Langmuir 2002, 18, 9937-9941
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Probing Microbial Cell Surface Charges by Atomic Force Microscopy Franc¸ ois Ahimou, Fre´de´ric A. Denis, Ahmed Touhami, and Yves F. Dufreˆne* Unite´ de chimie des interfaces, Universite´ catholique de Louvain, Croix du Sud 2/18, B-1348 Louvain-la-Neuve, Belgium Received July 19, 2002. In Final Form: October 4, 2002 Most microorganisms possess a negative surface charge under physiological conditions due to the presence of anionic carboxyl and phosphate groups. Cell surface charge plays an important role in controlling cell adhesion and aggregation phenomena, as well as antigen-antibody, cell-virus, cell-drug, and cell-ions interactions. We have used atomic force microscopy (AFM) with chemically functionalized probes to investigate the surface charges of yeast cells. Force-distance curves and adhesion maps recorded with probes terminated with ionizable carboxyl groups were strongly influenced by pH: while no adhesion was measured at neutral/alkaline pH, multiple adhesion forces were recorded at pH e 5. Three pieces of evidence indicated that these changes were related to differences in the ionization state of the cell surface functional groups. First, the adhesion force vs pH curve was correlated with microelectrophoresis data, the pH of the largest adhesion force corresponding to the cell isoelectric point, i.e., pH 4. Second, treating the cells with Cu(II) ions caused a reversal of the cell surface charge at neutral pH and promoted the adhesion toward the negatively charged probe. Third, control experiments using nonionizable hydroxylterminated probes indicated that the changes in adhesion forces were not simply due to the titration of the probe surface charges. This study shows that AFM with chemically modified probes is a valuable approach in microbiology and biophysics for probing the local electrostatic properties of microbial cell surfaces.
Introduction During the past 40 years, the importance of the microbial cell surface in biology, medicine, industry, and ecology has been increasingly recognized. Because they constitute the frontier between the cells and their environment, the walls of bacterial, fungal, and yeast cells play several key functions, including supporting the internal turgor pressure of the cell, protecting the cytoplasm from the outer environment, imparting shape to the organism, acting as a molecular sieve, and controlling interfacial interactions such as cell adhesion and aggregation. These biointerfacial processes have major consequences, which can be either beneficial, such as in biotechnology (wastewater treatment, bioremediation, cell immobilization in reactors), or detrimental, such as in industrial systems (biofouling, contamination) and in medicine (interactions of pathogens with animal host tissues, accumulation on implants and prosthetic devices).1 Hence, there is considerable interest in improving our current understanding of the functions of microbial cell surfaces. Most microorganisms possess a negative surface charge under physiological conditions due to the presence of anionic surface groups such as carboxyl and phosphate.2,3 The density and distribution of these charges within the cell wall have important consequences for its architecture, physical properties, and interactions with the environment. Indeed, surface charges play a role in controlling cell adhesion and aggregation phenomena, as well as * Corresponding author. Telephone: (32) 10 47 36 00. Fax: (32) 10 47 20 05. E-mail:
[email protected]. (1) Savage, D. C., Fletcher, M., Eds. Bacterial adhesion; Plenum Press: New York and London, 1985. (2) Mozes, N.; Marchal, F.; Hermesse, M. P.; van Haecht, J. L.; Reuliaux, L.; Leonard, A. J.; Rouxhet, P. G. Biotechnol. Bioeng. 1987, 30, 439. (3) James, A. M. In Microbial Cell Surface Analysis: Structural and Physicochemical Methods; Mozes, N.; Handley, P. S., Busscher, H. J., Rouxhet, P. G., Eds. VCH Publishers: New York, 1991; p 221.
antigen-antibody, cell-virus, cell-drug, and cell-ions interactions. In addition, surface charges enable microbial cells to remove heavy metals from aqueous solutions, which makes microbial biomass an attractive technology for the treatment of polluted waters.4,5 A variety of methods are available to establish the presence of ionizable groups at the cell surface, among which microelectrophoresis, colloid titration, isoelectric focusing of cells, ion-exchange chromatography, and surface conductivity.3,6 These techniques probe the overall electrochemical properties of cells, i.e., they provide averaged information obtained on a large ensemble of cells. Therefore, developing complementary tools for probing cell surface charges on the nanoscale is a highly relevant challenge in cellular microbiology and biophysics. With its high spatial resolution and capacity to work under aqueous solutions, atomic force microscopy (AFM) has become a valuable technique for probing the structure and properties of microbial cell surfaces. The structure of reconstituted microbial cell surface layers made of 2-D protein crystals (hexagonally packed intermediate (HPI) layers, purple membranes, porin OmpF, and aquaporin crystals) has been imaged at a lateral resolution of 0.5-1 nm.7 Recent studies have also demonstrated the power of the technique for visualizing the surface structure of living microbial cells and for probing their physical properties.8 Pioneering investigations have shown that AFM can be used to measure surface charges and electrostatic forces.9,10 More recently, chemically modified AFM probes were shown to be highly sensitive to the ionization state of (4) Kapoor, A.; Viraraghavan, T. Bioresour. Technol. 1995, 53, 195. (5) Sagˇ, Y. Sep. Purif. Methods 2001, 30, 1. (6) Pedersen, K. FEMS Microbiol. Lett. 1980, 12, 365. (7) Engel, A.; Mu¨ller, D. J. Nat. Struct. Biol. 2000, 7, 715. (8) Dufreˆne, Y. F. J. Bacteriol. 2002, 184, 5205. (9) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Nature (London) 1991, 353, 239. (10) Butt, H.-J. Biophys. J. 1991, 60, 1438.
10.1021/la026273k CCC: $22.00 © 2002 American Chemical Society Published on Web 11/06/2002
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surface functional groups.11,12 To our knowledge, the use of chemically modified probes to investigate the surface charges of living cells has not been reported. Here, we show that AFM with carboxyl-terminated probes can be used to probe the electrostatic properties and, more specifically, the isoelectric point of microbial cell surfaces at high spatial resolution. First, the functionalization of the surfaces is validated by surface analysis and by recording multiple force-distance curves between modified probes and model substrata. Then, carboxyl-terminated probes are used to record multiple force-distance curves at various pH at the surface of Saccharomyces cerevisiae cells. The yeast S. cerevisiae was selected because of the vast amount of biochemical data available on this microorganism (cell wall structure and composition) and of its great potential for industrial applications (e.g., beer brewing, water treatment). Different experiments are performed to validate the AFM measurements (microelectrophoresis, treatment of the cells with Cu(II), use of hydroxyl-terminated probes).
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Figure 1. AFM force-distance curves recorded at pH 10 and pH 3 between probes and model substrata terminated with COOH groups. As shown in the drawing, the differences observed according to pH can be related to a change of ionization state of the surfaces. Similar results were obtained in different spots and with independent preparations.
Materials and Methods The brewing yeast strain S. cerevisiae MUCL 38475 (top fermenting strain) was kindly supplied by Prof. A. M. Corbisier (Unite´ de Microbiologie, Universite´ catholique de Louvain, Belgium). The cells were grown as described by Dengis et al.13 Yeast cells were stored in 25% (v/v) glycerol at -20 °C. They were plated in a Petri dish on solid medium (1% [w/v] yeast extract, 3% [w/v] glucose, 2.5% [w/v] agar) and incubated at 30 °C for 3 days. The plates were stored at 4 °C and repitched every 3 weeks. The culture broth contained 2% (w/v) yeast extract and 5% (w/v) glucose. Two or three colonies from the solid medium plate were used as inoculum for the preculture. Cells of the exponential growth phase were harvested after 10 h by centrifugation, washed three times with cold deionized water and resuspended in 1 mM KNO3 to a concentration of ∼106 cells/mL. Silicon nitride cantilevers with spring constants of ∼0.06 N/m (Nanoprobes, Digital Instruments, Santa Barbara, CA) and ∼0.01 N/m (ThermoMicroscopes, Sunnyvale, CA), and silicon wafers (Wacker Chemitronic, Burghausen, Germany) were coated by electron beam thermal evaporation with a 4 nm thick Ti layer followed by a 30 nm thick Au layer.14 The spring constants of gold-coated cantilevers, determined by measuring the free resonance frequency in air, were found to be in the range of values specified by the manufacturers for noncoated cantilevers. The coated surfaces were functionalized with alkanethiol selfassembled monolayers (SAMs) terminated with carboxyl or hydroxyl groups. To this end, samples were first cleaned for 5 min by UV/ozone treatment (Jelight Co., Irvine, CA) and rinsed with ethanol. The cleaned samples were then immersed for 18 h in 1 mM solutions of HS(CH2)10COOH (Aldrich; used as received) or HS(CH2)11OH (Aldrich; used as received) in ethanol and then rinsed with ethanol. Sonication was briefly applied to remove alkanethiol aggregates that may be adsorbed. Functionalized probes and substrata were always used immediately after they were prepared. The quality of the surface functionalization was assessed by characterizing the substrata using contact angle measurements and X-ray photoelectron spectroscopy (XPS) analysis as described earlier.15 The electrophoretic mobility of the cells was measured as a function of pH, adjusted with KOH or HNO3 solutions, with a Pen Kem Laser Zee Meter (Bedford Hills, NY). AFM measurements were made at room temperature (20 °C), in 1 mM KNO3 solutions of defined pH (adjusted with KOH or (11) van der Vegte, E. W.; Hadziioannou, G. Langmuir 1997, 13, 4357. (12) Vezenov, D. V.; Noy, A.; Rozsnyai, L. F.; Lieber, C. M. J. Am. Chem. Soc. 1997, 119, 2006. (13) Dengis, P. B.; Ne´lissen, L. R.; Rouxhet, P. G. Appl. Environ. Microbiol. 1995, 61, 718. (14) Dufreˆne, Y. F. Biophys. J. 2000, 78, 3286. (15) Denis, F. A.; Hanarp, P.; Sutherland, D. S.; Gold, J.; Mustin, C.; Rouxhet, P. G.; Dufreˆne, Y. F. Langmuir 2002, 18, 819.
Figure 2. Force-distance curves recorded in 1 mM KNO3 solutions of varying pH between the surface of a single S. cerevisiae cell and a COOH-terminated probe. The differences observed with pH can be related to a change of ionization state of the functional groups of the probe and cell surfaces. The data shown at various pH were obtained using the same cell/probe combination. Small variations in the absolute values of the adhesion force were observed when using different probes and independent preparations, but the same pH dependence was always found. HNO3 solutions), using an optical lever microscope equipped with a liquid cell (Nanoscope III, Digital Instruments, Santa Barbara, CA). Cells were immobilized by mechanical trapping in a polycarbonate membrane (Millipore) with pore size similar to the cell size.16 The main advantage of this immobilization procedure is that it does not involve chemical treatments or drying which would cause rearrangement/denaturation of the surface molecules. After the cell suspension (10 mL) was filtered, the filter was gently rinsed with deionized water, carefully cut (1 cm × 1 cm), turned upside down, and attached to a steel sample puck (Digital Instruments, Santa Barbara, CA) using a small piece of adhesive tape, and the mounted sample was transferred into the AFM liquid cell. Force data were obtained at various pH values using the same cell/probe combination. To avoid the use of an O-ring, KNO3 solutions of different pH were exchanged as follows. The liquid cell was gently separated from the sample and flushed with the KNO3 solution of interest; a few drops of KNO3 solution of defined pH were added on top of the mounted sample; the liquid was then gently aspired and drops of the solution were added again; this procedure was repeated several (16) Dufreˆne, Y. F.; Boonaert, C. J. P.; Gerin, P. A.; Asther, M.; Rouxhet, P. G. J. Bacteriol. 1999, 181, 5350.
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Figure 3. Adhesion force maps (left) and adhesion histograms (right) recorded in 1 mM KNO3 solutions of varying pH between the surface of S. cerevisiae and a COOH-terminated probe. Multiple (n ) 256) force-distance curves were recorded over 250 nm × 250 nm areas and adhesion maps were created by calculating the adhesion force for each force curve and displaying adhesion force values as gray levels. Data were obtained using the same cell/probe combination. Similar trends were observed using different probes and independent preparations. times. For studying the influence of Cu(II) ions, drops of a 10 mM CuCl2 solution (pH 7) were deposited on the mounted filter; after 10 min, the sample was rinsed with a 1 mM KNO3 solution using several aspiration/addition cycles as described above. The sensitivity of the AFM detector was estimated using the slope of the retraction force curves, in the region where probe and sample are in contact. Adhesion maps and histograms were obtained by recording 16 × 16 force-distance curves on areas of given size and calculating the pull-off force measured for each force curve.
Results and Discussion Interaction Forces between Carboxyl-Terminated Surfaces. Gold-coated AFM probes and model substrata were functionalized with alkanethiol self-assembled monolayers (SAMs) terminated with carboxyl groups (COOH). The quality of the surface functionalization was assessed using contact angle measurements and XPS. The water contact angle determined for COOH-terminated substrata was smaller than 10°, in agreement with previous studies.17 The C:O:S ratio determined by XPS was 77:19:4, which is very close to values reported in the literature.17 These data are consistent with the formation of closely packed, COOH-terminated monolayers at the gold surface. (17) Bain, C. D.; Troughton, E. B.; Tao, Y. T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321.
Force-distance curves were recorded between functionalized probes and substrata in 1 mM KNO3 solutions of varying pH. As shown in Figure 1, the curves showed differences according to pH: while no adhesion was observed at pH 10, significant adhesion forces, of 1.4 ( 0.2 nN magnitude, were measured at pH 3. Curves recorded in the 3-10 pH range showed adhesion forces for pH values e 5. In addition, it can be seen in Figure 1 that a long-range repulsion force starting at 40 ( 4 nm was probed at pH 10, while a jump to contact was noted at pH 3. Multiple (n ) 256) force curves recorded on 250 nm × 250 nm areas yielded similar results, indicating that the substratum surface properties were fairly homogeneous. The repulsion force at high pH may be attributed to electrostatic double layer forces between negatively charged COO-/COO- surfaces. Indeed, the measured range is in agreement with that measured by others in similar conditions.12 The jump to contact at low pH is due to attractive van der Waals forces and indicates a lack of repulsion between uncharged COOH/COOH surfaces. In the same way, the strong adhesion observed at low pH values can be attributed to hydrogen bonding between COOH/COOH groups while the lack of adhesion at high pH would reflect the electrostatic repulsion between the negatively charged COO-/COO- surfaces. These data,
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Figure 4. AFM “titration curve” obtained by plotting the adhesion force measured between S. cerevisiae and a COOHterminated probe as a function of pH.
consistent with previous studies,11,12 show that the functionalized probes are sensitive to changes of ionization states of surface functional groups. Probing Cell Surface Charges. Having validated the surface properties of the COOH-terminated probes, these were used to record force-distance curves on S. cerevisiae cells in aqueous solution. To this end, topographic images were first recorded to identify a single cell16 and confirm homogeneity and smoothness of the cell surface morphology,8 after which 16 × 16 force-distance curves were recorded over 250 nm × 250 nm areas. As shown in Figure 2, the shape of the force-distance curves was strongly influenced by pH: while no adhesion was observed at neutral and alkaline pH, multiple adhesion forces were observed at pH e 5. Adhesion maps were created by calculating the maximum adhesion force for each force curve and displaying adhesion force values as gray levels. As can be seen in Figure 3, left, the maps always showed fairly homogeneous contrast, indicating that the cell surface properties were homogeneous. The resulting adhesion histograms (Figure 3, right) showed fairly narrow distributions and yielded mean adhesion force values of 0.5 ( 0.2, 1.3 ( 0.2, and 0.9 ( 0.2 nN, at pH 5, 4, and 3, respectively. The change of adhesion forces measured as a function of pH can be related to a change of cell surface electrostatic properties. The surface macromolecules of yeast cells, i.e., glucans and mannoproteins, are known to contain three main types of ionized groups: phosphodiesters, carboxyl, and amino groups.18 The strain investigated here is poor in mannans and rich in proteins, and consequently its electrical properties are mainly determined by a balance of carboxyl and amino groups.19 Hence, the lack of adhesion measured at pH > 6 is likely to reflect the electrostatic repulsion between the negatively charged COO-terminated probe and surface macromolecules bearing negatively charged COO- groups. In contrast, the adhesion force observed at pH < 6 can be attributed to hydrogen bonding between the COOH probe and protonated cell surface macromolecules. Besides surface chemistry, the radius of the functionalized AFM probe (here ∼50 nm) is also expected to affect the adhesion force values. The influence of this parameter could be investigated by varying the thickness of the gold layer. A striking difference between model substrata and yeast cells is that the later showed complex, multiple adhesion peaks along with nonlinear elongation forces and rupture lengths ranging from 50 to 500 nm. Similar features have (18) Eddy, A. A.; Rudin, A. D. Proc. R. Soc. London, B. 1958, 149, 419. (19) Dengis, P.; Rouxhet, P. G. Yeast 1997, 13, 931.
Ahimou et al.
Figure 5. Electrophoretic mobility vs pH curve obtained for S. cerevisiae cell suspensions.
Figure 6. AFM force-distance curves recorded at pH 7 with a COOH-terminated probe for the native S. cerevisiae surface and for the S. cerevisiae surface after treatment with Cu(II).
been reported for fungal spores20 and bacterial cells.21 They can be ascribed to the stretching of flexible cell surface macromolecules, i.e., glucans and mannoproteins in the present work.22,23 Interestingly, we also note that the mean rupture length was correlated with the adhesion force value, i.e., largest rupture lengths being observed at pH 4, suggesting that the adhesiveness of cell surface polymer chains may depend on their conformation. We provide three pieces of evidence indicating that the changes of adhesion forces as a function of pH reflect differences in the ionization state of cell surface functional groups. First, a correlation was obtained between the “titration curve” constructed by plotting the adhesion force vs pH (Figure 4) and the electrophoretic mobility vs pH curve obtained by classical microelectrophoresis (Figure 5). As expected, the microelectrophoresis curve showed an isoelectric point of 4 consistent with the presence of an aminocarboxyl surface.13 At pH values smaller than the isoelectric point, the surface is positively charged due to the presence of NH3+ groups while above pH 4, an increase in the negative value of the mobility is noted which is essentially due to the dissociation of the weak COOH groups. Comparison of Figures 4 and 5 reveals a good agreement between the position of the maximum of the titration curve and the isoelectric point of the cells, suggesting that the modified probes are sensitive to local isoelectric points. It is unclear why lower adhesion forces were observed at pH 3 compared to pH 4. Although the probe surface is expected to be uncharged at pH 3, one (20) van der Aa, B. C.; Michel, R. M.; Asther, M.; Zamora, M. T.; Rouxhet, P. G.; Dufreˆne, Y. F. Langmuir 2001, 17, 3116. (21) Abu-Lail, N. I.; Camesano, T. A. Langmuir 2002, 18, 4071. (22) Pringle, A. T.; Forsdyke, J.; Rose, A. H. J. Bacteriol. 1979, 140, 289. (23) Fleet, G. H. In The yeasts: yeast organelles; Rose, A. H., Harrison, J. S., Eds.; Academic press: London, 1991; p 199.
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Figure 7. Control experiment using nonionizable OH-terminated probes: AFM force-distance curves (A) and adhesion maps (B) recorded between the S. cerevisiae surface and a OH-terminated probe at pH 7 and pH 4.
cannot rule out surface contamination by adsorption of positively charged species, which would result in decreased adhesion forces due to electrostatic repulsion between probe and cell surface. Second, dramatic changes were noted when the cells were treated with Cu(II) ions. At neutral pH, a reversal of the cell surface charge was noted, the mobility of Cutreated cells being 2.5 × 10-8 m2 V-1 s-1 (data not shown) compared to -1.5 × 10-8 m2 V-1 s-1 for untreated cells. This result may be related to earlier biosorption studies,24,25 which have shown that yeasts are particularly effective in removing Cu(II) and that the removal process involves an initial, rapid surface binding of Cu(II). Figure 6 shows that the force-distance curves recorded at pH 7 on Cu(II)-treated cells were different from those obtained on native cells: they displayed shorter repulsion range and adhesion forces of 0.6 ( 0.1 nN. The decrease of the repulsion range may reflect the collapse of the negatively charged surface macromolecules by the Cu2+ cations, while the adhesion forces would essentially be due to Coulomb interactions between the positively charged groups at the cell surface and the negatively charged probe. Although the ability of microbial cells to bind heavy metals is attracting more and more interest in biotechnology for the treatment of polluted waters,4 little is known about the molecular mechanisms of this process. Because of its high sensitivity and high resolution, we believe that the approach presented here has a promising potential for future biosorption studies. Third, one may argue that the changes in adhesion forces essentially reflect the titration of the surface charges of the probe and not of the cells. To address this issue, forcedistance curves were recorded using OH-terminated probes, which are also hydrophilic but do not exhibit a pH-dependent change in ionization. As illustrated in Figure 7, similar pH-dependent behaviors were observed when comparing OH and COOH-terminated probes: that (24) Huang, C.-P.; Westman, D.; Quirk, K.; Huang, C.-P.; Morehart, A. L. Water Sci. Technol. 1988, 20, 369. (25) Huang, C.-P.; Huang, C.-P.; Morehart, A. L. Water Res. 1990, 24, 433.
is a lack of adhesion at neutral pH and the occurrence of multiple adhesion forces at low pH values. This demonstrates that the changes in adhesion forces are related, at least in part, to differences in the ionization state of cell surface functional groups. It is interesting to note that the adhesion force values obtained at pH 4 with OHterminated probes, 0.2 ( 0.06 nN, were much smaller than those obtained with COOH-terminated probes, i.e., 1.3 ( 0.2 nN. This difference is consistent with previous studies on model surfaces in which the forces between OH/COOH surfaces were shown to be much smaller that between COOH/COOH surfaces.12 This behavior is likely to originate from differences in hydrogen bond formation. In summary, we have shown that AFM with chemically functionalized probes is a valuable approach for probing the electrostatic properties of microbial cell surfaces. Force-distance curves recorded between the yeast cell surface and carboxyl-terminated probes show marked differences according to pH, large adhesion forces being observed at pH values smaller than 5. Adhesion maps recorded on 250 nm × 250 nm always show homogeneous contrast, indicating homogeneity of the cell surface properties. The AFM titration curve is correlated with microelectrophoresis data. Treating the cells with Cu(II) ions causes a reversal of the cell surface charge at neutral pH and promotes the adhesion toward the negatively charged probe. Acknowledgment. The support of the National Foundation for Scientific Research (FNRS), of the Foundation for Training in Industrial and Agricultural Research (FRIA), of the Interuniversity Poles of Attraction Program (Federal Office for Scientific, Technical and Cultural Affairs) and of the Research Department of Communaute´ Franc¸ aise de Belgique (Concerted Research Action) is gratefully acknowledged. The authors thank Prof. P. Grange for the use of the atomic force microscope, P. Bertrand for the use of the thermal evaporator, S. Derclaye and Y. Adriaensen for technical assistance, and P. Rouxhet for valuable discussions. LA026273K
