Article pubs.acs.org/Langmuir
Competition of Bovine Serum Albumin Adsorption and Bacterial Adhesion onto Surface-Grafted ODT: In Situ Study by Vibrational SFG and Fluorescence Confocal Microscopy Emilie Bulard,*,† Marie-Pierre Fontaine-Aupart,† Henri Dubost,† Wanquan Zheng,† Marie-Noel̈ le Bellon-Fontaine,‡ Jean-Marie Herry,‡ and Bernard Bourguignon† †
Institut des Sciences Moléculaires d’Orsay, ISMO−CNRS, Université Paris Sud, Bât. 350 91405 Orsay cedex, France INRA AgroParisTech, UMR 1319 MICALIS, 91300 Massy, France
‡
ABSTRACT: The interaction of hydrophilic and hydrophobic ovococcoid bacteria and bovine serum albumin (BSA) proteins with a well ordered surface of octadecanethiol (ODT) self assembled monolayer (SAM) has been studied in different situations where proteins were either preadsorbed on ODT or adsorbed simultaneously with bacterial adhesion as in life conditions. The two situations lead to very different antimicrobial behavior. Bacterial adhesion on preadsorbed BSA is very limited, while the simultaneous exposure of ODT SAM to proteins and bacteria lead to a markedly weaker antimicrobial effect. The combination of sum frequency generation spectroscopy and fluorescence confocal microscopy experiments allow one to draw conclusions on the factors that govern the ODT SAM or BSA film interaction with bacteria at the molecular level. On the hydrophobic ODT surface, interaction with hydrophobic or hydrophilic biomolecules results in opposite effects on the SAM, namely, a flattening or a raise of the terminal methyl groups of ODT. On an amphiphilic BSA layer, the bacterial adhesion strength is weakened by the negative charges carried by both BSA and bacteria. Surprisingly, preadsorbed BSA that cover part of the bacteria cell walls increase the adhesion strength to the BSA film and reduce hydrophobic interactions with the ODT SAM. Finally, bacterial adhesion on a BSA film is shown to modify the BSA proteins in some way that change their interaction with the ODT SAM. The antimicrobial effect is much stronger in the case of a preadsorbed BSA layer than when BSA and bacteria are in competition to colonize the ODT SAM surface.
1. INTRODUCTION All abiotic or biotic surfaces exposed to a fluid can be contaminated by biomolecules, microorganisms, and/or macromolecules as proteins. Their adsorption, often named primary or conditioning film, can occur through biospecific or non specific interactions such as van der Waals, Lewis acid/base and electrostatic interactions.1 More particularly, protein or peptide adsorption on materials (e.g., polymers,2 metals,3 ceramic materials,4 etc.) has a great interest in the biomedical and food industry communities owing to the fact that it could change biodegradability, biocompatibility,5 alter equipment functionalities (such as heat exchangers), or modify the efficiency of cleaning and disinfection processes. Therefore, in recent years, numerous experimental and theoretical studies have been focused on the study of the interactions between proteins and materials. They include contact angle measurements to evaluate the effect of the protein conditioning film on the surface wettability,6−8 adsorption isotherms to identify the different steps of protein adsorption,9 circular dichroism to provide information on protein conformation,10 differential scanning calorimetry to study the effect of protein content on the thermal properties of the surface,11 neutron reflection to determine the thickness of the protein layer,12 surface plasmon © 2012 American Chemical Society
resonance to measure the refractive index changes induced by biomolecules on surfaces,13,14 atomic force microscopy to quantify the adhesion Gibbs free energy,15,16 and mathematical models to predict the strength of adhesion.17 In the biological domain, studies have revealed that protein coated surfaces may reduce pathogenic bacterial adhesion or conversely may provide a better grip.13,18−20 Although a definite conclusion cannot be drawn owing to the too small number of cases studied so far, a tendency that seems to emerge is that protein coating would be unfavorable to bacterial adhesion if bonding between proteins and bacteria is nonspecific. For example, fibronectin or fibrinogen are known to form ligand/receptor bonds with Staphilococci, and they lead to an increase of bacterial adhesion on protein-coated surfaces such as PMMA21 or self-assembled monolayers (SAMs) terminated by isophtalic acid,22 methyl, hydroxyl, carboxylic acid, or tri(ethylene oxide) groups.19,23 On the contrary, bovin serum albumin interacts nonspecifically with bacteria, and protein precoating of SAM,16,22 silicone,20 or Received: July 23, 2012 Revised: November 2, 2012 Published: November 16, 2012 17001
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polyurethane surfaces24 leads to a decrease of bacterial retention. In natural conditions, proteins and bacteria are most often concomitantly present in complex biological fluids, and thus, a competition or a synergism between protein adsorption and microorganism adhesion could be expected. To our knowledge, this competition has never received attention while it is of fundamental interest for public health to improve the engineering of surfaces such as antimicrobial polymers,25,26 ordered brushes,27,28 or SAMs16,22 for better control of biofouling, biomaterial contamination, and decontamination. In this work, we are interested to study such competitive effect of protein adsorption and bacterial adhesion by femtosecond IR-visible broad band sum frequency generation (BBSFG) spectroscopy. The technique has proven to be well adapted to investigate at the molecular level, modifications of the interface in contact with biological fluids29−37 not easily accessible by other conventional techniques. The model interface that we used is composed of a SAM of hydrophobic methyl-terminated octadecanethiol (ODT) molecules grafted on a gold surface.38,39 SAMs are considered as antiadhesion materials.27,40 They are also convenient surfaces for studies of cell-surface interactions by SFG spectroscopy. We have previously demonstrated that the hydrophilic/hydrophobic cell wall character of two strains of Lactococcus lactis bacteria had an impact on the ODT conformation in an aqueous medium.41,42 The same two bacterial strains43,44 were considered in this study. Their adhesion was studied in the presence of the wellknown bovine serum albumin (BSA) plasma protein.45−47 Information on molecular conformation of the ODT-SAM was obtained through the molecular IR spectral signature of the surface in the C−H stretching region.48−50 SFG data were complemented by contact angle measurements, electrophoretic mobility measurements,51 and fluorescence confocal microscopy to highlight bacteria−BSA−ODT interactions. As will be shown, the unique ability of SFG to monitor molecular conformation allows to show that proteins make a film below the bacteria when the SAM is exposed to bacteria and proteins simultaneously, and that simultaneous or sequential exposure to proteins and bacteria does not result in the same adhesion strength.
The main physicochemical difference between the two strains is their cell surface properties: the Lactococcus lactis MG1363 has a hydrophilic polysaccharide pellicle on its cell wall,53 and the Lactococcus lactis MG1363 PRTP+ has a hydrophobic external envelope with anchored proteinase PRTP.54 The technique of microbial adhesion to solvents (MATS) was performed on the two strains to evaluate the hydrophobic/hydrophilic character of the cell surface.41 2.3. Proteins. At the pH value of ∼5.5 used in the experiments, the BSA is known to be in its folded form6 where its projected area on a surface is ∼2 × 10−17 m2. Considering the concentration of bacteria used in this study (4 × 109 UFC/mL) and its surface area (∼3 × 10−12 m2), the cell wall saturation by BSA can occur above a protein concentration of 0.06 mg/mL. Thus, most of the bacterial adhesion experiments were performed using a BSA concentration of 0.1 mg/mL to guarantee that all bacteria could be solvated by BSA. Contact angle measurements were also performed at BSA (Acros) concentrations of 0.5, 0.1, 0.05, and 0.01 mg/mL in distilled water to control the protein adsorption on the ODT SAM. 2.4. Adsorption and Adhesion assays. For protein adsorption studies, the ODT SAM substrates were immersed in BSA solutions at different protein concentrations during 90 min at 22 °C and then washed three times with distilled water to remove all nonadsorbed proteins. An ODT SAM immersed in distilled water under the same conditions was used as a control. In a previous study, we determined the optimal bacterial concentrations and deposition conditions.41 Briefly, ∼200 μL of the bacterial suspension at 4 × 109 cells/mL in distilled water was deposited over the ODT SAM. The solution was allowed to incubate for 90 min. The bacterial surface coverage on the ODT SAM was controlled by staining bacteria with acridin orange (0.01% in water) for 15 min in the dark at room temperature; the dye solution was then washed and replaced by pure water before mounting the sample under a LeicaDM2microscope equipped with an Olympus Camedia C5060WZ digital camera. Three protocols were used to investigate the ODT SAM conformational response to the competition between bacterial adhesion and protein adsorption. In protocol 1, the ODT SAM was exposed to proteins and bacteria simultaneously. A suspension (∼200 μL) of bacteria (4 × 109 cells/mL) mixed with BSA proteins (0.1 mg/ mL) was deposited over the substrate and incubated as described above for bacterial solutions. It must be noted that, with this protocol, proteins are in contact with bacteria in the suspension and thus can adhere to cell walls as well as the solid substrate. It was also checked for each strain that bacterial multiplication does not occur in the presence of BSA in the suspension, indicating that the protein is not a nutriment for L. lactis bacteria (data not shown). The interpretation of the SFG data has required to develop two additional protocols. In protocol 2, BSA is adsorbed before bacteria adhesion: the ODT SAM was immersed in a BSA solution at 0.1 mg/ mL in distilled water during 90 min. After washing to remove the nonadherent proteins, ∼200 μL of the bacteria suspension (cell concentration 4 × 109 cells/mL) was deposited over the BSA−ODT SAM support for 90 min. In protocol 3, BSA was only present on the bacteria cell walls: the solution containing both bacteria (4 × 109 cells/mL) and BSA proteins (0.1 mg/mL) was centrifuged (10 min, 7000g) to remove all free proteins. The pellet of bacteria solvated by BSA was resuspended in distilled water, added on the ODT SAM during 90 min and then washed to remove the nonadherent BSA solvated bacteria. For each protocol, care was taken to prevent bacteria from being exposed to air, because this was found to damage the homogeneity of the bacterial deposit.41,42 2.5. Contact Angle Measurements. The wettability of the surface before and after protein adsorption was evaluated from water contact angle measurements using the sessile drop technique with a “picoLiter goniometer” (Goniometer DSA100M, Krüss, Palaiseau, France). Pico-droplets of deionized distilled water (300 pL) were automatically placed onto the substrate surface by using a piezo dosing unit. Contact angles were monitored during 2 s through a fast CCD
2. MATERIALS AND METHODS 2.1. The ODT SAM. Borosilicate glass substrates (1 cm2) coated with 250 nm of polycrystalline gold film were annealed at 600 °C during 30 s and used within 3−4 weeks of preparation. A selfassembled monolayer of octadecanethiol was obtained by immersion of the gold-coated substrate in a 0.1 mM ODT solution in absolute ethanol during 3 h, rinsed in absolute ethanol, and dried under nitrogen flow.52 In these conditions, a continuous ODT SAM film of thickness ∼2 nm was obtained. The quality of the surface topography was characterized by atomic force microscopy (AFM) as described previously.41 2.2. Lactococcus lactis Bacteria. Bacteria used in this study were L. lactis ssp. cremoris strains MG1363 and its mutant PRTP+ expressing the PrtP protease.53,54 Bacteria were stored at −20 °C in M17 broth (Difco) containing 0.5% (w/v) of glucose and 50% (v/v) of glycerol. They were subcultured twice in M17-glucose broth at 30 °C, until stationary phase was reached and last cultivated overnight at 30 °C. A volume of 90 mL of this final bacterial working culture was harvested by centrifugation (10 min, 7000g, 4 °C) and washed twice. The pellet of bacteria was resuspended in ∼25 mL of distilled water at a final cell density of 4 × 109 cells/mL. 17002
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bacteria and proteins (∼5 μm). The term χNR eiφ is the constant nonresonant response of Au. The phase φ is characteristic of the surface electronic structure and may depend on the chemical nature of adsorbates or the medium surrounding the surface. Aν, ων, and Γ are the Lorentzian amplitude, frequency, and half width of mode ν, respectively. Γ is related to the decay of coherence and of vibrational energy to the surface, supposed to be the same for all CH modes. The vibrational modes are expected to have the same relative phases when vibronic coupling is absent as is the case with alkyl chains. χNR and Aν are effective second order nonlinear susceptibilities, which include geometric and Fresnel factors and several components of the second order susceptibility tensor, according to the surface symmetry. In our fitting procedure, the well-known CH stretch frequencies are fixed and the spectral width is assumed to be the same for all modes. The value of the phase in the different media is also fixed and corresponds to an average obtained over ∼50 experiments. This reduction of the number of fitted parameters avoids obtaining unphysical solutions and allows to obtain reproducible results.56 We have observed a phase change from air (290°) to water solutions (260°), which is most probably related to the fact that the first water layers affect electrostatically the Au substrate. In order to characterize the CH3 intensities, we define a phenomenological parameter R equal to the intensity ratio between the symmetric (including the Fermi resonance which borrows its intensity from the symmetric stretch) and the asymmetric bands: R = (Isym + IFermi)/Iasym. The variations of R directly reflect changes of the methyl tilt angle, which in turn are due to slight changes of the ODT chain twist angle Φ (Figure 1). The relationship between the two angles is obtained through the model described below.48
camera associated to a (×4) zoom and a (×20) microscope objective. An automatic procedure of image acquisition was used to map surfaces of typically 5 × 5 mm2 with one droplet deposited every 0.25 mm. The experiments were conducted at 22 °C. Just before contact angle measurements, substrates (ODT SAM and BSA−ODT SAM) prepared as described above were quickly dried in sterile conditions. An additional control sample was realized by immersing an ODT SAM in distilled water for 90 min (as for BSA adsorption procedure) and then drying it. 2.6. Electrical Charge Determination. In order to evaluate protein adsorption on bacteria cell walls, electrophoretic mobility experiments as a function of pH were carried out at 22 °C with a Zetaphoremeter II (CAD Instrument, Les Essarts le Roi, France), using a 2 mm cell and applying an electric field of 5 V cm−1. BSA (0.1 mg/mL) was mixed with bacteria (4 × 109 cells/mL) during 15 min. Just before zeta measurements, 100 μL of the suspension was added to ∼25 mL of physiologic water (NaCl at 1.5 × 10−3 M) to obtain a sufficiently diluted suspension to measure the electrophoretic mobility of a single bacterium. Experiments were also realized using only planktonic bacteria. The pH of the different suspensions was adjusted by adding nitric acid. 2.7. SFG Spectroscopy. SFG Spectra. Details about our broad band SFG setup can be found in our previously published work.52 Tunable IR pulses (4 μJ, 145 fs, and 150 cm−1 bandwidth) and “visible” pulses (800 nm, 2 μJ, adjustable duration, and bandwidth 1−6 ps ≡ 15−2.5 cm−1) were superimposed onto the ODT SAM (as prepared above) in a collinear copropagating configuration at the incident angle of ∼66° in p polarization. The laser spot size on the sample surface was ∼100 μm. For the samples immersed in water, a CaF2 plate was added onto the substrate to ensure a uniform ∼5 μm thin water layer above the surface sample41 in order to limit absorption of the infrared laser by liquid water. The beam emitted at the sum frequency was focused on the entrance slit of a spectrometer of resolution 0.4 cm−1 at 650 nm, equipped with a cooled CCD camera. The SFG spectra were collected during 30 and 150 s to obtain an acceptable signal-to-noise ratio. SFG vibrational spectra were recorded in the IR wavenumber range 2800−3050 cm−1 with a spectral resolution determined by the width of the visible laser. The spectra mainly consist of three strong vibrational bands assigned to symmetric and asymmetric stretching vibrations of the ODT CH3 group (2877 and 2962 cm−1, respectively) and the Fermi resonance of the CH3 symmetric mode with two quantas of the CH3 bending mode (2936 cm−1) (Figure 4). The bands belonging to CH2 groups at 2851 and 2918 cm−1 (corresponding to the symmetric and asymmetric stretching vibrations of the CH2 groups all along the chain) and at 2905 and 2973 cm−1 (corresponding to the symmetric and asymmetric stretching vibrations of the CH2 group near the sulfur atom at the bottom of the chain) give rise to a very weak SFG intensity. This is due to a combination of local centrosymmetry and to molecular orientation: the dipole moments of pairs of CH2 in trans conformation cancel, while those of unpaired CH2 near the surface remain close to the surface plane and have a small SFG intensity.43 The relative CH3 band intensities depend directly on the orientation of the CH3 group symmetry axis with respect to the surface normal.43 The symmetric [respectively asymmetric] stretch intensity decreases [respectively increases] as the CH3 symmetry axis tilts away from the surface normal. SFG spectral Analysis. The vibrational bands are superimposed on a so-called nonresonant background which exhibits the spectral profile of the IR laser and arises from the broad resonant response of the gold surface electronic states. In order to deconvolute the vibrational bands from this nonresonant background, experimental spectra are fitted to the standard formula:48,55 ISFG(ωIR ) ∝ g (ωIR ) χNR eiφ +
∑ ν
ωIR
Aν − ων + i Γ
Figure 1. Angles that characterize the ODT molecules. Molecules are all-trans except near the S and two first C atoms, implying that most of the C backbone is planar. Black filled circles represent carbon atoms. Hydrogen atoms are omitted for clarity. The gray filled circle represents the sulfur atom. Θ and Φ are the molecular tilt and twist angles, respectively. The directions of the dipole moments of CH3 (symmetric μs3, asymmetric in plane μas3ip, and out of plane μas3op) and CH2 nearest to the surface (symmetric μs2 and asymmetric μas2) are also indicated. All unexplicited angles correspond to trans tetrahedral conformation, except around the S and the two C atoms nearest to the surface. The angles around these three atoms are determined by Θ, Φ, and ψ1. SFG Spectrum Modeling. Molecular conformation changes of the ODT chains are calculated with the SFG spectrum modeling according to the adsorption model proposed by Bourguignon and coworkers,41,48 which allows one to calculate the SFG spectrum of adsorbed alkanethiol molecules for any conformation of the alkyl chain, taking into account optical parameters (such as Fresnel coefficients, refractive index, polarization of laser beams, incident
2
where g(ωIR) is the IR laser spectral profile recorded on a GaAs reference sample in air which provides only a non resonant SFG signal, corrected from absorption by the water layer that surrounds the 17003
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Figure 2. Mapping of the side view of water droplets deposited on the ODT SAM obtained with the “picoLiter goniometer” (two images are spaced by 250 μm) (A) without BSA and (B) after immersion for 90 min in BSA solution of 0.1 mg/mL in distilled water. The spread of the water droplet is correlated to the hydrophilicity of the substrate. It is clear that the substrate is much more hydrophilic when BSA is present. For each mapping, water droplets have the same shape showing the surface homogeneity. angles) and ab intio calculated molecular hyperpolarizability. Molecular conformations are obtained by comparison of calculated and experimental relative intensities of the C−H stretching modes. When the ODT chains are self-assembled, their conformation is constrained to nearly all trans, except near the substrate. The conformation of the ODT chain is then characterized by three independent angles: Θ, the tilt angle of the all-trans part of the alkyl chain with respect to the surface normal, Φ, the “twist” angle characterizing the rotation of the molecular C backbone around the average chain axis, and ψ1, the dihedral angle about the C−C bond nearest to the surface that allows to adapt the chain geometry to the available adsorption sites (Figure 1). All the remaining angles that describe the chain geometry are determined by tetrahedral geometry and all-trans conformation of bonds. The angles about the S atom can be calculated from Θ, Φ, and ψ1. The CH3 tilt angle, that is, the tilt of the CH3 symmetry axis with respect to the surface normal, depends only on Θ and Φ. Furthermore, the ODT SAM structure is characterized by the presence of two types of molecular conformation, subscripted A and B. The C backbones of the two conformations are perpendicular to each other (ΦB = ΦA + 90°).48 In this work, we consider only small modifications of the SAM structure, because the covalent bonding of the ODT chains with the gold surface and the ODT−ODT interactions are very strong with respect to the SAM−BSA or SAM−bacteria interactions. BSA and bacteria are assumed to adsorb on top of the SAM. They cover a large number of ODT chains: ∼235 ODT chains for BSA in its folded form (∼80 × 30 Ǻ 2)46 and ∼2200 ODT chains for bacteria. Insertion of BSA or bacteria would therefore destroy the assembly and would lead to the vanishing of the ODT vibrational bands in the SFG spectrum, which is not observed. As a result, the Θ angle is fixed to the value of 30°: decreasing Θ would imply a compression of the chains while increasing it would break the assembly. So the observed methyl tilt angle changes are considered to be related to changes of the Φ angle alone. 2.8. Confocal Fluorescence Microscopy. The bacterial adhesion and biofilm development on the ODT SAM in the absence and in the presence of BSA proteins was evaluated using confocal laser scanning microscopy (CLSM) (Leica SP2 microscope, Leica Microsystems, France, implemented at the MIMA2 platform of Massy). For these experiments, bacteria were labeled with SYTO9 dye (Invitrogen, France) at a concentration of 4.2 μM. After the bacterial adhesion phase (90 min as described above), the subsequent biofilm formation was achieved after an additional bacterial growing step in a M17glucose broth during 24 h at 30 °C. In each case, the sample was reversed and directly placed under the microscope equipped with a 10× air objective. The fluorescence excitation was obtained using the 488 nm line of a CW Ar+ laser, and the emitted signal was recorded within the range 500−600 nm. Each fluorescence image was processed with a macro-instruction written under ImageTool (UTHSCSA, San Antonio, TX). The percentage of bacteria adhering to the surface was determined after thresholding, binarization, and contour analysis.
3. RESULTS 3.1. Hydrophilic/Hydrophobic Character of the Substrate in Air and in Contact with BSA. The water contact angle measurement for the ODT SAM surface gives rise to a value of Θ = 109 ± 2° corresponding to a high hydrophobicity as expected for a SAM with alkane tails.15,57 It is constant over the entire surface (Figure 2A), demonstrating that the ODT functionalization of the gold surface is homogeneous. The same water contact angle value was obtained when the ODT SAM was first immersed in water during 90 min (the same experimental protocol as for protein adsorption), revealing that water does not modify the functionalized surface (data not shown). When the ODT SAM was exposed to the different concentrations of BSA, a smaller initial contact angle Θ = 64.4 ± 5.3° was obtained (Table 1) (average over random locations Table 1. Initial Water Contact Angles Measured with the picoLiter Goniometer for the ODT SAM Exposed to Air and to BSA Solutions contact angle ODT ODT ODT ODT ODT
in air + BSA + BSA + BSA + BSA
0.5 mg/L 0.1 mg/L 0.05 mg/L 0.01 mg/L
110.4 63.3 65.7 62.5 66.2
± ± ± ± ±
2° 5° 4° 4.3° 3.1°
on the surface, Figure 2B). These results ascertain that, in the whole range of protein concentration used, at least one microscopically homogeneous monolayer of BSA adsorbs on the surface and that the protein coating increases the hydrophilic character of the ODT SAM as previously reported2,45 3.2. Electrostatic State of Bacteria in Contact with Proteins. The interpretation of SFG data requires checking possible interactions between BSA and bacterial cell wall. For this purpose, electrophoretic mobility was performed on suspensions of the two bacterial strains with and without BSA proteins as a function of pH (Figure 3). The presence of proteins modified the isoelectric point, which increased from pH 2 to 3.25 for the hydrophilic bacteria and from 3.0 to 3.75 for the hydrophobic ones. These values were smaller than the isoelectric point of free BSA46 (∼4.5 in distilled water for T = 20 °C) which revealed that the protein adsorbed on the cell wall of the two bacterial strains but did not saturate the bacterial surface even when the protein concentration was in excess (see section 2.3). It must be noted that, at pH = 5.5 (pH of the experiments), the bacterial cell wall is negatively charged 17004
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Figure 3. Electrophoretic mobility as a function of pH: (A) in the case of hydrophilic L. lactis and (B) in the case of hydrophobic L. lactis PRTP+ bacteria. Black points correspond to bacteria alone, and red points to bacteria in contact with BSA proteins.
Figure 4. Experimental (blue dots), fitted SFG spectrum (red line), and deconvoluted vibrational bands (green lines) of an ODT SAM (A) in distilled water; (B) exposed to BSA solution of 0.1 mg/mL in distilled water; (C) exposed to a solution containing both BSA at 0.1 mg/mL and subsequently to hydrophilic L. lactis at 4 × 109 cells/mL in distilled water; (D) exposed to a solution containing both BSA at 0.1 mg/mL and hydrophobic L. lactis PRTP+ in distilled water at 4 × 109 cells/mL. For all spectra, the immersion time of the SAM was 90 min. The SFG reference spectrum (purple lines) is also represented.
in the presence or absence of BSA: the charge of hydrophilic and hydrophobic bacteria deduced from the measurements is −3.3 × 10−13 C and −1.8 × 10−13 C, respectively. 3.3. Effect of BSA Adsorption onto the ODT SAM Probed by SFG. SFG vibrational spectra were first collected as a control from the ODT-SAM in air (solid/air interface) and in distilled water (solid/liquid interface) (Figure 4A) for comparison to our previous report.41 The R ratio increases from R = 3.7 ± 0.5 for the ODT SAM in air to R = 5.8 ± 0.5 for the substrate in water, showing a decrease of the methyl tilt angle (2.6 and 3.9° for A and B molecules respectively) (Table 2) and confirming that in an aqueous environment the CH3 symmetry axis is closer to the surface normal than in air, corresponding to a “brush effect” on ODT.41
The BSA-ODT SAM SFG spectrum obtained at the solid/ liquid interface and its deconvoluted bands are shown in Figure 4B. This deconvolution sums up the contributions of “free” ODT and ODT in contact with BSA. The best fit was obtained for a BSA coverage of ∼95%, in good agreement with the experimental total coverage estimated from the water contact angle measurements. The corresponding R parameter, Φ, and CH3 tilt angles (see section 2.7) are almost equal to the values obtained for ODT exposed to distilled water (Table 2). BSA coating has a similar effect of straightening the methyl terminal part of the alkyl chain as water. It has been reported that the presence of CH3 groups in the BSA amino acids can give rise to specific additional SFG signal33,58 on silicone or gold surfaces. Therefore, we have 17005
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Table 2. Results of SFG Spectrum Deconvolution (intensity R ratio between symmetric and asymmetric CH3 bands, see text) and Modelling (Φ angles, CH3 tilt angles of A and B molecules in the adsorption model) support ODT ODT ODT ODT ODT ODT ODT ODT ODT ODT ODT
in air in distilled water with BSA in distilled water with hydrophilic bacteria in distilled water with BSA + hydrophilic bacteria in distilled water with BSA then hydrophilic bacteria in distilled water with hydrophilic bacteria solvated by BSA in distilled water with hydrophobic bacteria in distilled water with BSA + hydrophobic bacteria in distilled water with BSA then hydrophobic bacteria in distilled water with hydrophobic bacteria solvated by BSA in distilled water
R parameter
Φ angle A/B (deg) (±3°)
CH3 tilt angle A/B (°) (±1.0°)
± ± ± ± ± ± ± ± ± ± ±
242/332 250/340 248/338 260/350 264/354 (±4°) 256/346 262/352 238/328 256/346 (±6°) 263/353 (±4°) 250/340
55.1/15.8 52.5/11.9 53.2/12.9 48.9/7.5 47.5/6.5 (±1.5°) 50.6/9.4 48/6.7 55.8/16.8 50.4/9.4 (±2.5°) 47/6.6 (±2°) 52.5/11.9
3.7 5.8 5.5 8.8 10.5 7.7 10 3.8 8 10 5.8
0.5 0.5 0.3 0.5 1.5 0.9 0.6 0.5 2 2 0.5
Figure 5. Deconvoluted vibrational bands of an ODT SAM exposed during 90 min to (i) a bacterial suspension in distilled water (pink dashed lines), (ii) a solution containing both BSA at 0.1 mg/mL and bacteria at 4 × 109 cells/mL in distilled water (green lines), and (iii) a BSA solution at 0.1 mg/mL, then washed and exposed to L. lactis bacteria in distilled water (4 × 109 cells/mL) (blue lines); (A) hydrophilic bacteria and (B) hydrophobic bacteria PRTP+.
them completely; in addition, the suspending fluid is a mixture of BSA-solvated bacteria, free BSA. Whatever the hydrophobic or hydrophilic character of the bacterial cell wall, the relative vibrational CH3 band intensities are equal, and significantly different from those obtained for the ODT SAM exposed to BSA alone (Figure 4B). The R parameter increases in the presence of bacteria, corresponding to a decrease of the CH3 tilt angle (Table 2), by comparison to the case of BSA alone. Similar data were obtained (Figure 5, Table 2) when BSA was adsorbed first on the ODT SAM, before bacterial suspension was added (protocol 2). By contrast, the deposition of a solution of bacteria solvated with BSA (protocol 3) on the ODT SAM has only a small effect on the substrate conformation with respect to that of bacterial adhesion alone (Table 2). A small increase of the R value was significant only in the case of hydrophobic bacteria. 3.5. Impact of Proteins on Bacteria−Surface Interactions. After bacterial adhesion or biofilm development, the substrates were reversed and examined by confocal fluorescence microscopy (see Materials and Methods). Figure 6A shows the coverage of hydrophilic and hydrophobic L. lactis PRTP+ bacteria still adherent onto the surface of ODT SAM after the reversal, in the absence and in the presence of BSA: the
performed BSA adsorption onto a deuterated ODT SAM to remove the contribution of the SAM to the C−H signal and be able to detect CH3 from the BSA: we found that in our experimental conditions the contribution of CH3 groups from BSA is below our detection limit (spectrum not shown). Several hypotheses can be considered: either BSA proteins are not ordered onto the SAM (as already observed for high concentrated BSA adsorption on gold30) or there is a local inversion symmetry of the CH3 groups within BSA proteins (as already reported for BSA adsorption onto fused silica34). It has been reported that the SFG signal of BSA adsorbed on hydrophobic surfaces (as is the case for the ODT SAM) is much smaller than on hydrophilic surfaces;35 it was proposed that a loss of α-helical structures on the hydrophobic surfaces leaded to a more random arrangement of the methyl groups.37 3.4. Competition between BSA Adsorption and Bacterial Adhesion-SFG. Figure 4C and D depicts the experimental SFG spectra and the corresponding deconvoluted vibrational bands of the CH3 groups collected upon deposition of a solution containing both proteins and bacteria on the ODT SAM (protocol 1). It must be recalled that even though BSA adheres to both hydrophobic and hydrophilic bacteria as indicated by electrophoretic mobility, the proteins do not cover 17006
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Figure 6. (A) Confocal epifluorescence images (92.6 × 91.5 μm2) of L. lactis adhesion on the ODT SAM substrates after 90 min of adhesion and sample reversal (right) and its corresponding bacterial recovery (left). (B) Confocal epifluorescence images (92.6 × 91.5 μm2) representative of the attachment of biofilms formed by (1) hydrophobic L. lactis PRTP+ bacteria, (2) hydrophobic L. lactis PRTP+ bacteria mixed with BSA in solution, (3) hydrophobic L. lactis PRTP+ bacteria on the primary BSA film/ODT SAM, (4) hydrophilic L. lactis bacteria, (6) hydrophilic L. lactis bacteria mixed with BSA in solution, and (6) hydrophilic L. lactis bacteria on the primary BSA film/ODT SAM after 24 h of incubation at 30 °C after reversal of the substrate.
hydrophobic bacteria coverage was larger than that of hydrophilic bacteria, and the presence of the proteins decreased strongly the bacterial coverage. The coverages of Figure 6A must be compared to the coverage of 60% estimated from SFG spectra on substrates that were not reversed:34 about 30% of hydrophobic and more than 50% of hydrophilic bacteria were detached from the ODT SAM by sample reversal. There remained even less bacteria when BSA was present in the solution during adhesion and almost none when BSA was preadsorbed. Similar results were obtained after biofilm formation (Figure 6B). The biofilm covered the entire surface only in the case of hydrophobic bacteria on ODT SAM. The presence of BSA during adhesion decreased the biofilm coverage and thickness down to a few bacteria in the case of hydrophilic bacteria. The preadsorption of BSA completely prevented the biofilm formation in the case of hydrophilic bacteria, and left only scarce hydrophobic bacteria.
Hydrophilic and hydrophobic bacteria have opposite effects on ODT, and their behavior can be rationalized on the basis of hydrophobicity: ODT responds to hydrophilic (hydrophobic) bacteria by limiting (increasing) the contact area with bacteria and methyl groups raise (flatten). When the ODT SAM is covered by BSA alone, the ODT methyl groups adopt an orientation intermediate between the ones they have in the presence of hydrophilic and hydrophobic bacteria. This is not unexpected because the surface of proteins is partly hydrophobic and partly hydrophilic. When the ODT SAM is exposed to bacteria that were previously solvated by BSA (protocol 3), the methyl orientation angle of ODT remains different for the two types of bacteria. BSA covered hydrophobic bacteria have a smaller flattening effect than the bare ones, while hydrophilic bacteria with adsorbed BSA have a very similar effect as bare bacteria. This can be interpreted in a straightforward manner: part of the hydrophobic bacteria cell wall is now hydrophilic. In the other cases (preadsorption of a BSA film on the ODT SAM or simultaneous exposure of the substrate to BSA and bacteria, protocols 2 and 1, respectively), the effect produced on the SAM is the same for hydrophobic and hydrophilic bacteria. Contact angle and SFG measurements show that the
4. DISCUSSION SFG experiments show that the ODT SAM conformation is affected by the presence of BSA and bacteria. The effect of bacteria alone has been studied in our previous study.41,42 17007
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Figure 7. Schematic adhesion/adsorption processes onto the ODT SAM surface of bacterial adhesion: (A) on the primary BSA film and (B) mixed with BSA in distilled water. It should be noted that, in the case of bacterial colonization on the primary BSA film, after sample reversal, the presence of the proteic film was not controlled.
preadsorbed BSA film is homogeneous and is at least one monolayer thick. In the case of simultaneous exposure to BSA and bacteria, adsorption and adhesion are probably limited by transport. Bacteria diffuse much more slowly than BSA: the diffusion coefficient of nonmotile bacteria59 is around 10−12 m2·s−1, whereas the BSA diffusion coefficient is about 6.10−11 m2·s−1 in aqueous solvents at room temperature.60,61 Therefore, it is expected that BSA adsorbs before bacteria adhere. Thus, we may reasonably propose that protocols 2 and 1 produce a similar BSA film. This is consistent with the finding that hydrophobic and hydrophilic bacteria have nearly the same effect on the SAM, independent of the protocol and of the hydrophobic−hydrophilic character of bacteria: the SAM is in direct contact with BSA and not with bacteria. What was not anticipated is that the effect produced on ODT by BSA is different depending on the presence or absence of the bacteria. It implies that bacteria modify the BSA film which in turn has a different interaction with ODT. Further SFG experiments probing BSA through the amide function33,37,47,49 are required to prove protein conformational change induced by their interaction with bacteria. Fluorescence confocal microscopy allows one to gain more insight on bacterial adhesion onto ODT in the absence or in the presence of BSA. However, it requires one to reverse the samples, which may detach weakly bounded bacteria. Indeed the analysis of epifluorescence images of ODT with bare bacteria leads to an estimation of bacterial coverage smaller than by SFG, the coverage of hydrophilic bacteria being most affected by the sample reversal. In the case of protocol 2 (preadsorption of BSA), epifluorescence shows an even more dramatic effect of sample reversal: there remains no hydrophilic bacteria and very few hydrophobic ones on the sample. This implies that SFG is sensitive to bacteria that are too weakly bonded to resist the effect of sample reversal.
The observed antimicrobial effect of BSA is consistent with the literature.16,20,22,24 The striking new result of the present study is that the coexposure to BSA and bacteria (protocol 1) results in only a moderate antimicrobial effect with respect to the adhesion on preadsorbed BSA; the antimicrobial effect is very limited in the case of hydrophobic bacteria. This implies that the measurement of the antimicrobial effect on surfaces precoated with proteins is not sufficient to evaluate the practical efficiency of the proteins, which may depend on the exposure conditions. This might explain contradictions in the literature. The results of fluorescence confocal microscopy imply that the adhesion strength of hydrophobic bacteria is significantly stronger than that of hydrophilic bacteria. On the bare ODT SAM, hydrophobic interactions are believed to be larger than hydrophilic−hydrophobic ones. In the presence of BSA, hydrophobic and hydrophilic interactions are probably surpassed by electrostatic interactions. Our electrophoretic measurements show that BSA and bacteria are negatively charged, and that the charge of hydrophobic bacteria is twice smaller than that of hydrophilic bacteria: this suggests that the adhesion strength may be weakened by repulsive electrostatic interactions when BSA is present. This hypothesis is supported by other studies highlighting steric repulsion between negatively charged bacteria cell walls and negatively charged proteins such as BSA.20 Finally, the stronger adhesion of bacteria in the case of simultaneous exposure of the SAM to BSA and bacteria is most probably related to the presence of preadsorbed BSA on the bacteria cell walls: preadsorbed BSA on the bacteria would increase the adhesion strength of bacteria to the BSA film (Figure 7), implying that BSA−BSA interactions are stronger than BSA−bare-bacteria interactions. We have seen above that preadsorbed BSA on the bacteria cell walls has also a 17008
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measurable effect in the case of adsorption on the bare ODT SAM (protocol 3).
5. CONCLUSION The interaction of bacteria and proteins with a well ordered ODT SAM has been studied in different situations where proteins were either preadsorbed on the bacteria, preadsorbed on the sample, or adsorbed simultaneously with bacterial adhesion. The combination of SFG and fluorescence confocal microscopy experiments allows one to draw conclusions on the factors that govern the interactions of bacteria and proteins with the ODT SAM at the molecular level. Hydrophobic and hydrophilic bacteria have not the same adhesion strength. Preadsorbed proteins on the bacteria cell walls change the adhesion. Coating the sample with proteins has a strong effect on adhesion strength, but protein precoating and simultaneous exposure to proteins and bacteria lead to very different antimicrobial behavior. Bacterial adhesion on preadsorbed BSA is very limited, while the simultaneous exposure of ODT SAM to proteins and bacteria lead to a markedly weaker antimicrobial effect. This implies that the evaluation of the antimicrobial effect of protein films should take into account the adsorption conditions of the proteins. In life conditions, proteins and bacteria are simultaneously present in fluids, which is more favorable to bacterial adhesion than protein precoating.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. These authors contributed equally. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors wish to thank the MIMA2 platform (INRA, Massy, France) for allowing us to use the scanning electron microscope and the CPBM platform of LUMAT (Orsay, France) for the use of their biological infrastructures.
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