The Study On C-di-GMP Signaling Pathways And RgpAc Gene Regulating Streptococcus Mutans Cariogenic Properties

Dental caries is one of the most common chronic diseases afflicting humans and has a polymicrobial etiology. Streptococcus mutans (S. mutans) is one of the human oral caries bacteria, as well as the prime pathogen agents in the development of dental caries. An important virulence property of S. mutans is to form dental plaque biofilm on the tooth surface. Bacteria in the biofilm grow, reproduce, metabolize and constitute a special micro ecological environment.Comparative genomic study found a class of GGDEF/EAL domain protein widespread in various types of bacteria. Further studies showed that its main function is about synthesis and degradation of cyclic di(3’â†'5’)-guanylic acid (c-di-GMP). The GGDEF/EAL domain could catalyze GTPs to c-di-GMP. The GGDEF and EAL domain families have1,601and1,016members, respectively, in the Pfam protein family database in July2005. Proteins containing those domains are involved in motility and exopolysaccharide-biofilm production in a variety of bacteria. Some GGDEF-domain proteins control biofilm formation and/or cell aggregation by enhancing the levels of the novel second messenger c-di-GMP. Intracellular concentrations of c-di-GMP are regulated by multiple domain proteins. C-di-GMP is synthesized from GTP by diguanylate cyclases (DGCs) proteins containing the amino acid motif GGDEF domain. C-di-GMP is degraded by phosphodiesterases (PDEs) that contain one of two conserved domain families:one defined by the EAL motif and the other by an HD-GYP motif. C-di-GMP plays an important role in the physiology of many bacteria.Recent studies show that c-di-GMP is one of the second messager signaling molecules. It could regulate many processes to adapt to environment in bacteria. It involves in the biofilm formation, virulence, communication and the interaction between the host cells and bacteria. It is one of the key regulatory factors in bacterial survival and metabolism.There is c-di-GMP signaling pathway in Streptococcus mutans. Adding exogenous c-di-GMP or reducing endogenous c-di-GMP will inhibit the biofilm formation of Streptococcus mutans. But the mechanisms are unknown. Further research on these issues will help to deepen the role of c-di-GMP signaling pathways in Streptococcus mutans and explore a new method of anticaries. Construct gcp gene mutation strains of Streptococcus mutans. Then extract the RNA of mutation strain and wild strain. Compare the differences by microarray chip and screen the biofilm-related genes to analysis the signaling passways.This paper includes the following three chapters:Chapter I:Constructed the gcp gene mutation strain of S. mutans UA159Designed the upstream primers and downstream primers of gcp gene by Primer Premeier5.0software according to the whole genome sequence of S. mutans UA159in NCBI. The upstream fragment and downstream fragment were amplified. Constructed a vector containing spectinomycin resistance (spe+) pFW5-gcp-UP-DOWN. Constructed S. mutans UA159gcp gene mutation strain which was named gcp-KO by homologous recombination. Under an optical microscope, S. mutans UA159was G+, arranged in chains with different lengths. The gcp-KO strain was G+too, arranged in short chains and more sparse arrangement compared to the wild strain. This might be due to genetic defects. Gel electrophoresis was used to identify the upstream and downstream of gcp gene and the spectinomycin gene in wild strain and mutation strain. The results showed that the wild strain didn’t contain the spectinomycin gene but contain the gcp gene. The mutation strain could amplify upstream and downstream of gcp gene and amplify the spectinomycin gene, but it couldn’t amplify the gcp gene. Real-time PCR showed gcp gene a down-regulation of the gcp-KO strain. All the results showed the mutation strain was gcp gene defect strain. The sequencing results also showed that the gcp gene was replaced by the spectinomycin gene.Chapter â…¡:Microarray chip experiments in gcp-KO strain and wild strain of S. mutans UA159Extracted the RNA of wild strain and gcp-KO strain. Then synthesized "cDNA" by the instructions of "TaKaRa1st Strand cDNA Synthesis Kit" and marked the control group by Cy5-CTP and experimental group by Cy3-CTP. Immediately absorbed the appropriate amount of hybridization mixture on the slide and put the slide into NimbleGen MS200gene chip scanner. Laboratory assistant collected the data with MS200software.Compared the Cy5fluorescence signal strength and Cy3signal strength of the two groups and got the ratio by Cy5than Cy3. For the same probe, if the ratio was2times or more than2times, gene expression of this probe was downregulated. If the ratio was0.5times or less than0.5times, gene expression of this probe was upregulated. Finally got411genes with198gene upregulated and213gene down regulated. After GO analysis, COG analysis and KEGG analysis, choose8genes for Real-time PCR. Think about the previous experiments results, RgpAc gene is selected as the target gene in the following studies.Chapter III Construction and function studies of RgpAc mutation strainThis chapter includes two aspects:"Construction of RgpAc gene mutation strain" and "Function studies of RgpAc gene mutation strain"1. Construction of RgpAc gene mutation strainDesigned the upstream primers and downstream primers of RgpAc gene by Primer Premeier5.0software according to the whole genome sequence of S. mutans UA159in NCBI. The upstream fragment and downstream fragment were amplified. Then constructed a suicide vector containing spectinomycin resistance (spe+) pFW5-RgpAc-UP-DOWN. Finally construct S. mutans UA159RgpAc gene mutation strain which was named RgpAc-KO by homologous recombination. Gel electrophoresis was used to identify the upstream and downstream of RgpAc gene and the spectinomycin gene in wild strain and mutation strain. The experimental results showed that the wild strain contained the RgpAc gene, not the spectinomycin gene. The mutation strain could amplify upstream and downstream of RgpAc gene and the spectinomycin gene, but it couldn’t amplify the RgpAc gene. Real-time PCR showed RgpAc gene downregulation in the RgpAc-KO strain. All the results showed the mutation strain was RgpAc gene defect strain.2. Function studies of RgpAc gene mutation strainThis study compared the extracellular polysaccharide synthesis capacity of RgpAc gene mutation strain and wild strain by modified anthrone colorimetry. SPSS13.0statistical software was used to analysis the results. The formation of water-soluble glucan of RgpAc-KO strain decreased by55%, exopolysaccharide by41%. They were significantly lower than wild strain. But there was no significant difference in the ability to form water-insoluble glucan between RgpAc-KO strain and wild strain. The RgpAc gene was glycosyltransferase in S. mutans UA159. Glycosyltransferases catalyzed the transfer of sugar moieties to specific acceptor molecules and formed glycosidic bonds. This glycosyltransferase mainly relates to water-soluble dextran a-1,6. When RgpAc gene was knocked out, the ability of synthetic dextran a-1,6was inhibited and water-soluble glucan was decreased.Designed the experiment about biofilm formation in different strains. RgpAc-KO strain was experimental group and wild strain was control group. Then added200μM and400μM c-di-GMP into wild strain and RgpAc-KO strain. Both strains grown aerobically at37℃in a5%CO2atmosphere under static conditions for48h. Measured the absorbance value at575nm. The biofilm formation of S. mutans UA159was inhibited after adding c-di-GMP to the culture. However, no major differences were observed between different concentrations of c-di-GMP. The results showed that the RgpAc-KO strain was less efficient55%at forming biofilm than wild strain. The biofilm formation of RgpAc-KO strain was promoted after adding c-di-GMP to the culture. In this study, the biofilm formation of RgpAc-KO was decreased. However, the biofilm formation of RgpAc-KO strain with200μM c-di-GMP and400μM c-di-GMP was increased. Water-soluble and water-insoluble glucan are the biofilm matrix. The ability of the water-soluble glucan decreased after RgpAc gene knocked out. The biofilm formation was inhibited because lack of matrix. When extracellular c-di-GMP was added in the RgpAc-KO strain, it increased the binding of other receptors and promoted the biofilm formation.To observe the biofilm structure of RgpAc-KO strain with the c-di-GMP, prepared the enamel pieces and simulated the vivo situation to form artificial biofilm on the enamel pieces. Then biofilm structure was detected by a combination of scanning electron microscopy. Under low magnification, thick and compact biofilm was observed of sild strain, RgpAc-KO strain with200μM c-di-GMP and RgpAc-KO strain with400μM c-di-GMP. However, thin and loose biofilm was observed of RgpAc-KO strain. Under high magnification, cells with large extracellular matrix arranged regularly of sild strain, RgpAc-KO strain with200μM c-di-GMP and RgpAc-KO strain with400μM c-di-GMP. Cells with little extracellular matrix arranged irregularly of RgpAc-KO strain. RgpAc-KO strain reduced the synthesis of polysaccharide, especially glucan. Glucan and other polysaccharide were energy substances and metabolic substrates, as well as promoted bacteria adhesion and aggregation. When extracellular c-di-GMP was added in the RgpAc-KO strain, it played a role of endogenous second message and finally increased the biofilm formation. But RgpAc-KO strain reduced the addition of extracellular polysaccharide and suppressed the ability of biofilm synthesis. Under scanning electron microscope, loose extracellular matrix and less bacteria were observed.In conclusion, this study constructs gcp gene knockout strain of Streptococcus mutans UA159by homologous recombination. Microarray analysis finds the RgpAc gene is upregulation. Then construct RgpAc gene knockout strain by the same way and design studies about its functions. The results will provide new methods and ideas for caries prevention.