Article pubs.acs.org/Langmuir
Study of Bacterial Adhesion on Different Glycopolymer Surfaces by Quartz Crystal Microbalance with Dissipation Yinan Wang,†,‡ Ravin Narain,*,† and Yang Liu*,‡ †
Department of Chemical and Materials Engineering and ‡Department of Civil and Environmental Engineering, University of Alberta, 116 Street and 85 Avenue, Edmonton, Alberta T6G 2G6, Canada S Supporting Information *
ABSTRACT: Protein−carbohydrate interactions are involved in a wide variety of cellular recognition processes including cell growth regulation, differentiation and adhesion, the immune response, and viral or bacterial infections. A common way for bacteria to achieve adhesion is through their fimbriae which possess cellular lectins that can bind to complementary carbohydrates on the surface of the host tissues. In this work, we synthesized glycopolymers using reversible addition−fragmentation chain transfer (RAFT) polymerization which were subsequently immobilized on a sensor surface for studies of bacterial adhesion by quartz crystal microbalance with dissipation (QCM-D). Ricinus communis Agglutinin (RCA120), a galactose specific lectin, was first studied by QCM-D to determine the specific lectin interactions to the different glycopolymers-treated surfaces. Subsequently, Pseudomonas aeruginosa PAO1 (a Gram-negative bacterium with galactose-specific binding C-type lectin (PA-IL)) and Escherichia coli K-12 (a Gram-negative bacterium with mannose-specific binding lectin) were then used as model bacteria to study bacterial adhesion mechanisms on different polymer-treated sensor surfaces by the coupled resonance theory. Our results showed that lectin−carbohydrate interactions play significant roles in comparison to the nonspecific interactions, such as electrostatic interactions. A significantly higher amount of P. aeruginosa PAO1 could adhere on the glycopolymer surface with strong contact point stiffness as compared to E. coli K-12 on the same surface. Furthermore, in comparison to E. coli K-12, the adhesion of P. aeruginosa PAO1 to the glycopolymers was found to be highly dependent on the presence of calcium ions due to the specific C-type lectin interactions of PA-IL, and also the enhanced bacterial adhesion is attributed to the stiffer glycopolymer surface in higher ionic strength condition.
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INTRODUCTION Bacterial initial adhesion to a solid surface is important because it is the first step in biofilm formation which impacts both industry1,2 and public health.3,4 Exemplifying the former, formation of biofilms in pipelines causes clogging and corrosion1 and in watercraft reduces vessel speed, which affects many different forms of life after other marine organisms attach themselves on a biofilm-covered hull surface.2 Alternatively, key public health issues include bacterial adhesion on biomedical implants, believed to be one of the main factors for biomaterialcentered infections.3 Generally two major bacteria−substratum interactions are widely discussed these years, namely, nonspecific interactions including electrostatic forces and hydrophobic interactions and specific interactions such as carbohydrate−protein interactions.3,5,6 The nonspecific interactions have been widely analyzed in terms of the hydrophobicity and charges of the solid surface,7−9 the macromolecules on bacteria (e.g., lipopolysaccharide (LPS)),10 and the effects of solution chemistry (such as pH11 and ionic strength12). In some other studies,13−15 DLVO theory (the combined effect of van der Waals and double-layer forces) and the extended DLVO (XDLVO) theory were also established in © 2014 American Chemical Society
order to qualitatively and quantitatively evaluate the adhesion of bacteria on surfaces. In contrast, the specific bacteria−substratum interactions usually occur between carbohydrates and lectins on the tips of Gram-negative bacterial fimbriae.5,16 Different bacterial strains possess different lectins that can specifically interact with various carbohydrates. For example, Escherichia coli can bind to mannose on epithelial cells to cause urinary tract infections,17,18 while wild-type Pseudomonas aeruginosa PAO1 infects the respiratory tract using their D-galactose-specific PA-IL (lectin A) or L-fructose-specific PA-IIL (lectin B).19 Although various methods including biochemical tests,20 microscopy, immunosensing,21,22 and quartz crystal microbalance with dissipation (QCM-D)23−25 have been developed to study the mechanisms of bacterial adhesion, only a few studies have focused on comparing bacterial adhesion on different surfaces in terms of specific and nonspecific interactions.26,27 Therefore, our aim was to compare the different interactions Received: January 15, 2014 Revised: May 28, 2014 Published: June 2, 2014 7377
dx.doi.org/10.1021/la5016115 | Langmuir 2014, 30, 7377−7387
Langmuir
Article
RAFT Polymerization of LAEMA. RAFT polymerization was chosen to produce the well-defined glycopolymers.31,36 For a typical homopolymerization, 2-lactobionamidoethyl methacrylamide (LAEMA) (1 g, 2 mmol) was dissolved in 6 mL of distilled water in a 10 mL Schlenk tube with 1 mL of CTP (16 mg, 0. 057 mmol) and 4,4′azobis(4-cyanovaleric acid) (ACVA) (8 mg, 0.032 mmol) DMF stock solution. The tube was then sealed and degassed by purging it with nitrogen for 30 min. Polymerization was conducted in an oil bath (70 oC) for 24 h and followed by precipitation in acetone and subsequent washing with methanol to remove the monomers and residual RAFT agents. Conversion of the polymerization was determined by Varian 500 1H NMR using D2O. The polymer’s molecular weight and polydispersity were determined by aqueous GPC (Viscotek GPC system) at room temperature with a flow rate of 1.0 mL/min. RAFT Copolymerization of LAEMA with AEMA. Copolymerization was performed at 70 °C employing ACVA and CTP as the radical initiator and chain transfer agent, respectively. In a 10 mL Schlenk tube, LAEMA (0.5 g, 1.07 mmol) and AEMA (0.5 g, 3.02 mmol) were dissolved in 7 mL of double-distilled deionized water before addition of CTP (16 mg, 0.057 mmol) and ACVA (8 mg, 0.032 mmol) N,N′dimethylformamide (DMF) stock solution (1 mL). After degassing under nitrogen atmosphere for 30 min, the flask was placed in a preheated oil bath for 24 h. After precipitation in acetone, the polymer was extensively washed with methanol to remove any residual monomers and then dried under vacuum (Scheme 2). Conversion and composition of the copolymer were determined by Varian 500 1H NMR in D2O. The polymer molecular weight and molecular weight distributions were determined by aqueous GPC (Viscotek GPC system) at room temperature with a flow rate of 1.0 mL/min.30 Bacterial Cultivation. The stored bacterial strains (P. aeruginosa PAO1 and E. coli K-12) were streaked onto a Luria-Bertani (LB) agar plate and incubated at 37 °C overnight. A single colony was transferred into 5 mL of LB broth and grown overnight in a shaker incubator at 200 rpm and 37 °C. Stationary-phase bacterial cells were harvested by centrifugation at 4000 g and 4 °C for 5 min. After decanting the supernatant, the pellets were resuspended in 10 mM NaCl or 10 mM CaCl2 solution. The centrifugation and resuspension procedure was repeated at least twice to remove traces of growth media and suspended extracellular polymeric substances from the solution.37 The final bacterial concentration was adjusted to 107 cells/mL by the optical density (OD). Studying the Lectin−Polymer Interactions by QCM-D. QCM-D (Q-Sense, Sweden, E4 chamber) was applied in real time to study the interactions between lectin and gold-coated sensor chips (frequency, 4.95 MHz 50 kHz; cut, AT; diameter, 14 mm; thickness, 0.3 mm; surface roughness,
