1 Unité de Chimie des Interfaces, Université Catholique de Louvain, Croix du Sud 2/18, B-1348 Louvain-la-Neuve, Belgium
2 Laboratorium für Organische Chemie, HCI H 317, ETH-Hönggerberg, CH-8093 Zürich, Switzerland
Correspondence
Yves F. Dufrêne
dufrene{at}cifa.ucl.ac.be
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ABSTRACT |
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INTRODUCTION |
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Atomic force microscopy (AFM) (Binnig et al., 1986) has emerged as a powerful tool to measure intramolecular and intermolecular forces associated with biological systems (Lee et al., 1994
; Florin et al., 1994
; Hinterdorfer et al., 1996
; Benoit et al., 2000
). Recently, AFM force measurements were applied to microbial systems, providing new insight into microbial cell-surface functions (for recent reviews, see Dufrêne, 2002
, 2003
). In particular, force spectroscopy was used to investigate electrostatic and steric interactions associated with negatively charged bacterial strains (Camesano & Logan, 2000
); to measure the interaction forces between Escherichia coli strains and polymer biomaterials (Razatos et al., 1998
), between Aspergillus niger spores and mica (Bowen et al., 2000
) and between Shewanella oneidensis bacteria and mineral surfaces (Lower et al., 2001
); to probe the stiffness of bacterial cell wall components (Xu et al., 1996
; Yao et al., 1999
); to unzip single proteins from the hexagonally packed intermediate layer of Deinococcus radiodurans (Müller et al., 1999
); to map the distribution of yeast cell wall polysaccharides (Gad et al., 1997
); and to stretch macromolecules at the surface of fungal (van der Aa et al., 2001
) and bacterial (Abu-Lail & Camesano, 2002
) cells.
Here, we use AFM force spectroscopy to measure discrete lectin-carbohydrate interactions involved in the reversible aggregation of yeast cells, a process referred to as yeast flocculation which is of particular importance in fermentation processes such as brewing and wine-making (Calleja, 1989; Stratford, 1992
; Dengis et al., 1995
; Jin & Speers, 1998
). While cell flocculation has been examined for over a century, the flocculation mechanisms remain poorly understood at the molecular level. Biochemical studies have revealed the involvement of specific interactions between cell-surface lectins and mannose residues, based on the finding that sugars such as mannose and glucose can specifically inhibit flocculation (Miki et al., 1982
). However, direct measurement of these interactions has not been possible up to now.
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METHODS |
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Flocculation test.
Flocculation was quantified by measuring the optical density of yeast suspensions after agitation and subsequent undisturbed standing to allow floc settling (Dengis et al., 1995). Test tubes (16x100 mm) containing 5 ml of the following solutions were used: 10 mM sodium acetate/acetic acid buffer (pH 5·2), 0 or 100 mM D-(+)-mannose, and CaCl2 at various concentrations. Yeast cells were added as a concentrated suspension under whirl mixing to reach a final concentration of 108 cells ml-1. The tubes were agitated upside down at 25 r.p.m. for 15 min, left undisturbed for 15 min and the OD660 was then measured (Spectronic Instruments). Optical micrographs of the cells were obtained using a commercial optical microscope (Olympus BH-2).
Functionalization of AFM probes.
Self-assembled monolayers (SAMs) of thiol-terminated hexaamylose molecules were used to produce carbohydrate-coated AFM probes. The functionalization strategy and its validation using X-ray photoelectron spectroscopy and AFM imaging were described in a previous paper (Touhami et al., 2003). Briefly, commercial Si3N4 AFM cantilevers (ThermoMicroscopes) were coated by electron beam thermal evaporation with a 5 nm thick Cr layer followed by a 30 nm thick Au layer. They were cleaned for 5 min by UV/ozone treatment (Jelight), rinsed in ethanol and immersed for 3 h in 0·05 mM solution of the thiol-terminated hexasaccharide in a 1 : 1 solution of methylene chloride and ethanol. The functionalized cantilevers were then rinsed in three baths of methylene chloride, sonication being briefly applied during the rinsing step to remove loosely bound aggregates, and then dried in a gentle nitrogen stream. Using AFM imaging and force measurements, we recently showed that the carbohydrate-coated probes interact specifically with concanavalin A (Con A), a glucose/mannose-specific lectin (Touhami et al., 2003
).
Lectin-coated probes were created using carboxymethyl-amylose as a spacer to provide mobility to the protein and minimize non-specific adsorption (Johnsson et al., 1991). Gold-coated probes were cleaned for 5 min by UV/ozone treatment (UVO-Cleaner), rinsed in ethanol, immersed for 16 h in 1 mM solution of HS(CH2)2NH2 (Aldrich; used as received) and then rinsed with ethanol. A PBS solution (pH 7·2) of 10 mg carboxymethyl-amylose ml-1 (Sigma) was activated with 20 mg N-hydroxysuccinimide (NHS) ml-1 (Aldrich) and 50 mg 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) ml-1 (Sigma) for 5 min. Modified probes were then incubated with the NHS-activated amylose for 10 min, rinsed three times in PBS, incubated with Con A (Sigma) (0·5 mg ml-1 in PBS; pH 7·2) for 30 min and intensively rinsed with water to remove the unbound proteins. Water used in our experiments was HPLC grade produced by a MilliQ plus system from Millipore (MilliQ water).
AFM measurements.
AFM force-distance curves were obtained at room temperature, using a commercial microscope (Nanoscope III; Digital Instruments). Cells were immobilized by mechanical trapping in a polycarbonate membrane (Millipore) with a pore size similar to the cell size (Dufrêne et al., 1999; Dufrêne, 2002
). Measurements were performed in buffered solutions (sodium acetate/acetic acid; pH 5·2) containing 1 mM CaCl2 (carbohydrate-coated probe) or 1 mM CaCl2+1 mM MnCl2 (lectin-coated probe). Blocking control experiments were performed by adding 100 mM D-(+)-mannose to the solutions. The spring constants of functionalized cantilevers were found to be 8±0·4 mN m-1, as determined by measuring the free resonance frequency in air (Cleveland et al., 1993
). Retraction force curves were recorded at a rate of 0·5 µm s-1 (rate of the piezo displacement).
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RESULTS AND DISCUSSION |
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Using carbohydrate-coated probes, single cells were first imaged at high resolution. The surface structure was smooth and homogeneous, as demonstrated previously using silicon nitride probes (Ahimou et al., 2003). Multiple force-distance curves were then recorded at various locations at a rate of 0·5 µm s-1. For S. carlsbergensis in flocculating conditions (stationary phase; 1 mM Ca2+), single or multiple unbinding forces were observed in about 50 % of a total of 200 force-distance curves (Fig. 3
a), the remaining curves exhibiting no adhesion. The occurrence or lack of adhesion forces was found to depend essentially on the spot investigated, suggesting some heterogeneity in the distribution of the cell-surface molecules. The histogram of the largest unbinding forces (Fig. 3c
) displayed an asymmetric distribution centred at 121±53 pN (n=100). Adhesion histograms from independent experiments showed similar mean values and distribution. To demonstrate that this adhesion force reflects the specific interaction between cell-surface lectins and glucose residues on the probe, the same experiment was carried out in the presence of mannose (Fig. 4
a, b). Under these conditions, little or no adhesion was detected, indicating that mannose had blocked the lectin receptor sites. Interestingly, the measured force of 121 pN is in the range of force values (75200 pN) obtained by Gad et al. (1997)
between lectin-coated probes and yeast cells. It is also close to values typically reported for individual receptorligand interactions at fairly comparable rupture rates. By way of example, mean adhesion forces of 160, 112 and 96 pN were reported for the specific interaction between avidinbiotin (Florin et al., 1994
), antibodyantigen (Dammer et al., 1996
) and lectincarbohydrate (Touhami et al., 2003
). Taken together, these observations lead us to believe that the 121 pN force reflects the individual interaction between cell-surface lectins and glucose residues. There are several important questions to address in future research, including assessing the dependence of the unbinding forces on the pulling rate (dynamic force spectroscopy measurements) and determining whether the multiple unbinding events are due to the rupture of multiple receptorligand complexes or to some other phenomenon such as protein unfolding.
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Specific interactions between cell-surface mannose residues and lectin-coated probes
Flocculation of S. carlsbergensis through the lectin mechanism involves the binding of cell-surface lectins to mannose residues (mannan) of adjacent cells. Consequently, one would expect that AFM probes terminated with mannose-specific lectins should interact specifically with mannose residues of S. carlsbergensis cells. To test this hypothesis, force-distance curves were recorded between S. carlsbergensis cells in the stationary phase and Con A-coated probes. Note that measurements were performed in 1 mM Ca2++1 mM Mn2+ solutions, since both ions are required for Con A lectin activity. Most force-distance curves showed single or multiple unbinding forces (Fig. 5a) and the adhesion histogram (Fig. 5c
) revealed a mean adhesion force of 117±41 pN (n=100) which is close to the mean value obtained with the carbohydrate probe. We attribute this adhesion force to the specific binding between Con A and cell-surface mannose residues, which is supported by the finding that this adhesion force was not observed in the presence of mannose (Fig. 5d, e
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 18 April 2003;
revised 2 July 2003;
accepted 4 July 2003.