Surface-Induced Changes in the Conformation and Glucan Production of Glucosyltransferase Adsorbed on Saliva-Coated Hydroxyapatite

Abstract

Glucosyltransferases (Gtfs) from S. mutans play critical roles in the development of virulent oral biofilms associated with dental caries disease. Gtfs adsorbed to the tooth surface produce glucans that promote local microbial colonization and provide an insoluble exopolysaccharides (EPS) matrix that facilitates biofilm initiation. Moreover, agents that inhibit the enzymatic activity of Gtfs in solution often have reduced or no effects on surface-adsorbed Gtfs. This study elucidated the mechanisms responsible for the differences in functionality that GtfB exhibits in solution vs surface-adsorbed. Upon adsorption to planar fused-quartz substrates, GtfB displayed a 37% loss of helices and 36% increase of β-sheets, as determined by circular dichroism (CD) spectroscopy, and surface-induced conformational changes were more severe on substrates modified with CH3- and NH2-terminated self-assembled monolayers. GtfB also underwent substantial conformation changes when adsorbing to hydroxyapatite (HA) microspheres, likely due to electrostatic interactions between negatively charged GtfB and positively charged HA crystal faces. Conformational changes were lessened when HA surfaces were coated with saliva (sHA) prior to GtfB adsorption. Furthermore, GtfB remained highly active on sHA, as determined by in situ attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, producing glucans that were structurally different than GtfB in solution and known to increase the accumulation and virulence of biofilms. Our data provide the first insight into the structural underpinnings governing Gtf conformation and enzymatic function that occur on tooth surfaces in vivo, which may lead to designing potent new inhibitors and improved strategies to combat the formation of pathogenic oral biofilms.

Candida albicans mannans mediate Streptococcus mutans exoenzyme GtfB binding to modulate cross-kingdom biofilm development in vivo

“…However, recent elucidation of Gtf crystal structure combined with 3D modelling revealed several additional binding motifs [56–59]. Moreover, the Gtf protein-ligand mechanisms are rather complex, involving intricate conformational changes (e.g.…”

“…Moreover, the Gtf protein-ligand mechanisms are rather complex, involving intricate conformational changes (e.g. decrease in helical and increase in β-sheet structures) when adsorbing to solid or semi-solid surfaces [56]. Interestingly, GtfC binds less effectively than GtfB, while GtfD does not bind to Candida surface [18].…”

Dynamic cell–matrix interactions modulate microbial biofilm and tissue 3D microenvironments

“…For example, the oral pathogen Streptococcus mutans secretes EPS-producing exoenzymes termed glucosyltransferases (Gtfs) that can bind to both tooth and microbial surfaces, including fungi ( Candida albicans ) [17]. Intriguingly, these surface-bound Gtfs remain highly active despite major conformational changes [18], producing large amounts of EPS to provide bacterial binding sites that promote mixed-species (and even cross-kingdom) biofilm formation [19]. Motile bacteria ( Bacillus subtilis and Vibrio cholera ) appear to use flagella as mechanosensors to scan surfaces and initiate cell adhesion [5,20].…”

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“…The polysaccharides mainly comprise of insoluble glucans 3,5 and Gtf-I is identified as the key enzyme for glucan synthesis.…”

“…Thus Gtf-I is considered as the fundamental virulence factor of S. mutans 5 . Besides this, gtf-D codes for glucosyltrasferase (soluble) [Gtf-S] and gtf-C codes for Gtf-I and Gtf-S enzymes together 3 .…”