Article pubs.acs.org/Biomac
Interactions of Glycosphingolipids and Lipopolysaccharides with Silica and Polyamide Surfaces: Adsorption and Viscoelastic Properties Jenia Gutman,† Yair Kaufman,† Kazuyoshi Kawahara,‡ Sharon L. Walker,§ Viatcheslav Freger,∥ and Moshe Herzberg*,† †
Department of Desalination and Water Treatment, Zuckerberg Institute for Water Research, Albert Katz International School for Desert Studies, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede-Boqer Campus, Midreshet Ben Gurion 84990, Israel ‡ Department of Applied Material and Life Science, College of Engineering, Kanto Gakuin University, Yokohama, Japan § Department of Chemical and Environmental Engineering, University of California, 900 University Avenue, Riverside, California 92521, United States ∥ The Wolfson Department of Chemical Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel ABSTRACT: Bacterial outer membrane components play a critical role in bacteria−surface interactions (adhesion and repulsion). Sphingomonas species (spp.) differ from other Gram-negative bacteria in that they lack lipopolysaccharides (LPSs) in their outer membrane. Instead, Sphingomonas spp. outer membrane consists of glycosphingolipids (GSLs). To delineate the properties of the outer membrane of Sphingomonas spp. and to explain the adhesion of these cells to surfaces, we employed a single-component-based approach of comparing GSL vesicles to LPS vesicles. This is the first study to report the formation of vesicles containing 100% GSL. Significant physicochemical differences between GSL and LPS vesicles are reported. Compositiondependent vesicle adherence to different surfaces using quartz crystal microbalance with dissipation monitoring (QCM-D) technology was observed, where higher GSL content resulted in higher mass accumulation on the sensor. Additionally, the presence of 10% GSL and above was found to promote the relative rigidity of the vesicle obtaining viscoelastic ratio of 30−70% higher than that of pure LPS vesicles.
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Re LPS) that have the shortest sugar moiety.3,4 Clear graphical depictions of LPSs are shown elsewhere.5,6 Sphingomonas spp. differ from other Gram-negative bacteria in lack of LPS. Instead, the outer leaflet of the outer membrane in Sphingomonas spp. is composed of glycosphingolipids (GSLs)7 (Figure 1). The complete chemical structure of the GSL from S. paucimobilis IAM 12576 was first reported by Kawahara et al.8 The hydrophobic portion was found to be heterogeneous, with the oligosaccharide portion consisting of two main fractions, GSL-4A and GSL-1, Man-Gal-GlcNGlcA tetrasaccharide, and a GlcA monosaccharide, respectively. Interestingly, prokaryotic Sphingomonas spp.’s GSL resembles cell membrane components found in some eukaryotic cells. Mammalian glycosphingolipids begin with either glucose (glucosylceramides - GlcCer) or galactose (galactosylceramides - GalCer), attached to the 1-hydroxyl of Cer via a β-glycosidic bond.9 They are known to form lipid domains, often referred to as “rafts”, in the cell membrane, and their properties have been
INTRODUCTION The cell envelope of Gram-negative bacteria is composed of the cytoplasmic membrane, the peptidoglycan layer, and the outer membrane, which is strictly asymmetric with respect to its lipid composition. While the inner leaflet of the outer membrane contains only phospholipids, the outer leaflet is usually composed of lipopolysaccharides (LPSs).1 LPSs are composed of two main parts: the lipid part that anchors the LPS to the outer membrane (lipid A) and the hydrophilic oligo - or polysaccharide moiety. The core polysaccharide region of the LPSs, which is the portion immediately linked to the lipid A molecule, is two 2-keto-3-deoxyoctonate (KDO) monosaccharides. A core polysaccharide chain is extended from the KDO region. The outermost portion of the LPS assembly linked to the core polysaccharide region is the O-antigen. The exact size and composition of this polysaccharide is strain-specific. Gramnegative cells with LPSs terminating after KDO are referred to as “deep rough mutants” due to the unsmooth colony appearance on a Petri dish.2 LPS lacking the O-antigen but containing the core polysaccharide region characterize “rough mutants.” Because of experimental restrictions, most of the LPS investigations are limited to deep rough mutant LPS (termed as © 2014 American Chemical Society
Received: February 17, 2014 Revised: May 14, 2014 Published: May 16, 2014 2128
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Figure 1. Monosaccharide-type glycosphingolipid GSL-1 from Sphingomonas paucimobilis (GSL-1), deep rough mutant Re LPS from Salmonella Minnesota (Re LPS) according to Wiese and Seydel, 1999 and 1,2-dimyristoyl-sn-glycero-phosphoethanolamine-N-(lissamine-Rhodamine B sulfonyl)- (ammonium salt) modified chemical structure (Rh-PE).
extensively studied in the context of signal transduction,10 embryonic development, and immune receptor recognition.11 In addition, the levels of GlcCer/GalCer are altered by a wide spectrum of diseases, including neuronal disorders,12 cancer,13 diabetes,14 Gaucher disease15 and skin disorders.16 Like the work done with LPS, where studies mainly have been limited to mutants having the shortest sugar moiety, the GSL with the shortest sugar moiety, GSL-1, is within the scope of this and other scientific studies.3,4,17−19 In general, little is known about the properties of the outermembrane of Sphingomonas spp. and, more specifically about the bacterial GSL characteristics. In the 1990s, fundamental studies revealing GSL’s chemical structure,8 cell envelope structure,20 and physicochemical characterization21 were performed. Also, an electrophysiological test was carried out to determine the possible physiological roles of bacterial GSLs.3 Sphingomonas spp. have drawn scientific and biotechnological attention only recently, mainly due to their ability to mineralize or degrade recalcitrant compounds such as dioxin,22 nonylphenole,23,24 poly(ethylene glycol),25 pentachlorophenol,26 and other polycyclic aromatic hydrocarbons,27,28 yet the outer membrane component of Sphingomonas spp., prokaryotic GSL, has received little scientific attention.6 Interestingly, the Kikkoman Company patented industrial-scale Sphingomonas GSL production (patent number US6514744 B2). Because LPS and GSL are bacterial outer membrane components, at the interface of the cell and its environment, they are substantial and critical factors in bacterial adhesion,5,29 host−parasite interaction,30 and virulence determination.3,5,31 Because LPS and GSL are not the sole components of the exterior of bacterial outer membrane and because using the entire bacterial cell in adhesion experiments introduces significant complexity due to membrane components such as pili, flagella, and extracellular polymeric substances (EPSs), a single-componentbased approach is practiced in this study.
To investigate the differences between GSL and LPS in their contribution to cell attachment to surfaces, small unilamellar vesicles (SUVs) of pure GSL (100% GSL-1), 10% GSL-1 (with 90% Re LPS), 50% GSL-1 (with 50% Re LPS) and pure Re LPS vesicles (100% Re LPS) were produced and compared with respect to their size, charge, and adhesion to hydrophobic and hydrophilic surfaces. SUVs are an often-used model of cell membranes with maintained fluidity.28,32 The adhesion of the SUVs and SUV layer properties such as viscoelasticity were studied using a quartz crystal microbalance with dissipation (QCM-D)33,34 monitoring technology. In addition to the measurements of a bound mass, which is deduced from changes in the resonance frequency, Δf, of the piezoelectric sensor, the QCM-D technique also provides information on the rheology of biomolecular layers via changes in the damping, ΔD, of the crystal.34 In this way, adsorbed lipid vesicles, which induce low damping, can be distinguished from viscous supported membranes, which induce high damping.33,34 The method thus provides both real-time, label-free monitoring of the formation of close-packed layers of intact lipid vesicles and, via changes in the damping, information on the rigidity/softness of bound entities. This study reveals significant differences in the adhesion properties to hydrophobic and hydrophilic surfaces of the GSL1 vesicles in comparison with Re LPS as well as the effect of GSL-1 on SUV viscoelastic properties. Results open new avenues for future studies that can shed light on: (i) biochemical mechanisms promoting Sphingomonas spp.’s ability to biodegrade recalcitrant hydrophobic compounds27,35 and prevail in oligotrophic environments;36−38 (ii) biofouling phenomena in water reclamation processes, such as reverse osmosis (RO);39,40 and (iii) various biotechnological processes.41,42 Polyamide surface was chosen in this study to address the characteristics of hydrophobic surfaces pertaining to biofilms formed in water-treating systems, in which dominance 2129
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Figure 2. CryoTEM images of GSL-1 (A,C) and LPS Re (B,D), rehydration solution of 100 mM NaCl (upper row, A,B), and 200 mM NaCl bottom row (C,D). The scale bar is 100 nm. Within the dashed circle (D) and at its proximity the rough texture is an early ice formation in the sample. mixture was placed in 50 °C for 1 h, followed by bath sonication at 50 °C for1 h. The mixtures were periodically shaken by hand. Each liposome suspension was bath-sonicated, followed by a tip sonication (Sonics Vibra-Cell, Newtown, CT) at 60% of the sonicator net power (130 W, frequency 20 kHz) using four on and off cycles of 60 and 30 s, respectively. Samples were subjected to centrifugation (10 000 rpm for 10 min) to get rid of any titanium nanoparticles ablated from the tip sonicator. Fluorescent SUVs were obtained by the addition of 1% RhPE tagged phospholipid to the glycolipid mixture, prior to the evaporation of solvents by N2. Confirmation of Vesicle Formation-CryoTEM. Cryo-transmission electron microscopy (cryo-TEM) was applied to observe the vesicles formed in 100 and 200 mM NaCl. A drop of the solution was placed on a 300-mesh Cu grid coated with a porous carbon film (Lacey substrate, Ted Pella) in a 30 °C chamber. Excess liquid was removed, and the grid was plunged into liquid ethane using an automatic plunger (Leica EM GP) to vitrify the solution. Vitrified samples were transferred to a cryo-holder (Gatan 626) and were examined at −178 °C on an FEI Tecnai 12G2 TWIN TEM. The images were recorded on a Gatan 794 charge-coupled device camera at 120 kV in low-dose mode. ζ-Potential and Dynamic Light Scattering Measurements of Vesicles. ζ-Potential and dynamic light scattering (DLS) measurements were carried out using a Zetasizer Nano ZS (Malvern) at room temperature. The scattered intensity of the 633 nm laser beam was recorded at an angle of 173° relative to the incident beam. The size of the particles was determined using Zetasizer 7.01 software using the Smulochowski equation. The results were analyzed using ANOVA test by rank. Hydrophobicity/Hydrophilicity of the GSL-1 and Re LPS: Contact-Angle Measurements. The glass was first modified by polyethylenimine (PEI) using a published protocol to ensure uniform and complete coverage of the glass substrate by the different vesicles (pure or combined LPS and GSL).43,45 PEI is used to minimize electrostatic repulsion between the negatively charged biological
of Sphingomonas spp. was revealed. Additionally, this is the first study to examine the physicochemical properties of SUVs containing pure GSL.
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EXPERIMENTAL SECTION
Lipid Selection. LPSs (rough strains) from Salmonella enterica serotype minnesota Re 595 (Re mutant), LPS Re, were purchased from Sigma-Aldrich, L9764. The glycosphingolipids GSL-1 were extracted from Sphingomonas paucimobilis IAM12576 with the chloroform/methanol method,8 followed by elution from a silica gel column by stepwise increase in methanol fraction. 1,2-Dimyristoyl-snglycero-phosphoethanolamine-N-(lissamine-Rhodamine B sulfonyl) (ammonium salt), Rh-PE, were purchased from Avanti-Polar lipids, US (PL 810157P). The chemical structure of these lipids is shown in Figure 1. It is evident that the hydrophobic part of GSL-1 is heterogeneous with respect to the occurrence of two different sphingosine derivatives (erythro-1,3-dihydroxy-2-amino-octadecane and erythro-1,3-dihydroxy-2-amino-cis-13,14-methylene-eicosane), which are present in a ratio 1:4. Vesicle Preparation. A modified protocol of vesicle preparation was developed based on previously published methods.43,44 The sonication technique was preferred over extrusion due to the high adhesion of GSL-1 SUVs to the track-etched polycarbonate membrane of the extruder. For lipid stock solution, GSL-1 and LPS Re were dissolved in chloroform/methanol (10:1) solution, 1 mg of lipid powder to 1 mL of solution, followed by 1 h of incubation at 60 °C, resulting in a clear solution. The stock solutions were subsequently stored at 4 °C. For liposome preparation, the stock solution was placed in a glass vial, and the solvent was evaporated with N2. The glass vials were stored overnight under vacuum. For each 200 μg of lipids, 1 mL of hydration background solution (solution used for rehydration and swelling of the dry lipid layer, resulting in the formation of multilamellar vesicles) was added. Minimal ionic strength of 100 mM was required for a stable vesicles solution to form. The 2130
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Table 1. Particle Average Size (diameter, nm), As Measured by Dynamic Light Scattering (DLS), and the ζ Potential (mV) of Vesicles, as a Function of Ionic Strength and Lipid Composition, Standard Error of Five Experiments Is Presented 100 mM NaCl
200 mM NaCl
vesicle type
size
ζ potential
size
ζ potential
LPS Re 90% LPS Re 10% GSL-1 50% LPS Re 50% GSL-1 GSL-1
59.1 ± 3.9 53.5 ± 3.0
−34.5 ± 0.1 −32.9 ± 2.5
59.6 ± 8.4 63.6 ± 7.6
−25.7 ± 0.6 −25.8 ± 2.1
56.7 ± 3.3
−33.5 ± 1.2
50 ± 6.8
−26.5 ± 2.4
51.1 ± 0.5
−36.8 ± 4.3
54.6 ± 2.9
−30.9 ± 1.9
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molecules and negatively charged glass surface. The PEI surface was exposed to SUV mixtures for 1 h at room temperature, followed by gentle washing of the slides by hydration solution (100 mM NaCl). The contact angles were measured using a sessile drop of deionized water on an OCA-20 contact-angle analyzer (DataPhysics). Every measurement was repeated and averaged for at least five drops (25 μL) on each surface sample. The results were analyzed using t test. Fluorescent Microscopy. Images were acquired using an Axio Imager A1M upright microscope (Zeiss) equipped with a filter set 20 (Zeiss) (excitation 546/12; beam splitter 560; emission 575−640 nm) and an AxioCam MRm camera (Zeiss) using 10× objective. Confocal Microscopy. Microscopic observation and image acquisition were performed using Zeiss-Meta 510, a CLSM equipped with Zeiss dry objective LCI Plan-NeoFluar (63× magnification). The CLSM was equipped with detectors and filter sets for monitoring fluorescent phospholipid-containing SUVs (excitation and emission wavelengths of 560 and 583 nm, respectively). We mounted 200 μL of SUV solution for 2 h on a 1 cm × 1 cm nanofiltration (NF 270) flatsheet membrane surface (Dow-Filmtec), the upper layer of which is composed of polyamide. The samples were gently washed with 100 mM NaCl prior to observation. QCM-D Experiments and Analysis. QCM-D experiments were performed using AT-cut quartz crystals mounted in an E4 system (Qsense AB, Gothenburg, Sweden). Two types of crystals were used in this study: crystals coated with (1) a 50 nm silica layer and (2) a ∼50 nm polyamide layer. Both crystals have a resonance frequency of ∼5 MHz (Q-Sense AB, Västra Frölunda, Sweden). Before each measurement, the crystals were soaked in 2% (w/w) sodium dodecyl sulfate (SDS) solution for 30 min, thoroughly rinsed with double-distilled water, and dried with N2 gas, and the silica-coated sensors were further cleaned in a UV/Ozone ProCleaner (BioForce Nanoscience) for 30 min. All QCM-D experiments were performed under flow-through conditions using a digital peristaltic pump (IsmaTec, IDEX) operating in a sucking mode. The flow rate of the working solution in the QCMD flow cell was 100 μL/min. The following solutions were injected sequentially into the QCM-D system: (i) double-distilled water baseline, 20 min; (ii) background solution, 10 min; (iii) GSL or LPS vesicles, 0.2 mg/mL, in background solution, flow rate 10 μL/min, 150 min; and (iv) background solution, flow rate 10 μL/min, 180 min. The adsorption and dissipation kinetic curves were built by Q-Tools software (Q-SENSE, Sweden). The variations of frequency shift (Δf, Hz) and dissipation factor (ΔD) were measured for five overtones n = 3, 5, 7, 9, and 11. The viscoelastic properties of the SUV layers were calculated based on the Voigt model according to Voinova et al. 1999.46 The density and viscosity of the solution used in this model were 1 g/cm3 and 10−3 Pa·s, respectively. The density of the adsorbed layer was fixed at 1.030 g/cm3 following the recommendations of Gurdak et al. 2005.47 The best fitting values of the shear viscosity (η), shear modulus (μ), and Voigt thickness of the adsorbed layer were obtained by modeling the experimental data of Δf and ΔD for three overtones (5, 7, and 9) using the Q-Tools software provided by QSense AB (Sweden). The goodness of fit was in accordance with QTools guidelines, in which a good overlap between modeled and measured Δf and ΔD was observed as well as relatively low chi square values between the model and the experimental results.
RESULTS AND DISCUSSION GSL-1 and Re LPS Vesicle Characterization: Size, ζPotential, and Hydrophobicity. Figure 2 shows cryoTEM images of GSL-1 and Re LPS lipids confirming vesicle formation. The typical size of 50−60 nm was observed for both GSL-1 and Re LPS vesicles. Pure Re LPS and GSL-1 solutions resulted in a round-shaped vesicles (Figure 2), which is different from mammalian-source glycosphingolipids. These images are compared with the data reported by Varela et al. (2013). In the Varela et al. study, mammalian-sourced GSL vesicles, composed of up to 10% GlcCer in a mixture with 1palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) (10% GlcCer, 90% POPC), had a mean diameter of 100 nm and were round-shaped. With increasing fraction of GlcCer, vesicles tended to increase in size, aggregate, and form tubular structures. For very high GlcCer fraction (∼92%), crystal formation started to occur.48−50 Solutions of pure GlcCer showed a well-defined crystalline structure. This was explained by the highly ordered structures formed by GlcCer.48,51,52 This was not the case in the current study of Sphingomonas spp. glycolipids (GSL-1), and only vesicles and no crystal-like structures were observed for solution of pure GSL-1. The main difference between the two sources of glycosphingolipids is that Sphingomonas glycolipids contain α-linked glucuronic acid with the OH at the C-4′-position of the sugar in the equatorial position. The impact of length of the lipid chains is challenging for comparison because mammalian-source glycosphingolipids that were previously investigated obtain a range of acyl chains and sphingoid bases, all resulting in a structured gel-phase or crystalline structure once examined as a pure solution.53,54 The discussed dissimilarity between the eukaryotic and prokaryotic GSL is initially evident from the fact that in eukaryotic cells the physiological levels of GSL in the extracellular leaflet of the plasma membrane are very low (1 to 2%)55 and increase upon an apoptotic stimulus,56 whereas in Sphingomonas spp., the outer membrane leaflet is composed of 100% GSL,20,41,57,58 with a sustained fluidic structure. After verifying vesicle formation for a range of GSL-1 fractions, including pure GSL-1 solutions, further characterization was conducted by measuring the size distribution and ζ potential of the vesicles (Table 1). ζ potential is the electrical potential at the plane of the shear of the colloidal surface. The diameter of the SUVs ranged from 50 to 64 nm with similar size and smaller standard error at 100 mM NaCl, in comparison with the 200 mM condition. According to the relatively low standard errors of the DLS values and Figure 2, we could plausibly assume that both GSL and LPS vesicles were monodispersed, enabling us to delineate the vesicles ζ potential. It should be mentioned that the exposure of the charged moieties toward the bulk liquid will affect vesicle surface charge 2131
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surface)65 was achieved for GSL-1 (emission intensity of 60 ± 23 and 82 ± 16 au, respectively), the more hydrophobic compound. Re LPSs adhered to the silica surface in a patchy nonuniform manner, although both silica and Re LPSs are hydrophilic (emission a.u. of 13 ± 8 and 41 ± 18 for silica and polyamide, respectively). QCM-D results (Figures 4 and 5) confirmed the qualitative trends observed in Figure 3, where GSL-1 vesicles adhered more substantially to both the silica- and polyamide-coated surfaces. The change in the frequency of the crystal, Δf, indicative of the mass adsorption, on both polyamide (∼ −70 and −90 Hz after 200 min for Re LPS and GSL-1, respectively) and silica (∼ −2 and −7 Hz after 120 min for Re LPS and GSL1, respectively) surfaces, was greater for GSL-1 than for Re LPS for all analyzed overtones. Figures 4 and 5 also show the relative contribution of GSL-1 versus Re LPS to the adhesion behavior of mixed SUVs. The adhesion of pure Re LPS vesicles to the polyamide surface was the fastest and reached a plateau after ∼1 h (Figure 4). In contrast, vesicles containing any fractions of GSL-1 showed an adhesion to the polyamide surface steadily increasing throughout the experiment. The larger the fraction of GSL-1 was, the more mass adherence to the surface was observed. The trends of adhesion versus time showed no saturation and, likely, would have continued and surpassed the adhesion of pure Re LPS SUVs had the experiment been carried out longer. It was previously suggested that the strong nonselective adhesion of the GSL-1 to both hydrophilic and hydrophobic surfaces contributes, on the larger, environmental level, to the presence of Sphingomonas spp. in a variety of environments, including extreme and oligotrophic ones.36−38 Strong adhesion stabilizes the bacteria in a certain location, and the hydrophobic characteristics contribute to the ability to utilize hydrophobic recalcitrant compounds as a sole energy source.27,35 Changes in dissipation (ΔD) and in resonance frequencies (Δf) in Figures 4 and 5 can also provide an insight into the viscoelastic and inertial characteristics of the deposited SUV layers.66,67 A particularly beneficial feature of QCM-D is the availability of several overtone frequencies because elastic, inertial, and dissipative viscous contributions to frequency and dissipation shifts depend on frequency in a different manner.64 Thus, QCM-D shows how the adhered layers’ viscoelastic properties evolve with time.68,69 Note the fan-like spread of the curves for different overtones (n = 3, 5, 7, 9, 11) for each type of the SUV is a typical effect of viscoelastic properties on the shift of frequency and dissipation values.33 To be later discussed, the higher rigidity of the LPS layer indicated by the ΔD/Δf values in Figure 7 may also explain smaller dispersion of the frequency and dissipation for different overtones. Regarding the dissipation caused by different types of SUVs on the polyamide and silica surfaces (Figures 4 and 5), the GSL-1 fraction-dependent behavior was disrupted by SUVs containing 90% Re LPS and 10% GSL-1. Although this type of vesicle had a slower rate of attachment to the polyamide surface, as observed by the changing frequency slope, this layer dissipated significantly on the polyamide-coated sensor. These findings are enlightened in Figure 6, where the trends of dissipation (Figure 6A) and frequency (Figure 6B) changes are summarized as a function of the GSL-1 content. Figure 6 presents the composition-dependent behavior of the SUV layers on silica and polyamide with regard to the content of GSL-1: A higher content of GSL-1 in the vesicle results in higher mass accumulation (frequency changes) of the SUV on
and depends among others on vesicle size, composition, and interactions between the glycolipids. The results of size and ζ potential for all different vesicles types (at similar ionic strength) were not found to be statistically significant, with p = 0.15 and 0.48, respectively. Following an increase in the ionic strength of the rehydrating solution from 100 to 200 mM, the ζ potential decreased in agreement with the electrical doublelayer (EDL) theory.59 Indeed, at higher ionic strengths, the particles become less negatively charged due to the compression of the EDL and the reduction of the Stern potential.60,61 The ζ potential of LPS Re vesicles (∼25% reduction) was more strongly affected by the ionic strength than GSL-1 vesicles (∼15%). These observations are in line with previous findings, in which the ζ potentials of E. coli K12 cells containing LPS decreased with ionic strength, while Sphingomonas cells containing GSL showed ζ potentials nearly constant (and less negative than E. coli K12 cells) across the range of ionic strength tested.63 Corroborating with these results, although under different aquatic conditions, GSLs were found to be less negatively charged compared with LPS: the surface charge of GSL and LPS was 1.35 and 3.33 elementary charges/nm2, respectively, in 50 mM KCl supplemented with 5 mM MgCl2 at pH 7.4.4 In line with the hydrophobic nature of Sphingomonas spp. bacteria,62,63 GSL-1, the building block of their outer membrane, formed more hydrophobic SUVs in comparison with Re LPS, the building blocks of the rest of the Gramnegative bacteria (Table 2). The difference between the contact Table 2. Air−Water Sessile Drops and Contact Angles of the Pure Re LPS and GSL-1 SUV Layersa
a
Upper row, numerical values; middle row, visual image of the water sessile drop; bottom row, fluorescence images of PEI-modified-glass slide covered by SUVs. The scale bar is 10 μm.
angles of a water drop placed on PEI-modified glass slide was found to be significant (tdf=20 = 3.86; p = 0.001). This relative hydrophobicity might contribute to the outstanding abilities of the Sphingomonas spp. to biodegrade hydrophobic environmental pollutants, composed of chemically stable aromatic hydrocarbons.25,26,28 The greater affinity of the hydrophobic constituents of the outer membrane of Sphingomonas to the hydrophobic aromatic compound could be a factor facilitating biodegradation. GSL-1 and Re LPS SUV Adhesion to Surfaces. The light emission intensity of CLSM images in Figure 3 shows that a larger and, apparently, more uniform coverage of both silica (hydrophilic surface)64 and polyamide (a more hydrophobic 2132
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Figure 3. CLSM images of GSL-1 and Re LPS attached to silicon wafer (silica) and polyamide (PA) surfaces (online version in color). Scale bar is 10 μm.
Figure 5. Changes in frequency and dissipation as a function of time during vesicles adsorption to silica-coated QCM-D sensors for different overtones (n = 3, 5, 7, 9, 11) and vesicle composition (black: 100% GSL-1; gray: 50% GSL-1, 50% LPS Re; white: 10% GSL1, 90% LPS Re; red: 100% LPS).
Figure 4. Changes in frequency and dissipation as a function of time during vesicles adsorption to polyamide-coated QCM-D sensors for different overtones (n = 3, 5, 7, 9, 11) and vesicle composition (black: 100% GSL-1; gray 50% GSL-1, 50% LPS Re; white: 10% GSL-1, 90% LPS Re; red: 100% LPS).
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Figure 6. Dissipation (A) and frequency shift (B) of sensors with adsorbed vesicles with various LPS and GSL content to silica- (gray solid square) and polyamide-coated (■) QCM-D sensors measured at the seventh overtone after 150 min of adsorption. Zero values on the x axes indicate vesicles composed of 100% LPS at 100 mM NaCl.
the QCM-D sensor, regardless of its hydrophobic/hydrophilic character. Further analysis was performed only for the SUV layers on the polyamide surface due to the significantly lower levels of adhesion of Re LPS to silica (Figure 5) and the generally insufficient Re LPS-containing vesicles on the silica surface, which likely compose a nonhomogeneous layer. Viscoelastic Characterization of SUV Layers. Changes in the viscoelastic properties of the SUV layers, attributable to the GSL-1 fraction, were examined by comparing the dissipative behavior of the different SUV layers (Figure 7) as well as their shear modulus and shear viscosity (Figure 8). For characterizing the viscoelastic properties of the SUV layers, the sufficient
Figure 8. Modeling results of shear modulus (A), shear viscosity (B), and thickness (C) of the SUV layer formed on a polyamide QCM-D sensor as a function of time and vesicle composition (using shifts of F and D at three overtones: fifth, seventh, and ninth).
amount of homogeneous SUV layer is needed; therefore, only the SUV layers formed on the polyamide sensors were analyzed. When considering the trends for adherence of the two glycolipids and their mixtures to the polyamide-coated QCM-D sensor at the linear regime of the dissipation factor versus frequency shift (slope of ΔD/Δf, Figure 7), significant differences of the SUV layer dissipative characteristics were observed. The layer composed of 100% Re LPS was observed to be less dissipative than the pure GSL-1 layer per adsorbed material on the sensor, as indicated by the lower value of ΔD/ Δf for 100% Re LPS layer and higher ΔD/Δf value for 100% GSL-1 layer (Figure 7). While in the presence of only 10% of GSL-1 in the vesicle, the SUV layer had the highest dissipation properties (normalized to the frequency shift), and in the presence of 50% of GSL-1, the dissipation leveled off. This result is an evidence for the dominant effect of GSL-1 on the layer, which probably dictates its dissipative character. The shear modulus, shear viscosity, and thickness were deduced from the QCM-D dissipation factors and frequency shifts acquired at the fifth, seventh, and ninth overtones using the Voigt model (Voinova et al., 1999) implemented in the QTools software (Figure 8). The 100% Re LPS layer is shown to have both a higher shear modulus (Figure 8A, upper thin gray line) and shear viscosity (Figure 8 B, upper thin gray line) than the pure layer of GSL-1 (Figure 8A,B, black line). The higher values of the shear modulus for the Re LPS layer (Figure 8A)
Figure 7. Effect of the vesicle content on relative dissipative character of the vesicle layer on top of the QCM-D polyamide sensor. Slopes of the ΔD plotted against Δf were extracted and displayed against the vesicle content. Zero value on the x axis stands for 100% LPS. Only for 100% LPS Re vesicles, the slopes for the beginning and the end of the adhesion process largely differ; therefore, the last 70 min of adhesion period was analyzed. 2134
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CONCLUDING REMARKS In this study, we applied a single-component-based approach using SUVs of outer membrane components of Gram-negative bacteria, Re LPS and GSL-1, and a fundamental understanding of the adherence and viscoelastic properties of vesicles layers is provided. The SUV layer composed of 100% Re LPS was observed as the most rigid with the highest shear modulus and shear viscosity values, but once GSL was present, it dictated the SUV viscoelastic characteristics, mostly reducing the SUV rigidity at low concentrations of GSL (10%), a trend that was leveled off at 50 and 100% of GSL. In addition, the presence of GSL in the SUV layer was shown to elevate the fraction of the energy-conserving component of the layer’s viscoelastic properties. These results may elevate the adherence of Sphingomonas spp. and other species containing GSL in their outer membrane to surfaces. In addition to adherence, membrane elasticity is a property that might contribute to bacterial cell integrity, resistance, and overall survival.
corroborate with the higher rigidity (less dissipative characteristics) of the 100% Re LPS layer observed in Figure 7. While the highest values of the storage (shear) modulus for Re LPS can explain its higher contribution to the energy conservation of the sensor (and its lower dissipation), the increased shear viscosity (associated with the loss modulus) of the Re LPS layer (Figure 8 B) could cause the opposite effect and elevate the dissipation of the sensor due to viscous dissipation.70 Hence, Re LPS and GSL-1 adsorbed layers are not purely elastic or purely viscous. Thickness values of the SUV layers varied between ∼20 to 27 nm with higher thickness for the pure GSL-1 SUV. These values correspond with the cryo-TEM analysis for nonadhered SUV (Figure 2) and the DLS values (Table 1), taking into account the interaction of the SUV with the surface, which probably reduces their effective diameter toward the bulk solution (and analyzed thickness) by ∼50%. It has been reported that the behavior of ΔD/Δf (Figure 7) as a function of the layer’s viscoelastic properties and thickness (Figure 8) may be nonmonotonic.46 In this study, a highest shear modulus and shear viscosity is calculated for the layer of 100% LPS, while its ΔD/Δf is the lowest. Interestingly, close values of shear modulus and shear viscosity were calculated by the Voigt model for 50 and 100% GSL (Figure 8A,B), implying that the GSL has a dominant effect on both the viscosity and elasticity of the SUV layer and probably dictates the viscoelastic properties of the adhered vesicles. For the mixed SUVs, with both lipids, the presence of GSL in the SUV layer is also shown to elevate the fraction of the energy-conserving component of the layer’s viscoelastic properties (Figure 9), presented as the viscoelastic ratio (μ/ηω), when
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AUTHOR INFORMATION
Corresponding Author
*Tel: 972-8-6563520. Fax: 972-8-6563503. E-mail: herzberg@ bgu.ac.il. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to the staff at the Ilse Katz Institute for Nanoscale Science and Technology at Ben Gurion University for their kind assistance. This work was financially supported by the Israel Science Foundation (grant no. 1360/10).
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REFERENCES
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Figure 9. Effect of the vesicle content on the viscoelastic ratio (μ/ηω) of the adhered SUV layer. μ, η, are ω are the shear modulus, shear viscosity, and frequency at the seventh overtone, respectively.
μ, η, are ω are the shear modulus, shear viscosity, and frequency of the seventh overtone, respectively. The higher the viscoelastic ratio, the higher the relative contribution of elasticity (the energy-conservation component) in comparison with the energy loss (the viscous component). Even though the pure LPS layer (Figure 9) possessed the lowest viscoelastic ratio, it also had the highest shear modulus (highly rigid) and the smallest thickness (Figure 8A,C). Figure 9, derived from Figure 8, indicates that GSL-1 provided the vesicle with a higher ratio of elastic characteristics, compared with its viscosity (viscoelastic ratio: μ/ηω), when the most dramatic alteration from viscous to an increase in elastic characterization happened at the 10% GSL. 2135
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