Abstract
ABSTRACTThe extracellular matrix is a poorly studied, yet important component of dental biofilms. Fluorescence lectin-binding analysis (FLBA) is a powerful tool to characterize glycoconjugates in the biofilm matrix. This study aimed to systematically investigate the ability of 75 fluorescently labeled lectins to visualize and quantify extracellular glycoconjugates in dental biofilms. Lectin binding was screened on pooled supragingival biofilm samples collected from 76 subjects using confocal microscopy. FLBA was then performed with 10 selected lectins on biofilms grown in situ for 48 h in the absence of sucrose. For five lectins that proved particularly suitable, stained biovolumes were quantified and correlated to the bacterial composition of the biofilms. Additionally, combinations of up to three differently labeled lectins were tested. Of the 10 lectins, five bound particularly well in 48-h-biofilms: Aleuria aurantia (AAL), Calystega sepiem (Calsepa), Lycopersicon esculentum (LEA), Morniga-G (MNA-G) and Helix pomatia (HPA). No significant correlation between the binding of specific lectins and bacterial composition was found. Fluorescently labeled lectins enable the visualization of glycoconjugates in the dental biofilm matrix. The characterization and quantification of glycoconjugates in dental biofilms require a combination of several lectins. For 48-h-biofilms grown in absence of sucrose, AAL, Calsepa, HPA, LEA, and MNA-G are recommendable.
Highlights
The two most prevalent oral diseases – caries and periodontitis – are caused by biofilms [1]
Fluorescence lectin-binding analysis (FLBA) was performed with 10 selected lectins on biofilms grown in situ for 48 h in the absence of sucrose
The aims of the current study were: (1) to screen the binding of 75 fluorescently labeled lectins to pooled dental biofilm samples in order to identify a set of lectins that are generally useful for matrix staining in dental biofilms; (2) to test which of the lectins selected during the screening are suitable to visualize glycoconjugates in 48 h biofilms grown in situ in the absence of dietary carbohydrates; (3) to study the spatial distribution and quantify the biovolume of these lectins in 48 h biofilms from nine different subjects using confocal laser scanning microscopy (CLSM) and digital image analysis; and (4) to correlate lectin binding to the species composition of the biofilms, as determined by 16S rRNA gene sequencing
Summary
The two most prevalent oral diseases – caries and periodontitis – are caused by biofilms [1]. The extracellular matrix, a major biofilm component, plays a pivotal role in the development of these infections. It surrounds microbial cells and promotes bacterial adhesion and cohesion processes. It hinders the diffusion of antimicrobial agents, provides mechanical stability, prevents desiccation, and serves as a nutrient reservoir [2,3]. The binding of GtfB to bacterial surfaces converts other bacteria to glucan producers [7], yielding a rigid matrix that serves as a scaffold for biofilm architecture and contributes to the creation and preservation of highly acidic niches [8,9]
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