Ftf Expression Research Articles

Ca19Zn2(PO4)14 Nanoparticles: Synthesis, characterization and its effect on the colonization of Streptococcus mutans on tooth surface

The main cause of tooth decay is the biofilm formation of Streptococcus mutans. This study aimed to investigate the effect of the zinc oxide nanoparticles (ZnO NPs), hydroxyapatite nanoparticles (Ca10(PO4)6(OH)2) (HAP NPs), zinc oxide/hydroxyapatite nanocomposites (ZnO/HAP NCs), and Zn-substituted hydroxyapatite nanoparticles (Ca19Zn2(PO4)14 NPs) on the growth and biofilm formation, bacterial adherence, and the expression of ftf and gtf genes in Streptococcus mutans. The nanostructures were prepared via simple and fast co-precipitation route. Twelve isolates of Streptococcus mutans collected from children with dental caries referred to the dental clinic of Kashan University of Medical Sciences. All S. mutans isolates were susceptible to Ampicillin. The mean MIC for ZnO NPs, HAP NPs, Ca19Zn2(PO4)14, and ZnO/HAP NCs were 118, 260, 70.6, and 994 µg/mL, respectively. All prepared nanostructures significantly reduced biofilm formation at MIC and sub-MIC concentrations (p < 0.01). In biofilm and cell culture treated with nanoparticles, the expression of ftf and gtfC genes decreased. Results were shown that IC50 for the Ca19Zn2(PO4)14 was 8.5, and for non-toxic concentration, was 0.065 µg/mL. The attachment rate to the denture surface and HGF1 cell line treated with the Ca19Zn2(PO4)14 NPs has decreased. The results showed that the Ca19Zn2(PO4)14 NPs has a better effect than the ZnO/HAP NCs. It can therefore be used as a coating on dental surfaces. Investigation of Ca19Zn2(PO4)14 NPs form for covering dental teeth surface recommended in future study.

THE CORRELATION BETWEEN TERPENOID OF SARANG SEMUT (Myrmecodia pendans) WITH mRNA FRUCTOSYLTRANFERASE EXPRESSION OF Streptococcus mutans BIOFILM ATCC 25175

Background: Terpenoid isolate sarang semut (Myrmecodia pendans) is a natural material that has antibacterial power through the destruction of the cell membrane of Streptoccus mutans (S. mutans). Fructosiltransferase (ftf) is one of the extrapolisaccharides produced by S. mutans. The formation of Ftf is governed by the ftf gene. The aim of research was to know the correlation between terpenoid of Sarang semut administration with ftf mRNA expression of S. mutans biofilm ATCC 25175. Method: The study used mixed methods, namely descriptive explorative method to look at the isolation of ftf gene and ftf mRNA expression. This study began with making 1 day biofilm , then given terpenoid isolates of sarang semut and chlorhexidine. Next, isolation of ftf genes and ftf mRNA expression was carried out, then the differences in S. mutans ATCC 25175 mRNA ftf expression in sarang semut and chlorhexidine were given for 1 and 30 minutes using Real-Time PCR. Continued by looking at the relationship of the duration of administration of sarang semut terpenoids to the expression of S. mutans ATCC 25175 mRNA ftf biofilm. Statistical analysis used the t test with p-value &lt;0.05 and Pearson. Results: The results showed that ftf gene with 130 bp length and ftf mRNA expression could be isolated from S. mutans biofilm ATCC 25175 which were given terpenoid isolates from sarang semut. The expression of sarang semut mRNA ftf and chlorhexidine did not have a significant difference for 1 minute (p = 0.0761), whereas in 30 minutes the ant nest had a higher mRNA expression (p = 0.0186). There is a relationship between the duration of administration of terpenoid isolates of sarang semut with S. mutans ATCC 25175 mRNA ftf expression (r2 = 0.614), with a medium correlation coefficient. Conclusion: The conclusion of the study were that sarang semut terpenoid had the same effect as chlorhexidine for 1 minute on S. mutans biofilms, whereas at 30 minutes it was different. There is a relationship between terpenoids of sarang semut isolates and S. mutans biofilm ATCC 25175 mRNA expression.

The effect of Padma-hepaten herbal combination on the caries-inducing properties of Streptococcus mutans on orthodontic surfaces

Dental biofilm is recognized as the primary cause of oral infections, caries being the most common. Orthodontic appliances might increase the risk for dental caries and white spot lesions. Phytomedicinal methods were shown to inhibit dental biofilm formation. The goal of this study was to examine whether Padma hepaten (PH), a polyphenolic formula derived from traditional Tibetan medicine, could inhibit the caries-inducing properties of S. mutans. Biofilms of S. mutans were grown in the presence of PH. Formation of biofilms was tested spectrophotometrically. Exopolysaccharides (EPS) secretion was quantified using confocal scanning laser microscopy. Gene expression of caries-related genes was tested using qRT-PCR. Development of biofilm was also tested on an orthodontic surface (Essix) to assess biofilm inhibition ability on such orthodontic appliances. PH had a dose-dependent inhibition on biofilm formation, with no inhibition on planktonic bacterial growth. The minimal biofilm concentration needed to inhibit the formation by 50 % was 0.16 mg/ml PH. This concentration inhibited EPS secretion by 88 %. A strong reduction was found in the expression of gtfB, gtfC, ftf and brpA caries associated genes. These results demonstrate the inhibitory effect of natural PH polyphenolic extraction on S. mutans biofilm formation and EPS secretion. In an era of increased bacterial resistance to antibiotics, the use of herbal medicine to inhibit biofilm development, without killing host cells, may pave the way to a novel therapeutic concept, especially in children as orthodontic patients.

The antihypercholesterolemic effect of columbamine from Rhizoma Coptidis in HFHC-diet induced hamsters through HNF-4α/FTF-mediated CYP7A1 activation

The aim of this study was to investigate the antihypercholesterolemic activity and potential molecular mechanism of columbamine (COL) that was prepared by extraction from Rhizoma Coptidis in hamsters and HepG2 cells. The results displayed that the COL from Rhizoma Coptidis was a safe natural compound with a LD50 0f 1524.6mg/kg and no detectable toxic symptoms during the observation of chronic toxicity. COL dose-dependently reversed the abnormal lipid levels induced by HFHC diet. Specifically, COL(M) and COL(H) significantly reduced the blood lipid levels(TC, TG and LDL-c) and enhanced the fecal contents of TBA by 21.8% and 25.1% respectively in hamsters. COL up-regulated the genes of CYP8B1, CYP7A1 and LDLR in mRNA and protein level, and down-regulated those of HMGCR to a different degree. Especially, CYP7A1 were significantly up-regulated by COL in hamsters (p<0.01). Further analysis indicated that COL obviously activated the mRNA and protein expression of the transcription factors FTF, HNF-4α, and inhibited those of SHP. Promoter luciferase assay showed that COL induced the expression of FTF and HNF-4α, further transactivating CYP7A1, which accelerated the conversion of liver cholesterol to bile acids. It concluded that the COL showed high lipid-lowering activities through indirectly transactivating CYP7A1 by upregulating FTF and HNF-4α, and directly activating CYP7A1 catalytic activity by strongly interacting with receptor and ligand, therefore promoting cholesterol catabolism and accelerating the excretion of bile acids.

English

Streptococcus mutans is the bacteria most frequently associated with dental caries. One of its virulence factors is its ability to form biofilm. gtf, ftf and spaP are genes of S. mutans that are involved in adhesion and colonization of teeth surfaces and therefore biofilm formation. Exopolysaccharides are catalyzed by a group of bacterial enzymes termed glycosyltransferases (GTF) and fructosyltransferases (FTF), encoded by the genes gtf and ftf, respectively, which under certain conditions can be strongly associated with cell surfaces. Lactobacillus acidophilus is one of the pathogens associated with S. mutans. We carried out research to determine whether the presence of glucose and L. acidophilus promote adhesion mechanisms that increase the expression of gtf, ftf and spaP of S. mutans. Key words: Exopolysaccharides, dental caries, Streptococcus mutans, Lactobacillus acidophilus, glucose, gtf, ftf, spaP.

Sucrose Substitutes Affect the Cariogenic Potential of Streptococcus mutans Biofilms

Streptococcus mutans is considered the primary etiologic agent of dental caries and contributes significantly to the virulence of dental plaque, especially in the presence of sucrose. To avoid the role of sucrose on the virulence factors of S. mutans, sugar substitutes are commonly consumed because they lead to lower or no production of acids and interfere with biofilm formation. This study aimed to investigate the contribution of sugar substitutes in the cariogenic potential of S. mutans biofilms. Thus, in the presence of sucrose, glucose, sucralose and sorbitol, the biofilm mass was quantified up to 96 h, the pH of the spent culture media was measured, the expression of biofilm-related genes was determined, and demineralization challenge experiments were conduct in enamel fragments. The presence of sugars or sugar substitutes profoundly affected the expression of spaP, gtfB, gtfC, gbpB, ftf, vicR and vicX in either biofilm or planktonic cells. The substitution of sucrose induced a down-regulation of most genes involved in sucrose-dependent colonization in biofilm cells. When the ratio between the expression of biofilm and planktonic cells was considered, most of those genes were down-regulated in biofilm cells in the presence of sugars and up-regulated in the presence of sugar substitutes. However, sucralose but not sorbitol fulfilled the purpose of reducing the cariogenic potential of the diet since it induced the biofilm formation with the lowest biomass, did not change the pH of the medium and led to the lowest lesion depth in the cariogenic challenge.

Interleukin-1 Controls the Constitutive Expression of the Cyp7a1 Gene by Regulating the Expression of Cyp7a1 Transcriptional Regulators in the Mouse Liver

Our previous study using interleukin-1α/β-knockout (IL-1-KO) and wild-type (WT) mice demonstrated that IL-1 acts as a positive factor for constitutive gene expression of hepatic cytochrome P4507a1 (Cyp7a1). In this study, to clarify the role of IL-1 in the expression of the hepatic Cyp7a1 gene, we focused on Cyp7a1 transcriptional regulators such as α-fetoprotein transcription factor (FTF), liver X receptor α (LXRα), hepatocyte nuclear factor 4α (HNF4α) and small heterodimer partner (SHP) and examined the effects of IL-1 on their gene expression by real-time reverse-transcription polymerase chain reaction using IL-1-KO and WT mice. We observed no significant differences between sex-matched IL-1-KO and WT mice with regard to gene expression levels of FTF, LXRα, and HNF4α, all of which are positive transcriptional regulators for the Cyp7a1 gene. However, interindividual differences in hepatic FTF and LXRα expression were closely dependent on the gene expression level(s) of hepatic IL-1 and tumor necrosis factor-α (TNF-α), while interindividual differences in hepatic HNF4α were clearly correlated with the expression of IL-1, but not TNF-α. In contrast, the gene expression level of SHP, which is a negative transcriptional regulator of the Cyp7a1 gene through inhibition of FTF function, was higher in IL-1-KO mice than in sex-matched WT mice. These findings demonstrate that, like TNF-α, IL-1 positively controls the gene expression of Cyp7a1 transcriptional upregulators but, in contrast to the previously reported action of TNF-α, IL-1 also acts to downregulate SHP gene expression.

Prevention of Mother-to-child Transmission of Streptococcus mutans

Objective: Transmission of S. mutans, a major dental caries pathogen, occurs mainly during the first 2.5 years of age. Children appear to acquire S. mutans mostly from their mothers, but few studies have investigated preventive effect of xylitol to S. mutans transmission from mother to child. The aim of this study was to perform a follow-up evaluation the preventive effect of xylitol chewing gum of the S. mutans of children's oral cavities, which included the characteristics of vertical transmission from mother to child. Methods: The mothers voluntarily participating in a women's oral health prevention program were divided into two groups (a control and a xylitol group). The subjects were 20 mother-child pairs, who were monitored for 30 months. Xylitol chewing gum group had consumed 2 gum pellets, 3 times a day for 24 months, and then they were followed until 30 months. At baseline, 24 and 30 months whole stimulated saliva samples were collected from the mothers. Children were also recruited from 6 months to 30 months after birth and were collected their dental plaque samples. After isolation and identification, the analysis of the colony count, transmission electron microscopy and real-time RT-PCR were performed to analyze the characteristics of S. mutans. Results: The S. mutans counts decreased steadily in the xylitol group at 24 months, but increased at 30 months. The similar results were showed at their children. While the glucan synthesis was decreased at xylitol group both mother and child. The expression of gtfB, gtfD and ftf were significantly reduced in the xylitol group both mother and child (p&lt;0.05). Conclusions: These findings indicate that chewing xylitol gum over a long period may decrease the expression of the genes associated virulence and reduced the glucan synthesis of S. mutans, which can result the preventing the mother-to-child transmission of S. mutans.

α 1-Fetoprotein transcription factor (FTF)/liver receptor homolog-1 (LRH-1) is an essential lipogenic regulator

α 1-Fetoprotein transcription factor (FTF), also known as liver receptor homolog 1 (LRH-1) is highly expressed in the liver and intestine, where it is implicated in the regulation of cholesterol, bile acid and steroid hormone homeostasis. FTF is an important regulator of bile acid metabolism. We show here that FTF plays a key regulatory role in lipid homeostasis including triglyceride and cholesterol homeostasis. FTF deficient mice developed lower levels of serum triglyceride and cholesterol as a result of lower expression of several hepatic FTF target genes. Chenodeoxycholic acid repressed FTF expression resulting in a decrease in serum triglyceride in wild-type mice. The absence of chenodeoxycholic acid-mediated repression in FTF +/ − mice demonstrated the essential role of FTF in triglyceride metabolism. Taken together, our results identify the nuclear receptor FTF as a central regulator of lipid metabolism.

Effect of denture base materials on mRNA expression of the adhesion‐associated genes from the Streptococcus mutans biofilms

Dental caries is one of the most important reasons for the failure of removable partial denture. The adhesion-associated genes of Streptococcus mutans, such as gtfBCD, ftf and gbpB, might play an important role in bacterial adhesion to denture base materials. Therefore, the aim of this study was to investigate the influence of denture base materials (heat-curing acrylic resins, cobalt-chromium alloy and ceramics served as control) on the formation of S. mutans biofilms and the mRNA expression of adhesion-associated genes. The viability of the bacteria in biofilms was determined by colony-forming units at 24 and 72 h, meanwhile one sample of each group was prepared for scanning electron microscope (SEM) at 24 h. A real-time reverse transcription polymerase chain-reaction was used to quantify the mRNA expression levels of gtfBCD, ftf and gbpB genes in the presence of various materials. The results revealed that acrylic resins had more S. mutans adhesion than Co-Cr alloy and ceramics in terms of the bacterial viability and SEM observation. The level of gtfBCD expression was the highest in ceramics group and the lowest in acrylic resins group in 24 h biofilms; however, acrylic resins had more gtfBC transcription significantly than other materials at 72 h (P < 0.05). Ftf and gbpB were upregulated in 24 h biofilms on denture base materials compared with control (P < 0.05). These results suggested that the expression profiles of gtfBCD, ftf and gbpB were material-dependent and the physico-chemical properties of the materials might influence the transcription of these genes. Acrylic resins, which had the most bacterial adhesion, might affect biofilm formation via increased expression of ftf and gbpB at 24 h and gtfBC at 72 h.

A Pleiotropic Regulator, Frp, Affects Exopolysaccharide Synthesis, Biofilm Formation, and Competence Development in Streptococcus mutans

Exopolysaccharide synthesis, biofilm formation, and competence are important physiologic functions and virulence factors for Streptococcus mutans. In this study, we report the role of Frp, a transcriptional regulator, on the regulation of these traits crucial to pathogenesis. An Frp-deficient mutant showed decreased transcription of several genes important in virulence, including those encoding fructosyltransferase (Ftf), glucosyltransferase B (GtfB), and GtfC, by reverse transcription and quantitative real-time PCR. Expression of Ftf was decreased in the frp mutant, as assessed by Western blotting as well as by the activity assays. Frp deficiency also inhibited the production of GtfB in the presence of glucose and sucrose as well as the production of GtfC in the presence of glucose. As a consequence of the effects on GtfB and -C, sucrose-induced biofilm formation was decreased in the frp mutant. The expression of competence mediated by the competence-signaling peptide (CSP) system, as assessed by comC gene transcription, was attenuated in the frp mutant. As a result, the transformation efficiency was decreased in the frp mutant but was partially restored by adding synthetic CSP. Transcription of the frp gene was significantly increased in the frp mutant under all conditions tested, indicating that frp transcription is autoregulated. Furthermore, complementation of the frp gene in the frp mutant restored transcription of the affected genes to levels similar to those in the wild-type strain. These results suggest that Frp is a novel pleiotropic effector of multiple cellular functions and is involved in the modulation of exopolysaccharide synthesis, sucrose-dependent biofilm formation, and competence development.

Differential expression profiles of Streptococcus mutans ftf, gtf and vicR genes in the presence of dietary carbohydrates at early and late exponential growth phases

Dental caries is one of the most common infectious diseases that affects humans. Streptococcus mutans, the main pathogenic bacterium associated with dental caries, produces a number of extracellular sucrose-metabolizing enzymes, such as glucosyltransferases (GTFB, GTFC and GTFD) and fructosyltransferase (FTF). The cooperative action of these enzymes is essential for sucrose-dependent cellular adhesion and biofilm formation. A global response regulator (vicR) plays important roles in S. mutans ftf and gtf expression in response to a variety of stimuli. A real-time reverse-transcription polymerase chain-reaction was used to quantify the relative levels of ftf, gtfB, gtfC, gtfD and vicR transcription of S. mutans in the presence of various dietary carbohydrates: sucrose, d-glucose, d-fructose, d-glucitol (d-sorbitol), d-mannitol and xylitol. Ftf was highly expressed at late exponential phase in the presence of sorbitol and mannitol. GtfB was highly expressed in the presence of all the above carbohydrates except for xylitol at early exponential growth phase and glucose and fructose at late exponential growth phase. Similar to gtfB, the expression of gtfC was also induced with the presence of all the tested carbohydrates except for xylitol at early growth and glucose and fructose at late exponential phase. In addition, no effect of mannitol on gtfC expression at early exponential phase was observed. GtfD was less influenced compared to the gtfB and gtfC, demonstrating enhanced expression especially in the presence of sorbitol, glucose, mannitol and xylitol at early exponential phase and mannitol at late exponential phase. VicR expression was induced only at the presence of xylitol at late exponential phase, and a decrease in expression was recorded at early exponential phase. Our findings show that dietary carbohydrates have a major influence on the transcription of ftf, gtfB, gtfC and gtfD, but less on vicR. Sorbitol and mannitol, which are considered as noncariogenic sugar substitutes, may indirectly affect caries by promoting biofilm formation via enhanced expression of gtfs and ftf. These results suggest regulatory circuits for exopolysaccharide gene expression in S. mutans.

Effect of different iodine formulations on the expression and activity of Streptococcus mutans glucosyltransferase and fructosyltransferase in biofilm and planktonic environments

The glucosyltransferase (GTF) and fructosyltransferase (FTF) enzymes play a pivotal role in dental biofilm formation as they synthesize polysaccharides that act as the extracellular matrix of the biofilm. Iodine is a unique antibacterial agent that has distinct properties from other conventional antibacterial agents. In this study we have examined the effect of iodine and povidone iodine (PI) on gtf and ftf expression in biofilm and planktonic environments and on immobilized and unbound GTF and FTF activity. Real-time reverse transcription-PCR was used to investigate the effect of iodine and PI on ftf, gtfB and gtfC expression. The effect of iodine and PI on GTF and FTF activity was tested using radioactive assays. Our results indicate that iodine and PI in a tetraglycol carrier cause enhancement of expression of gtfB in Streptococcus mutans in biofilms but not in planktonic bacteria. PI in water induced expression of gtfB and gtfC in planktonic bacteria. However, iodine and PI strongly inhibit polysaccharide production by GTF and to a lesser extent by FTF activity. The inhibitory effect on GTF activity was similar in solution compared to its activity in the immobilized environment. This unique effect may be attributed to the distinct chemical properties of iodine compared with other antibacterial agents. This study indicates that iodine at sub-bactericidal concentrations demonstrates molecular and enzymatic effects that are highly associated with biofilm formation.

RegM is required for optimal fructosyltransferase and glucosyltransferase gene expression in Streptococcus mutans

Glucosyltransferases (Gtfs) and fructosyltransferase (Ftf), and the exopolysaccharides they produce, facilitate bacterial adherence and biofilm formation, and enhance the virulence of Streptococcus mutans. In this study, we used continuous chemostat cultures and reporter gene fusions to study the expression of ftf and gtfBC in response to carbohydrate availability and pH, and to asses the role of a protein similar to catabolite control protein A (CcpA), RegM, in regulation of these genes. Expression of ftf was efficient at pH 7.0 and 6.0, but was repressed at pH 5.0 under glucose-excess conditions. At pH 7.0, ftf expression was 5-fold lower under glucose-limiting conditions than in cells growing with an excess of glucose. Expression of gtfBC was also sensitive, albeit to a lesser extent, to pH and glucose availability. Inactivation of regM resulted in decreases of as much as 10-fold in both ftf and gtfBC expression, depending on growth conditions. These findings reinforce the importance of pH and carbohydrate availability for expression of two primary virulence attributes of S. mutans and reveal a critical role for RegM in regulation of expression of both gtfBC and ftf.

Regulation of the gtfBC and ftf genes of Streptococcus mutans in biofilms in response to pH and carbohydrate.

Streptococcus mutans produces a number of extracellular sucrose-metabolizing enzymes that contribute to the ability of the organism to cause dental caries, including three glucosyltransferases, the products of the gtfB, gtfC and gtfD genes, and a fructosyltransferase, encoded by the ftf gene. To better understand the regulation of the expression of these genes under environmental conditions that more closely mimic those in dental plaque, two strains of S. mutans harbouring fusions of the gtfBC (SMS102) and ftf (SMS101) promoters to a chloramphenicol acetyltransferase (CAT) gene were examined in biofilms formed in vitro. The strains were grown in a Rototorque biofilm reactor in a tryptone-yeast extract-sucrose medium. CAT specific activity in biofilm cells was measured at quasi-steady state or following additions of 25 mM sucrose or glucose, with or without pH control. After approximately 10 generations of biofilm growth, the ftf and gtfBC genes of S. mutans were found to be expressed at levels different from those reported for planktonic cells growing under otherwise similar conditions. The expression of these genes was induced by the addition of sucrose to the quasi-steady-state cultures. Expression of the gtfBC genes was influenced by environmental pH, since CAT specific activities in quasi-steady-state biofilms of strain SMS102 grown without pH control were twice those produced by cells grown with pH control. Moreover, addition of glucose to quasi-steady-state biofilms resulted in increased expression of the gtfBC-cat fusion, although the magnitude of the induction was less than that seen with sucrose. The effect of pH on ftf expression was negligible. A modest and transient induction of ftf was observed in biofilms pulsed with excess glucose and the kinetics and level of induction of ftf by excess carbohydrate were dependent on the pH of the biofilms. This study demonstrates that the type and amount of carbohydrate and the environmental pH have a major influence on transcription of the gtfBC and ftf genes when the organisms are growing in biofilms, and provides evidence for previously undisclosed regulatory circuits for exopolysaccharide gene expression in S. mutans.

Nuclear receptor-mediated repression of human cholesterol 7α-hydroxylase gene transcription by bile acids

Hydrophobic bile acids strongly repressed transcription of the human cholesterol 7α-hydroxylase gene (CYP7A1) in the bile acid biosynthetic pathway in the liver. Farnesoid X receptor (FXR) repressed CYP7A1/Luc reporter activity in a transfection assay in human liver-derived HepG2 cells, but not in human embryonic kidney (HEK) 293 cells. FXR-binding activity was required for bile acid repression of CYP7A1 transcription despite the fact that FXR did not bind to the CYP7A1 promoter. FXR-induced liver-specific factors must be required for mediating bile acid repression. Bile acids and FXR repressed endogenous CYP7A1 but stimulated α-fetoprotein transcription factor (FTF) and small heterodimer partner (SHP) mRNA expression in HepG2 cells. Feeding of rats with chenodeoxycholic acid repressed CYP7A1, induced FTF, but had no effect on SHP mRNA expression in the liver. FTF strongly repressed CYP7A1 transcription in a dose-dependent manner, and SHP further inhibited CYP7A1 in HepG2 cells, but not in HEK 293 cells. FXR only moderately stimulated SHP transcription, whereas FTF strongly inhibited SHP transcription in HepG2 cells. Results revealed that FTF was a dominant negative factor that was induced by bile acid-activated FXR to inhibit both CYP7A1 and SHP transcription. Differential regulation of FTF and SHP expression by bile acids may explain the wide variation in CYP7A1 expression and the rate of bile acid synthesis and regulation in different species. —Chen, W., E. Owsley, Y. Yang, D. Stroup, and J. Y. L. Chiang. Nuclear receptor-mediated repression of human cholesterol 7α-hydroxylase gene transcription by bile acids.

Characterization of the fructosyltransferase gene of Actinomyces naeslundii WVU45.

Oral actinomycetes produce fructosyltransferase (FTF) enzymes which convert sucrose into polymers of D-fructose, known as levans, and these polymers are thought to contribute to the persistence and virulence of the organisms. A gene encoding FTF was isolated from Actinomyces naeslundii WVU45; the deduced amino acid sequence showed significant similarity to known levansucrases of gram-negative environmental isolates but was less similar to FTFs from gram-positive bacteria. A transcriptional start site was mapped by primer extension 70 bp 5' from the putative start codon. Promoter fusions to a chloramphenicol acetyltransferase gene were used to confirm that there was a functional promoter driving ftf expression and to show that sequences located 86 to 218 bp upstream of the transcription initiation site were required for optimal ftf expression. Quantitative slot blot analysis against total RNA from cells grown on different sugars or from different growth phases revealed that ftf was constitutively transcribed. Thus, the A. naeslundii FTF is more similar in primary sequence and the regulation of expression to levansucrases of gram-negative bacteria than gram-positive bacteria.

Expression of the Streptococcus mutans fructosyltransferase gene within a mammalian host.

In vivo expression of the virulence-associated fructosyltransferase gene (ftf) of Streptococcus mutans has been examined. S. mutans ftf expression is affected by both the specific carbohydrate consumed and the age of the host animal.

Genetic regulation of fructosyltransferase in Streptococcus mutans

Streptococcus mutans possesses several extracellular sucrose-metabolizing enzymes which have been implicated as important virulence factors in dental caries. This study was initiated to investigate the genetic regulation of one of these enzymes, the extracellular fructosyltransferase (Ftf). Fusions were constructed with the region upstream of the S. mutans GS5 Ftf gene (ftf) and a promoterless chloramphenicol acetyltransferase (CAT) gene. The fusions were integrated at a remote site in the chromosome, and transcriptional activity in response to the addition of various carbohydrates to the growth medium was measured. A significant increase in CAT activity was observed when glucose-grown cells were shifted to sucrose-containing medium. Sucrose-induced expression was repressed immediately upon addition of phosphoenolpyruvate phosphotransferase system sugars to the growth media. Deletion analysis of the ftf upstream region revealed that an inverted repeat structure was involved in the control of ftf expression in response to carbohydrate. However, the control of the level of ftf transcription appeared to involve a region distinct from that mediating carbohydrate regulation. CAT gene fusions also were constructed with the ftf upstream region from S. mutans V403, a fructan-hyperproducing strain which synthesizes increased levels of Ftf. Sequence analysis of the upstream ftf region in this strain revealed several nucleotide sequence changes which were associated with high-level ftf expression. Comparison of the GS5 and V403 ftf expression patterns suggested the presence of a trans-acting factor(s) involved in modulation of ftf expression in response to carbohydrate. This factor(s) was either absent or altered in V403, resulting in the inability of this organism to respond to the presence of carbohydrate. The sequences of the ftf regions from three additional fructan-hyperproducing strains were determined and compared with that of V403. Only one strain displayed nucleotide changes similar to those of V403. Two additional strains did not have these changes, suggesting that several mechanisms for up-regulation of ftf expression exist.