In vitro binding interactions of oral bacteria with immobilized fructosyltransferase

AIMS: The objective of the present study was to explore the role of immobilized fructosyltransferase (FTF) in adhesion process. METHODS AND RESULTS: We investigated real-time biospecific interactions between several types of oral bacteria and recombinant FTF immobilized on a biosensor chip, using surface plasmon resonance technology. Streptococcus mutans, Streptococcus sobrinus and Actinomyces viscosus demonstrated significant binding to FTF. Actinomyces viscosus had a greater binding to FTF, with 373 Resonance Units (RU), than the other tested bacteria. The binding level to FTF of Strep. sobrinus was 320 RU, whereas Strep. mutans and Streptococcus salivarious show binding of 296 and 245 RU, respectively. The binding sensograms displayed different profiles for the tested bacteria at various cell density, suggesting a different affinity to immobilized FTF. CONCLUSIONS: The results from this study suggest that FTF may influence bacterial adherence and colonization of the dental biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: The biomolecular interaction analysis enables real-time monitoring of the interaction between adhesions of intact bacteria and their ligands, which might be crucial in the initial phase of biofilm development in vivo.     

Effect of chlorhexidine on molecular weight distribution of fructans produced by fructosyltransferase in solution and immobilized on surface

The effect of chlorhexidine (CHX), a potent antibacterial agent, was tested on the molecular weight distribution (MWD) of fructans synthesized by cell-free fructosyltransferase (FTF) in solution in comparison to FTF immobilized onto hydroxyapatite (HA). Size-exclusion chromatography (SEC) analysis has shown that cell-free FTF, both in solution and immobilized on HA, produces both low MW (1.9-2.2 kDa) and high MW (913-1047 kDa) fructans. CHX at a concentration of 0.02% altered the MWD of the fructans by reducing the polydispersity ratio and changing the MWD of the fructans synthesized both by immobilized FTF and by FTF in solution. These changes of the fructans in the presence of CHX adds a new prospective to the anticaries effect of CHX in addition to its antibacterial properties.     

Effect of oxazaborolidines on immobilized fructosyltransferase analyzed by surface plasmon resonance

Dental diseases are among the most prevalent afflictions of humankind. These diseases are associated with the formation of biofilms harboring pathogenic bacteria. Fructosyltransferases (FTF) are extra cellular enzymes of several oral bacteria. FTF are associated with the formation of extracellular polysaccharide matrix (fructans) which play a role in biofilm formation and oral bacteria physiology. Oxazaborolidines have been shown to inhibit biofilm formation. The purpose of this study was to examine if the anti-biofilm effect is, in part, an effect on the immobilized enzymes synthesizing the extra cellular polysaccharide participating in biofilm formation. Eight different oxazaborolidines (BNO1-BNO8) were synthesized and evaluated for their affect on the synthesis of fructans by FTF using the biomolecular interaction analysis (BIAcore) system which involves the use of real-time surface plasmon resonance (SPR) technique. The tested oxazaborolidines demonstrated a significant and immediate inhibitory effect on immobilized FTF activity. This effect was reversible. Our results show that oxazaborolidines can act as enzymatic inhibitors of FTF immobilized on the surface, also at levels lower than their MIC. Part of the anti-biofilm effect of BNOs may be accounted for this enzymatic inhibition.     

Regulation of fructosyltransferase activity by carbohydrates, in solution and immobilized on hydroxyapatite surfaces

We tested the effect of several carbohydrates on the activity of cell-free fructosyltransferases (FTF) in solution and immobilized onto hydroxyapatite (HA) and found an inhibitory dose-dependent effect of glucose on FTF activity, both on the surface and in solution. Glucose at 160 mM inhibits FTF activity by 75% both on HA and in solution. Fructose at 160 mM inhibited FTF activity by 25% in solution and by 15% on HA. Levan inhibited FTF activity by 30% in solution, while dextrans and inulin had a limited effect on FTF activity. Circular dichroism and infrared analysis demonstrated no major changes in the chemical structure of fructans synthesized by cell-free FTF on HA and in solution, in the presence or absence of glucose. However, as verified by size-exclusion chromatography, glucose inhibited the synthesis of high molecular-weight fructans. The results indicate that glucose, a byproduct of the FTF enzymatic reaction, is the main carbohydrate affecting FTF activity. Selective inhibition of high molecular-weight fructan production by glucose, may indicate that two mechanisms are involved in the synthesis of fructans, both in solution and on the surface.     

Effect of carbohydrates on fructosyltransferase expression and distribution in Streptococcus mutans GS-5 biofilms

Streptococcus mutans produces a fructosyltransferase (FTF) enzyme, which synthesizes fructan polymers from sucrose. Fructans contribute to the virulence of the biofilm by acting as binding sites for S. mutans adhesion and as extracellular nutrition reservoir for the oral bacteria. Antibodies raised against a recombinant S. mutans FTF were used to test the effect of glucose, fructose, and sucrose on FTF expression in S. mutans GS-5 biofilms. Biofilms formed in the presence of fructose and glucose showed a higher ratio of FTF compared to biofilms formed in the presence of sucrose. Confocal laser scanning microscopy images of S. mutans biofilms indicated a carbohydrate-dependent FTF distribution. The layer adjacent to the surface and those at the liquid interface displayed high amounts cell-free FTF with limited amount of bacteria while the in-between layers demonstrated both cell-free FTF and cells expressing cell-surface FTF. Biofilm of S. mutans grown on hydroxyapatite surfaces expressed several FTF bands with molecular masses of 160, 125, 120, 100, and 50 kDa, as detected by using FTF specific antibodies. The results show that FTF expression and distribution in S. mutans GS-5 biofilms is carbohydrate regulated.     

Secretion of fructosyltransferase by Streptococcus salivarius involves the sucrose-dependent release of the cell-bound form

Three strains of Streptococcus salivarius including a recent clinical isolate were found to possess Ca2(+)-dependent fructosyltransferase (FTF) activity. The extracellular FTF activity of cells grown on sucrose increased as much as 9-fold compared with cells grown on either glucose, fructose or galactose. This increase in activity was due not to induction of FTF by sucrose, but to the release of the cell-bound form of the enzyme. Studies with washed cells of S. salivarius ATCC 25975 showed that the extent of release of the cell-bound FTF activity was dependent upon the sucrose concentration up to 4 mM, at which concentration maximum release (95%) of cell-bound FTF occurred. Several lines of evidence suggested that either substrate binding or de novo synthesis of fructan is required for the release of the cell-bound FTF activity.     

N-Deglycosylation of fructosyltransferase and invertase from Fusarium oxysporum decreases stability but has little effect on kinetics and synthetic specificity

Most of the carbohydrate moiety of invertase and fructosyltransferase (FTF) from F. oxysporum IMI 172464 was removed by peptide-N-glycosidase F. The molecular weights of native invertase and FTF were 260 kDa and 210 kDa respectively. Deglycosylation lowered the molecular sizes by 42% and 23%, respectively. The K values for sucrose remained unchanged by deglycosylation. However the stability of both enzymes at their optimum pH (4.0 for invertase, 5.0 for FTF) and optimum temperature (45°C for invertase, 35°C for FTF) was decreased: their sensitivity to protease digestion was increased by 36% and 41%, respectively. The synthetic specificity of deglycosylated FTF remained unchanged. The carbohydrate moiety of invertase and FTF contributes to the stability of the enzymes but is not essential in their catalytic function and plays no part in determining their specificity.     

Immobilization of fructosyltransferase from Streptococcus mutans on hydroxyapatite surfaces induces the formation of multimeric complexes

AIMS: To investigate the formation of fructosyltransferase (FTF) complexes on hydroxyapatite (HA) surfaces. METHODS AND RESULTS: Cell-free extracellular FTF from Streptococcus mutans, purified from hyperproducing strain V-1995, was adsorbed onto HA and then eluted from the surface by means of a concentration gradient of potassium phosphate buffer. The FTF monomers loaded onto HA formed, upon adsorption, various complexes ranging from 200 to 700 kDa as demonstrated using native PAGE. All these complexes exhibited enzymatic activity. CONCLUSIONS: Adsorption of FTF onto HA induced the formation of stable and enzymatically-active complexes. SIGNIFICANCE AND IMPACT OF THE STUDY: The formation of these complexes may explain the change of FTF catalytic properties after adsorption onto HA. This study is another step in determining the properties of a-cellular constituents of the oral biofilm.