomic force microscopy (AFM). The mechanical force required to unbind the peptide-porphyrin complex was measured by vertical movement of the AFM tip. Peptide self-assembled monolayers were formed on both a gold-coated mica substrate and a gold-coated AFM tip. The TMpyPs could bind between the two peptide layers when the peptide-immobilized AFM tip contacted the peptide-immobilized substrate in solution containing TMpyP. In the retracting process a force that ruptured the interaction between TMpyPs and peptides was observed. The unbinding force values correlated to the concentration of TMpyP. A detection limit of 100 ng/mL porphyrin was obtained for the force measurement, and was similar to surface plasmon resonance sensor detection limits. Furthermore, we calculated the product of the observed force and the length of the molecular elongation to determine the work required to unbind the complexes. The obtained values of unbinding work were in a reasonable range compared to the binding energy of porphyrin-peptide.     

Discrimination of DNA hybridization using chemical force microscopy.

Atomic force microscopy (AFM) can be used to probe the mechanics of molecular recognition between surfaces. In the application known as "chemical force" microscopy (CFM), a chemically modified AFM tip probes a surface through chemical recognition. When modified with a biological ligand or receptor, the AFM tip can discriminate between its biological binding partner and other molecules on a heterogeneous substrate. The strength of the interaction between the modified tip and the substrate is governed by the molecular affinity. We have used CFM to probe the interactions between short segments of single-strand DNA (oligonucleotides). First, a latex microparticle was modified with the sequence 3'-CAGTTCTACGATGGCAAGTC and epoxied to a standard AFM cantilever. This DNA-modified probe was then used to scan substrates containing the complementary sequence 5'-GTCAAGATGCTACCGTTCAG. These substrates consisted of micron-scale, patterned arrays of one or more distinct oligonucleotides. A strong friction interaction was measured between the modified tip and both elements of surface-bound DNA. Complementary oligonucleotides exhibited a stronger friction than the noncomplementary sequences within the patterned array. The friction force correlated with the measured strength of adhesion (rupture force) for the tip- and array-bound oligonucleotides. This result is consistent with the formation of a greater number of hydrogen bonds for the complementary sequence, suggesting that the friction arises from a sequence-specific interaction (hybridization) of the tip and surface DNA.     

Hyaluronan conformations on surfaces: effect of surface charge and hydrophobicity

Extended, relaxed, condensed, and interacting forms of the polysaccharide hyaluronan have been observed by atomic force microscopy (AFM). The types of images obtained depend on the properties of the surfaces used. We have investigated several different surface conditions for HA imaging, including unmodified mica, mica chemically modified with two different kinds of amino-terminated silanes (3-aminopropyltriethoxysilane and N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride), and highly oriented pyrolytic graphite. We found the degree of HA molecular extension or condensation to be variable, and the number of bound chains per unit area was low, for all of the mica-based surfaces. HA was more easily imaged on graphite, a hydrophobic surface. Chains were frequently observed in high degrees of extension, maintained by favorable interaction with the surface after molecular combing. This observation suggests that the HA macromolecule interacts with graphite through hydrophobic patches along its surface. AFM studies of HA behavior on differing surfaces under well-controlled environmental conditions provides useful insight into the variety of conformations and interactions likely to be found under differing physiological conditions.     

Dengue virus capsid protein binding to hepatic lipid droplets (LD) is potassium ion dependent and is mediated by LD surface proteins

Dengue virus (DENV) affects millions of people, causing more than 20,000 deaths annually. No effective treatment for the disease caused by DENV infection is currently available, partially due to the lack of knowledge on the basic aspects of the viral life cycle, including the molecular basis of the interaction between viral components and cellular compartments. Here, we characterized the properties of the interaction between the DENV capsid (C) protein and hepatic lipid droplets (LDs), which was recently shown to be essential for the virus replication cycle. Zeta potential analysis revealed a negative surface charge of LDs, with an average surface charge of -19 mV. The titration of LDs with C protein led to an increase of the surface charge, which reached a plateau at +13.7 mV, suggesting that the viral protein-LD interaction exposes the protein cationic surface to the aqueous environment. Atomic force microscopy (AFM)-based force spectroscopy measurements were performed by using C protein-functionalized AFM tips. The C protein-LD interaction was found to be strong, with a single (un)binding force of 33.6 pN. This binding was dependent on high intracellular concentrations of potassium ions but not sodium. The inhibition of Na(+)/K(+)-ATPase in DENV-infected cells resulted in the dissociation of C protein from LDs and a 50-fold inhibition of infectious virus production but not of RNA replication, indicating a biological relevance for the potassium-dependent interaction. Limited proteolysis of the LD surface impaired the C protein-LD interaction, and force measurements in the presence of specific antibodies indicated that perilipin 3 (TIP47) is the major DENV C protein ligand on the surface of LDs.     

Functionalization of amino-modified probes for atomic force microscopy

The probes for atomic force microscopy (AFM) functionalized with bovine serum albumin (BSA) were obtained; they can be used for molecular recognition studies. The procedure of modification and functionalization of the AFM probe included three stages. First, amino probes were obtained by modification in vapors of an amino silane derivative. Then, a covalent bond was formed between the surface amino groups of the probe and a homobifunctional aminoreactive crosslinker. Finally, the probe with a covalently attached crosslinker was functionalized with BSA molecules. The AFM probes were characterized by force measurements at different stages of the modification; the adhesion force and the work of adhesion force were determined. The modification process was confirmed by visualization of BSA and supercoiled pGEMEX DNA molecules immobilized on the standard amino mica and on amino mica modified with a crosslinker.