Molecular Recognition Forces between Immunoglobulin G and a

Guelph, Ontario, Canada N1G 2W1. ReceiVed October 2, 2006. In Final Form: ... Excellence (AFMnet-NCE), University of Guelph. § Department of Molecula...
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Langmuir 2007, 23, 2755-2760

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Molecular Recognition Forces between Immunoglobulin G and a Surface Protein Adhesin on Living Staphylococcus aureus Ahmed Touhami,† Manfred H. Jericho,*,†,‡ and Terry J. Beveridge‡,§ Department of Physics and Atmospheric Science, Dalhousie UniVersity, Halifax NoVa Scotia, Canada B3H 3J5, AdVanced Food and Materials Network - Networks of Centres of Excellence (AFMnet-NCE) and Department of Molecular & Cellular Biology, College of Biological Science, UniVersity of Guelph, Guelph, Ontario, Canada N1G 2W1 ReceiVed October 2, 2006. In Final Form: December 4, 2006 We report AFM measurements of binding events between immunoglobulin G (IgG) and protein A (PA) on the surface of live Staphylococcus aureus bacteria. The experiments were carried out with IgG molecules tethered via CM-amylose linkers to thiol SAMs on gold-coated AFM tips. For comparison, a model system consisting of protein A molecules tethered to thiol SAMs on gold-coated silicon substrates was also investigated. Histograms of binding forces for the PA-IgG bond showed comparable rupture forces of 59 and 64 pN for the model system and live bacteria, respectively. We suggest that linker molecules with a length comparable to the AFM tip radius should make it possible to detect specific binding events on the surface of live bacteria with a lateral resolution of a few tens of nanometers. Furthermore, because S. aureus is an important human pathogen, especially methicillin-resistant strains (MRSA), it is possible that additional virulence factors beyond PA can be probed using this technique.

1. Introduction Surface proteins on bacteria, such as Staphylococcus aureus, perform a variety of functions, from facilitating the secretion of molecules to the expression of virulence. One of the important functions of these proteins is to interact with the surrounding environment and often to adhere to a substrate. This adherence can be to host tissues, other bacteria (to form flocs), and specific immune system components.1-3 S. aureus has always been an important human pathogen, being responsible for a diverse number of respiratory, gastrointestinal, and skin diseases and implicated in toxic shock syndrome. Its resistance to β-lactam antibiotics is well known and methicillin resistance is currently a worldwide problem (i.e., MRSA). Protein A (PA) is a virulence factor because it binds immunoglobulins and it is a well-characterized grampositive cell wall-anchored protein. It interacts with several specific host molecules during infection, resulting in adhesion to host tissues and escape from the host’s immune system.4-7 PA’s amino acid sequence consists of five (in some strains four) homologous domains that bind to the Fc region of mammalian Abs, especially that of immunoglobulin G (IgG).6,7 Molecular recognition between PA and IgG molecules is an important concern for rational drug design and provides a model system for understanding the atomic detail of interactions between R-helical proteins and their IgG-like receptors.7 Therefore, direct * Corresponding author. E-mail: [email protected]. Phone: (902) 4942316. Fax: (902) 494-5191. † Dalhousie University. ‡ Advanced Food and Materials Network - Networks of Centres of Excellence (AFMnet-NCE), University of Guelph. § Department of Molecular & Cellular Biology, University of Guelph. (1) Fischer, J. R.; Parveen, N.; Magoun, L.; Leong, J. M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7307. (2) Samuelson, P.; Gunneriusson, E.; Nygren, P. A° .; Sta˚hl, S. J. Biotechnol. 2002, 96, 129. (3) Orndorff, P. E.; Dworkin, M. J. Bacteriol. 1982, 149, 29. (4) Navarre, W. W.; Schneewind. O. Microbiol. Mol. Biol. ReV. 1999, 63, 174. (5) Darwin, O. V.; Alonso, D. O. V.; Daggett, V. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 133. (6) Gouda, H.; Shiraishi, M.; Takahashi, H.; Kato, K.; Torigoe, H.; Arata, Y.; Ichio Shimada, I. Biochemistry 1998, 37, 129. (7) Graille, M.; Stura, E. A.; Corper, A. L.; Sutton, B. J.; Taussig, M. J.; Charbonnier, J. B.; Silverman, G. J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5399.

Figure 1. AFM images of modified surfaces for the model system. (A) Bare gold surface. (B) Gold surface modified with SAMs, CMamylose, and protein A (PA). (C) The surface shown in B exposed to IgG molecules. There was no major increase in surface roughness after IgG attachment. Image area ) 3 µm × 3 µm.

measurement of these interactions at the molecular level is of considerable interest from both the basic and applied points of view. In recent studies, optical tweezers and micropipette aspiration techniques have been used as sensitive force transducers to reveal valuable details of the statistical distribution of yield forces for single PA-IgG bonds.8-10 Atomic force microscopy (AFM) has emerged as a versatile tool for imaging biological structures under physiological conditions with subnanometer resolution, and the possibility of employing it as a powerful tool for probing the intermolecular forces between various ligands and receptors with high spatial resolution and sensitivity in the piconewton range is of great interest.13 Single-molecule force spectroscopy on membranebound proteins on live bacteria, for example, was previously performed by Lower et al.14 These authors used the adhesion of proteins to bare Si3N4 AFM tips to stretch surface molecules. (8) Stout, A. L. Biophys. J. 2001, 80, 2976. (9) Simson, D. A.; Strigl, M.; Hohenadl, M.; Merkel, R. Phys. ReV. Lett. 1999, 83, 652. (10) Strigl, M.; Simson, D. A.; Kacher, C. M.; Merkel, R. Langmuir 1999, 15, 7316. (11) Touhami, A.; Hoffmann, B.; Vasella, A.; Denis, F. A.; Dufreˆne, Y. F. Langmuir 2003, 19, 1745. (12) Dupres, V.; Menozzi, F. D.; Locht, C.; Clare, B. H.; Abbott, N. L.; Cuenot, S.; Bompard, C.; Raze, D.; Dufreˆne, Y. F. Nat. Methods 2005, 2, 515. (13) Hinterdorfer, P.; Dufreˆne, Y. F. Nature Methods 2006, 3, 347. (14) Lower, B. H.; Yongsunthon, R.; Vellano, F. P., III; Lower, S. K. J. Bacteriol. 2005, 187, 2127-2137.

10.1021/la0628930 CCC: $37.00 © 2007 American Chemical Society Published on Web 01/24/2007

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demonstrate the specificity of the measured interaction forces by blocking the binding sites of PA with free rabbit IgG molecules. 2. Materials and Methods

Figure 2. Force spectrum obtained after a bare tip contacted a gold surface modified with a SAM and CM-amylose linker molecules. The force curve is characteristic of spectra obtained from polysaccharide polymers. The inset is an AFM image of the surface; image area ) 3 µm × 3 µm.

The polymer rupture length was then used to identify outer membrane protein A molecules on E. coli bacteria. The detection and measurement of forces between molecules by AFM is less direct and is accomplished by physically bringing the two interacting biomolecules close to one another until molecular interaction can be perceived. One biomolecule is attached to the AFM tip (the very small tip is mounted at the end of a sensitive cantilever), and the second biomolecule, to a solid substrate such as an appropriately prepared gold surface. By periodically approaching and retracting the functionalized AFM tip to and from the functionalized substrate, bonds between the two biomolecules are alternately created and disrupted. An analysis of AFM force curves recorded for many approach and retraction cycles permits one to calculate the force required to break the bond between the two biomolecules and calculate its strength. This method has been successful in measuring interaction forces between several different proteins.15 However, successful experiments require that the binding of the biomolecules to both probe and substrate is much stronger than the intermolecular force being studied. This is typically achieved by using the chemisorption of alkanethiols on gold or the covalent binding of silanes on silicon oxide. In these cases, the biomolecules of interest are then covalently attached using cross-linkers.11,16 Amylose is often employed as a flexible linker polymer for the biomolecules, being highly biocompatible and often used as a tether for force AFM spectroscopy.17,18 The tethers between both biomolecules has the added advantage of moving the specific interactions away from both the tip and substrate surface, which improves the fidelity of the force measurements.15,16 Our strategy in the present study was to modify the solid substrate and AFM tip, respectively, with rabbit IgG and PA. We first measured unbinding forces for a model system using only purified biomolecules, rabbit IgG-PA. In the second part of this study, force-distance curves were recorded directly between rabbit IgG-terminated AFM probes and the PA on living bacteria. The results indicated that such probing is capable of identifying target molecules among a heterogeneous population of molecules on a cell surface. Control experiments were performed to (15) Kienberger, F.; Ebner, A.; Gruber, H. J.; Hinterdorfer, P. Acc. Chem. Res. 2006, 39, 29. (16) Ratto, T. V.; Langry, K. C.; Rudd, R. E.; Balhorn, R. L.; Allen, M. J. Biophys. J. 2004, 86, 2430. (17) Granbois, M.; Dettmann, W.; Benoit, M.; Gaub, H. E. J. Histochem. Cytochem. 2000, 48, 719. (18) Touhami, A.; Hoffmann, B.; Vasella, A.; Denis, F. A.; Dufreˆne, Y. F. Microbiology 2003, 149, 2873.

2.1. Solid Substrate and AFM Probe Functionalization. PA and rabbit IgG were covalently linked onto gold substrates and AFM tips, respectively, by using a procedure similar to that used in a previous paper.18 Silicon wafers and Si3N4 AFM cantilevers were coated by thermal evaporation of a 4-nm-thick Cr layer followed by a 40-nm-thick Au layer. They were rinsed in ethanol and immersed for 16 h in a 1 mM solution of HS(CH2)2NH2 (Aldrich). The functionalized cantilevers and substrates were then rinsed in three baths of ethanol. During the rinsing step, sonication was briefly applied to remove loosely bound alkanethiol aggregates and then dried in a gentle nitrogen stream. In the second step, a phosphatebuffered saline solution (PBS, pH 7.4) of 10 mg/mL carboxymethylamylose (CM amylose) (Sigma) was activated with 20 mg/mL N-hydroxysuccinimide (NHS) (Aldrich) and 50 mg/mL 1-ethyl- 3-(3dimethylamino-propyl) carbodiimide (EDC) (Sigma) for 5 min in a 1:1:1 proportion (vol/vol/vol).

The amino-functionalized cantilevers and substrates were then incubated with the NHS-activated amylose for 10 min and rinsed three times in PBS. In the final step, the modified surfaces were incubated with 0.1 mg/mL protein A (recombinant protein A, Sigma) or 0.15 mg/mL rabbit IgG (rabbit anti-mouse IgG, Sigma) in PBS, pH 7.4, for 30 min and intensively rinsed with deionized water to remove the unbound molecules. The quality of the functionalized surfaces was assessed using AFM imaging and surface analysis by XPS. 2.2. Growth Conditions and Immobilization of Bacteria. S. aureus D2H was maintained on trypticase soy agar slants (Difco) until experimentation. A slant culture initiated an overnight trypticase soy broth culture that was used for the mid-exponential growth phase (optical density at 600 nm ) 0.2) used for study. All liquid cultures were grown at 37 °C, which was optimal for this bacterial strain. For AFM measurements, bacterial cells were immobilized by mechanical trapping in a polycarbonate filter membrane (Millipore) with a pore size comparable to the cell size (diameter 1.2 µm). After filtering the cell suspension (10 mL), the filter was gently rinsed with deionized water to remove surface debris from cells. The filter was then turned upside down and attached to a glass substrate using a small piece of adhesive tape. The mounted sample was then transferred into the AFM liquid cell. This protocol produced many filter pores that were plugged with single cells, exposing their surface for AFM. Moreover, the main advantage of this immobilization procedure is that it does not involve chemical treatments or drying, which could cause rearrangement or denaturation of the cell surface molecules.19 After filtration, the cells were immediately transferred to the AFM where the collection of force spectra was completed in about 2 h. 2.3. AFM Measurements. AFM images and force measurements were recorded in contact mode at room temperature using a Molecular Imaging Microscope. V-shaped cantilevers with oxide-sharpened Si3N4 tips were used with spring constants of 0.008 N/m. Spring constants of the coated cantilevers were determined by measuring the tip deflections for known applied forces as described by Jericho et al.20 The change in the spring constants after the deposition of (19) Touhami, A.; Jericho, M. H.; Beveridge, T. J. J. Bacteriol. 2004, 186, 3286. (20) Jericho, S. K.; Jericho, M. H. ReV. Sci. Instrum. 2002, 73, 2483.

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Figure 3. (A) Example of force spectra obtained with an IgG-functionalized tip over a protein A-functionalized surface. (B) Histogram of rupture forces from 85 force curves showing a mean rupture force of 59 nN with SD ) 34 pN. (C) Corresponding histogram for rupture length showing a mean rupture length of 96 nm with SD ) 49 nm.

3. Results

Figure 4. Histograms of (A) rupture force and (B) rupture length after blocking the binding sites on the surface with extra IgG antibodies. The inset in A shows typical force spectra after the blocking of binding sites.

Figure 5. AFM image of a single Staphylococcus aureus bacterium trapped in a filter pore. Image area ) 3 µm × 3 µm, image height ) 600 nm.

∼40 nm of gold on the tip side was about 12% and was within the measurement uncertainty. Measurements were performed in PBSbuffered solutions. Force curves were measured with a z velocity of 0.5 µm/s both on approach and retraction and with an interaction time range of 0.1 to 0.5 s. In all measurements the maximum loading force was 1.5 nN. All force curves in the figures show extension data where the piezo displacement was corrected by the deflection of the cantilevers. Blocking control experiments were performed by adding free IgG (10 µM) to the solutions.

3.1. Model System. The functionalization procedure resulted in a low frequency of nonspecific interactions between the IgG probe and PA substrates. The morphology of both surfaces (PA and IgG) was homogeneous and stable upon repeated AFM scanning (Figure 1B,C). The presence of the biomolecules on the functionalized substrates was indicated by an increase in AFM surface roughness. The surface texture was described by calculating Rq, the rms of the Zi deflections over the image area where Rq ) (Σ(Zi Zav)2/Np)1/2 and Zav is the deflection averaged over Np pixels. For bare gold substrates (Figure 1A), Rq was 0.18 nm, whereas surfaces coated with IgG and PA had average roughness values of 0.45 and 0.35 nm. The thickness of the biomolecular layers was evaluated by using the cantilever tip to scrape a small area (1 × 1 µm) of the functionalized surfaces down to the substrate. (For details, see ref 11.) From this procedure, we estimated the IgG and PA layers to be respectively 5 ( 1 and 4 ( 1 nm thick. The N/C ratios, determined by XPS, for the amylose-covered thiol layers was 0.05. A small value is expected for this ratio because the thiol nitrogen is buried beneath the amylose layer. The CO ratio, however, increased from 0.14 (for NH2/Au) to 0.32 (for amylose/NH2/Au). With PA molecules attached to the amylose, the C/N ratio increased to 0.13 and then increased further to 0.18 with the addition of the IgG. These data are consistent with the formation of protein layers at the amylose surface. A key prerequisite for the successful investigation of molecular recognition forces by AFM is that receptor and ligand molecules are firmly anchored to solid surfaces while keeping sufficient mobility for proper molecular interaction. As shown in the inset in Figure 2, there is no significant change in surface morphology after the modification of the SAMs substrate with amylose polymer. Importantly, when the AFM tip was brought into contact with the sample and kept in contact for several seconds (allowing the individual polymers to adsorb onto the tip), a typical force signature for amylose could be recorded when the cantilever was pulled away from the surface to stretch the polymer (Figure 2). The observed plateau region is a characteristic feature of the single-molecule force spectrum of amylose.21,22 This finding clearly confirms the presence of the amylose polymer on the SAMs surface. (21) Hongbin, L.; Rief, M.; Oesterhelt, F.; Gaub, H. E.; Zhang, X.; Shen, J. Chem. Phys. Lett. 1999, 305, 197.

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Figure 6. (A) Example of force curves obtained on a single cell with an IgG-functionalized tip interacting with the surface of a live S. aureus bacterium trapped in a filter pore. (B) Histogram of rupture forces from 100 force curves showing a mean rupture force of 64 pN with SD ) 28 pN. (C) Corresponding histogram of rupture length showing a mean rupture length of 74 nm with SD ) 40 nm.

3.1.1. Dissociation of IgG-PA Bonds. After validation of the surface-functionalization procedure, we measured the forces required to dissociate single IgG-PA pair bonds between the two proteins. Hundreds of force measurements were performed over several lateral positions on the experimental surface. After analysis, a distribution of the recorded rupture forces is given as a force histogram in Figure 3A. A significant fraction (about 75%) of the retraction curves displayed single or multiple peaks. The histogram of the unbinding forces, shown in Figure 3B, shows an asymmetric distribution centered at 59 pN. Adhesion histograms from other independent experiments showed similar mean values and distributions. The occurrence of unbinding forces was found to depend on the spot investigated, and multiple curves recorded at the same location of the substrate yielded reproducible behavior. Importantly, Figure 3C reveals that most unbinding events were accompanied by nonlinear elongation forces along with rupture lengths ranging from 0 to 250 nm. We suggest that these elongation forces and large rupture lengths are due to the stretching of the long, flexible amylose spacer. The measured force of 59 pN for this system is typically in the range of the unbinding forces reported by Stout8 between several IgG species and PA. It is also close to the values typically reported for individual receptor-ligand interactions at fairly comparable rupture rates.23,24 To demonstrate that this adhesion force reflects the specific interaction between IgG and PA, the same experiment was carried out in the presence of free IgG (10 µM). As shown in Figure 4A, under these conditions, a dramatic reduction of adhesion events was seen, indicating that the free IgG had blocked most receptor sites on the PA. Adhesion forces were measured but with a much smaller magnitude, (i.e., the mean value was e10 pN), and most rupture lengths were in the 0-20 nm range (Figure 4B). (22) Marszalek, P. E.; Oberhauser, A. F.; Pang, Y. P.; Fernandez, J. M. Nature 1998, 396, 661. (23) Berquand, A.; Xia, N.; Castner, D. G.; Clare, B. H.; Abbott, N. L.; Dupres, V.; Adriaensen, Y.; Dufreˆne, Y. F. Langmuir 2005, 21, 5517. (24) Dettmann, W.; Grandbois, M.; Andre´, S.; Benoit, M.; Wehle, A. K.; Kaltner, H.; Gabius, H. J.; Gaub, H. E. Arch. Biochem. Biophys. 2000, 383, 157. (25) Umeda, A.; Ikebuchi, T.; Amako, K. J. Bacteriol. 1980, 141, 838. (26) Hertadi, R.; Gruswitz, F.; Silver, L.; Koide, A.; Koide, S.; Arakawa, H.; Ikai, A. J. Mol. Biol. 2003, 333, 993. (27) Stroh, C.; Wang, H.; Bash, R.; Ashcroft, B.; Nelson, J.; Gruber, H.; Lohr, D.; Lindsay, S. M.; Hinterdorfer, P. Proc. Natl. Acad. Sci. U.S.A. 2004, 01, 12503. (28) Neuert, G.; Albrecht, C.; Pamir, E.; Gaub, H. E. FEBS Lett. 2006, 580, 505. (29) Friedsam, C.; Wehle, A. K.; Kuehner, F.; Gaub, H. E. J. Phys.: Condens. Matter 2003, 15, S1709. (30) Hanke, F.; Kreuzer, H. J. Biointerphases 2006, 1, 11-17.

3.2. IgG-PA Interactions Using Living Bacteria. The mechanical trapping of bacterial cells in a porous polymer membrane appears to be the most reliable method for their immobilization. This method does not use drying or chemical fixation, thereby making it possible to investigate living cells while preserving the native macromolecular architecture of the surface. As shown in Figure 5, AFM imaging revealed a smooth and homogeneous cell surface on S. aureus, as demonstrated previously at this magnification.19 Repeated imaging can be performed without cell detachment or surface distortion. Using the same loading rate as in the pure molecule system (0.5 µm/s retraction velocity), about 65% of the force-distance curves recorded across the cell surface using the IgG probe showed single or multiple unbinding forces (Figure 6A). Topographical AFM imaging always confirmed that measurements were made only on single bacteria and that the bacteria were not dislodged. Force spectra were repeated on 5 to 10 different cells from fresh cultures. The corresponding histogram of the unbinding forces again displayed an asymmetric distribution but now with a mean value of 64 pN, which was close to the value obtained with the model system. The rupture lengths for the unbinding events were also in the same range (0-200 nm) but with a smaller mean value. A smaller value is expected because for these measurements amylose linker molecules were present only on the AFM tip. For the model system, both PA and IgG were tethered with CM-amylose. To confirm that the measured forces were related to the specific binding between the IgG-PA pair, a control experiment with the same IgG-functionalized tip was performed after adding free IgG to the buffer to block PA-reactive sites on the bacterial surface. A significant reduction was found in binding (Figure 7A), and most of the rupture events occurred when the tip was still close to the bacterial surface (Figure 7B), which can be caused by nonspecific adhesion between IgG or amylose and different surface components. For the control experiments for our PA-IgG model system about 40% of force curves showed small adhesion and a very short rupture length, whereas the corresponding number for the IgG-cell system is 57%.

4. Discussion Molecular spacers, such as amylose, and linking agents, such as SAMs with their thiol-terminated amino groups, hold great promise for the determination of forces between important

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Figure 7. Histograms of the (A) rupture force and (B) rupture length after blocking binding sites on the bacterial surface with extra IgG antibodies.

biomolecules, especially those important for adsorption such as PA and IgG. Although we were particularly interested in determining the bonding forces between an antibody and a cell (i.e., IgG and S. aureus), a preliminary experiment using purified components (IgG and PA) was necessary. From the surface morphology in aqueous solution shown in Figure 1, the presence of IgG and PA at the solid substrate surface was clearly evident. After functionalization, dot-like features were distributed across the surface, and the roughness of the underlying gold substrate increased. Surface chemistry analysis by XPS confirmed that the biomolecules were present by their C and N signatures. These findings, combined with the AFM thickness measurements, were consistent with the presence of a thin layer of IgG or PA on the solid substrate. We were able to confine specific and nonspecific binding events to different regions of our force curves, enabling us to measure the distribution of IgG-PA bond rupture forces. The primary goal of this model system was to see how well the force-induced dissociation of single specific bonds between IgG and PA could be measured. The mean value of the rupture force from the histogram was 59 pN, corresponding to the most probable value of the unbinding force. As discussed by Simons et al.,9 the dissociation of a molecular bond (such as that between IgG and PA) increases logarithmically with loading rate, and our value of 59 pN compares favorably with previous data for IgG-PA bonds measured at a loading rate similar to ours.8 It is difficult to determine how many individual proteins formed a bond with the IgG probe. As stated above, we used a cantilever with a relatively sharp tip (∼20 nm), which would enhance singlemolecule interactions. However, the branched structure of our amylose linkers could have encouraged multiple binding events per tether, which makes it difficult to determine the rupture force per bond. To examine the effects of a branched tether on the force curves, we calculated the force signature of a tether with three branches, each branch with a different length as shown in the inset in Figure 8. The stretching force of each tether as a function of its extension was calculated with the wormlike chain model (WLC)31 and is given by

F(x) )

[

1 kT 1 x - + p 4(1 - x/ )2 4 L L

]

(1)

In eq 1, k is Boltsmann’s constant, T is the absolute temperature, L is the polymer contour length, and p is the persistence length. The force applied by the AFM cantilever is shared between the tether branches such that the shorter the branch, the larger the (31) Bustamante, C.; Marko, J.; Siggia, E.; Smith, S. Science 1994, 265, 15991600.

Figure 8. Force spectra calculated with the wormlike chain model for polymer extension showing the effects of linker molecule branching on the rupture force measurement. The model considered three branches each of length L1, L2, and L3 (inset), an assumed bond rupture force of 100 pN (dashed-dotted line), and a linker molecule persistence length of 0.5 nm as they interact with a receptor molecule such as PA. When the branches have comparable lengths (L1 ) 70 nm, L2 ) 80 nm, L3 ) 90 nm), then closely spaced rupture peaks are obtained that overestimate the rupture force per branch (dotted line). With a broader spread in branch length (L1 ) 80 nm, L2 ) 130 nm, L3 ) 200 nm), rupture peaks are spaced far apart, and they measure the bond rupture force more closely (solid lines).

force it applies to the substrate and therefore to its bond to PA. The net applied force in this example is thus the sum of three forces where each force component is determined by the contour length, Li, of each tether. If in the above model the IgG-PA bond on a tether breaks at a particular tip-substrate separation, then the force curve collapses abruptly to a new curve that is now characteristic of the two remaining tether branches. Figure 8 shows the result of force spectra calculations for two sets of tether branch lengths. In the first set, the spread in contour length of the branches was small compared to the average contour length, whereas in the second set the spread was comparable to the average. The lengths chosen were comparable to rupture lengths shown in Figures 3 and 6. The bond rupture force in the calculation was set to 100 pN. As expected and as shown in Figure 8, if the tether branches are of comparable length, then a closely spaced rupture triplet is obtained and the cantilever force at the break of the first bond is much larger than the actual bond rupture force. If the length difference between branches is comparable to their length, however, then more isolated rupture peaks are obtained and the cantilever force at rupture is now much closer to the actual bond rupture force. Most of our force spectra were of this second type, and we therefore conclude that polymer branching and multiple attachments did not have a major affect on the histogram data. However, the observed high number of binding events can be attributed to the structural nature of amylose, which allows the binding of multiple protein molecules per tether, as well as to the fact that each PA can bind to at least four IgG molecules. The IgG probe was also used to detect PA on the surface of living S. aureus cells. The quantitative measurements of the adhesion strength on a whole cell revealed an average unbinding force of 64 pN. This value is comparable to the one found for our model system and is consistent with other reports of the unbinding force for IgG-PA using similar loading rates. Remarkably, binding events were detected across the whole surface of a bacterium, which indicated that the PA was distributed over the entire cell surface, confirming a previous report.23 When PA was blocked with excess IgG, most of the force curves again

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showed force spectra characteristic of nonspecific interactions between the IgG probe and additional cell wall components. It is interesting that the adhesion signatures in Figure 6A for live bacteria had a better signal-to-noise ratio than those for the single-molecule-functionalized surfaces. Both surfaces, however, showed a similar number of adhesion events that in general were limited to four or five events per retraction cycle. Our results clearly show that tips functionalized with appropriate antibodies are effective in detecting the presence of the corresponding antigens on surfaces. The presence of the linker molecules will unfortunately limit the spatial resolution obtainable. The spatial resolution will depend on the tip radius and the length of the linker molecules and their conformation on the AFM tip. For linkers that are short compared to the tip radius, the lateral resolution should be comparable to the tip radius. However, for linker molecules that are substantially longer than the tip radius, a reduction in the lateral resolution is inevitable, and the resolution may be on the order of the linker length. From Figure 6C, we estimate a lateral resolution of about 70-100 nm for our experiments. Because many rod-shaped bacteria are several micrometers in length, this resolution may be adequate to decide whether certain surface molecules occur in patches or are more uniformly distributed over their surface. In our experiments, adhesion events occurred completely and randomly over the surface of S. aureus. This suggests that no areas much larger than 100 nm2 were devoid of PA. Shorter and less branched linker molecules such as PEG (∼6 nm) could improve the lateral resolution. Short linkers were used by Hertadi et al.26 to study the unfolding of an outer membrane protein from Borrelia burgdorferi.26 Short PEG linkers with attached antibodies were also successfully used to obtain adhesion maps of surfaces that contained antigen deposits.27 However, when linker molecules become too short, the bond rupture signature might become indistinguishable from nonspecific interactions between the AFM tip and the bacterial surface. We estimate that a useful resolution should be obtainable with linkers in the 20 nm length range. The 64 pN strength of the bond between PA and IgG is consistent with values reported by Simson et al.9 A more quantitative discussion of bond strength in the context of this experiment is complicated. As discussed by other authors,9,28,29 the bond rupture strength and the rupture force distribution depends on the rate at which the force on the bond increases. At low rates, the distribution is narrow, and mean rupture forces are small. As the force rate increases, the rupture force distributions broaden, and the mean rupture force increases logarithmically with force rate. The force rates near rupture were in the range of 1400 pN/s in our experiments. This high force rate is consistent

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with a broad distribution9 and with the relatively high mean rupture force of 64 pN. A number of authors have developed theoretical expressions for the force histograms and have expressed the force-dependent bond dissociation rate in terms of ∆x, the width of the potential barrier, and the natural bond dissociation or off rate, koff.29,30 The resulting force-dependent probability function for bond rupture, however, also involves an integral over dF/dt, the rate at which the force is applied. In the case of tethered molecules, the rate at which the force is applied to the bond varies with time and reaches its maximum value at bond rupture, as is evident from Figures 3 and 6. This greatly complicates the analysis of the force histograms. The extraction of bond parameters, such as ∆x and koff, requires a force spectra signal-to-noise ratio that is better than was achieved in our experiment. The importance of a high signal-to-noise ratio in constant velocity retraction experiments was emphasized by Hanke et al.30 The use of single polymer strand linkers such as PEG, for example, should allow a more detailed analysis of the force spectra and associated histograms and should make it possible to probe surface proteins on bacteria in a more quantitative way.

5. Conclusions The functionalization of Si3N4 AFM tips with SAM and amylose allowed us to probe the binding capacity of PA to the Fc fragment of IgG. To examine the probe’s molecular recognition properties, we tested it on a gold surface coated with PA. The unbinding force was about 59 pN before blocking of the antibody sites but dropped to a mean of less than 10 pN after blocking. This functionalization method also allowed us to measure the specific interactions between IgG-PA pairs on living bacteria. Our measurements were performed directly in aqueous solution without any cell pretreatment, thus preserving the native organization and conformation of the surface molecules. We suggest that with linker molecules with a length comparable to the AFM tip radius it should be possible to detect specific binding events on the surface of live bacteria with a lateral resolution of a few tens of nanometers. Because S. aureus is an important pathogen, especially methicillin-resistant strains (MRSA), it is possible that additional virulence factors beyond PA can be probed. Acknowledgment. We thank the National Science and Engineering Research Council of Canada (NSERC) for financial support of this work and AFMnet for providing travel funds between the Dalhousie and Guelph laboratories. LA0628930