ProtocolsAntibacterial properties of sophorolipid-modified gold surfaces against Gram positive and Gram negative pathogens
Graphical abstract
Introduction
Carbohydrates are an interesting group of functional compounds with a variety of properties and applications in pharmacology (vaccines, drugs) [1], [2], [3] and diagnosis (glyco-microarrays for antibody recognition and cell adhesion) [4]. Their use as antimicrobials is of particular interest, with glycolipids or polysaccharides having broad activities for prevention of microbial contamination either via antiadhesive or biocidal properties [5]. For instance, amphotericin B [6] a complex glycolipid, is a commonly employed antifungal, while exopolysaccharides [7] are used for their antiadhesive properties. Other bioderived glycolipids like rhamnolipids, cellobioselipids or sophorolipids [8] are well-known for their antimicrobial properties. Their biocidal effect seems to depend on the synergistic effect of the sugar and lipid moieties in the so-called “surfactant-effect” [8], [9], [10], which is characterized by plasma membrane disruption, causing lysis and possible leakage of cytoplasm material [11], [12]. Antiadhesive properties are also commonly observed for polysaccharides and glycolipids which are either immobilized on a substrate (glycoarray) or free in solution [5], [13], [14].
The antimicrobial effects of carbohydrate-based compounds are worth studying because of their mild/low cytotoxicity and low ecotoxicological effects and their lower cost (compared to other antimicrobial agents like peptides) [15] which make them very appealing for large scale application, such as protection of outdoor infrastructure for instance. Moreover the diversity in this carbohydrate family promotes a broad spectrum of activity [16]. Despite these advantages and the extensive use of these compounds as antimicrobial agents, the role of the carbohydrate moiety, and in particular its interaction with the cell envelope is still not very clear. The interactions between carbohydrates and bacterial membranes have been studied for decades [17]. However, only recently, modeling studies have tried to put in evidence the impact of simple mono- and disaccharides on the liquid crystalline order of phospholipid bilayers [18], although experimental data are still lacking. In our recent work, we have shown for the first time that a self-assembled monolayer (SAM) composed of glucose β1,2 glycolipid stabilized on gold is able to pierce the cell envelope, and in particular the plasma membrane, of the non-pathogenic bacterial strain Listeria ivanovii [19]. This work has shown the important membrane-disrupting role of the disaccharide, which cannot be related to the so-called “surfactant-effect” since the glycolipid is covalently bond to the surface. However, we showed the impact of the disaccharide orientation on the efficiency of bacterial membrane disruption.
In the present work, we studied the effect of glycolipid grafting density on its biocidal properties. Indeed, sugar concentration appears as a key factor in sugar-membrane interactions [20] and grafting may enable to reach high concentrations in the vicinity of bacteria, thus enhancing its antimicrobial effects [3]; we also want to explore the spectrum of activity of such grafting towards several Gram positive and Gram negative bacterial strains.
Section snippets
Materials and methods
Cysteamine (cys), N-hydroxysuccinimide (NHS), 1-(3-(dimethylamino)propyl)-N-ethylcarbodiimide hydrochloride (EDC), glutaraldehyde and sodium chloride (NaCl) were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). Sophorolipids (SL) were derived from a commercial acidic and lactone mixture of sophorolipids purchased from Soliance (France) (Sopholiance S; batch number, 11103A; dry content, 60 ± 6%). To obtain a high purity form of the nonacetylated acidic sophorolipids only (>90 mol%,
Variation of SL grafting density
In order to explore the effect of glycolipid grafting density on biocidal properties, gold surfaces were functionalized with solutions at different sophorolipid concentrations. The first step was to assess the possible influence of the initial concentration on the modification of gold surfaces. The lowest concentration used was 5 mg L−1, ca. 0.8 μmol L−1; at this concentration, the solution contains theoretically enough molecules to fully saturate the surfaces with SL molecules (ca. 5 × 1015
Conclusion
In a recent publication we have shown that a monolayer of sophorolipids, microbial-derived glycolipids, can be chemically grafted on top of a gold substrate using a cysteamine primer and that such a material exhibits antibacterial properties against Listeria ivanovii, a non-pathogenic Gram+ bacterium. In this work, we go further and show that gold substrates engineered to tether modified-sophorolipids also damage the membrane of a series of pathogenic Gram+ (Enterococcus faecalis,
Acknowledgments
The authors would like to acknowledge the French state funds managed by the ANR within the Investissements d’Avenir program under Reference ANR-11-IDEX-0004-02 and more specifically within the framework of the Cluster of Excellence MATISSE for a Ph.D. Grant for CV. The authors acknowledge IMPC (Institut des Matériaux deParis Centre, FR2482) and the C’ Nano projects of the Region Ile-de-France, for SEM-FEG and Omicron XPS apparatus funding. The authors would also like to thank Dr. Souhir Boujday
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