Cloning, Expression And Functional Study Of Streptococcus Mutans GGDEF Protein Domain

Dental caries is one of the most common bacterial infections in human beings. Streptococcus mutans has shown to be the causative agent of this disease. Research into the etiology of dental caries and caries prevention established the key role of S. mutans cariogenic factors in caries occurrence and progression. The main virulence factors associated with cariogenicity include adhesion, acidogenicity, and acid tolerance. Streptococcus mutants (S. mutans) is thought to be one of the primary causative agents of dental caries depends on a biofilm lifestyle. Even though dental plaque consists of numerous bacterial species, but the presence of S. mutans has been consistently linked with the production of dental caries.Recently, as the completion of the majority bacterials genomic sequencing, according to the research of comparative genomics, GGDEF is widespread in bacteria but is not found the outside bacterial kingdom, The observation that GGDEF domains are often associate with domains involved in signal perception or signal transduction, argued for the existence of a dedicated regulatory network that converts a variety of different signals into the production of the second messenger c-di-GMP. Recent studies showed that it has effects on bacterial cell differentiation,biofilm formation,virulence factor expression,cell-cell communication and host cell interaction, etc.. It is one of key regulatory factors in bacterium exist and metabolism , however, the research in its streptococcus mutans is still a blank. Is there any way of c-di-GMP acting existing in streptococcus mutans vital movement? What are functions of the genes similar to GGDEF in streptococcus mutans gene group? Does the intervention to this protein affect the changes of phenotypic and expression of virulence factor? The answers of these questions will help to strengthened the comprehension of bacterium signal transduction pathway, and provide the evidence for exploring new anticarious methods。This study will give evidence on the effect of c-di-GMP on s.mutans metabolism and cariogenic activity, which might theoreticallyindicate a new way in caries prevention.This research consists of three parts as follows.Partâ… Cloning, prokaryotic expression of S. mutans gcp gene andpurification of the recombinant Gcp proteinS. mutans UA159 was routinely cultured at an anaerobic atmosphere and its genomic DNA was isolated. The encoding region of gcp, excluding the signal sequence, was amplified by polymerase chain reaction (PCR). The relevant fragment was ligated with a pMD-19T simple vector before transformed into E. coli competent cells. Positive clones were selected and sequenced. The sequencing result was found completely consistent with that in GenBank.The fragment was then inserted into a prokaryotic expression vector pP_ROEX HTb such that the coding sequence of the gcp fragment was in-frame with an N-terminal His₆ tag, to construct the plasmid pP_ROEX HTb-gcp. The plasmid was then used to transform E. coli DH5αcompetent cells. Positive clones were selected and identified by enzyme restriction analysis, and then induced by IPTG to express the His-tagged GGDEF fusion protein, named Gcp protein. The supernatant of cell lysate was analyzed by SDS-PAGE. A new band with 77KDa appeared in the SDS-PAGE gel and was approximately the same as expected, which indicated that we successfully obtained the soluble expression of Gcp protein in E. coli.By fermenter, We get large amount of recombinant Gcp fusion protein. The harvested cells were sonificated and the supernatant of cell lysate were purified by Ni²⁺-NTA affinity chromatography. The purified rGcp protein was utilized to test interaction with c-di-GMP by HPLC. Its reactivity to anti-His antibody was tested by western-blot. The results indicated that we successfully obtained the purified rGcp protein possessing activity.Partâ…¡Partâ…¡Construction and identification of an insertion mutant of gcp and examination of its cariogenic propertiesA 873bp internal fragment of the gcp gene was amplified from S. mutans UA159 chromosomal DNA by PCR and ligated with the pMD-19T vector. After enzyme restriction analysis, the fragment was subcloned into the suicide vector pVA8912 to give plasmid pVA8912/gcp. S. mutans UA159 cells were transformed with plasmid pVA8912/gcp using natural transformation method. The positive clones were selected and their genomic DNA was isolated. The correct location of the insertion was confirmed by PCR and enzyme restriction analysis, as well as western-blot analysis. The results showed that plasmid integrated into the gcp gene region on the chromosome. These results indicated that the gcp mutant was successfully constructed.In order to examine the cariogenic properties of gcp mutant, several tests were performed.The growth, acidogenicity and cariogenicity of the gcp mutant and wildtype cultures were evaluated during the 48 h inoculation. The results showed that the gcp mutant was indistinguishable from the wildtype with respect to growth rate,but distinguishable with acidogenicity and acidodurance.The abilities of gcp mutant and wildtype cells to form stable biofilms were assessed by growing the cells in 96-well, flat-bottom microtiter plates in an aerobic atmosphere for 48 h. The adherent bacteria were stained with crystal violet and the bound dyes were extracted by using ethanol/acetone (8:2, v/v) mixture. Biofilm formation was then quantified by measuring the optical density of the solution at 575 nm. The results showed that the biofilms formed by the mutant had reduced numbers of cells compared to the wild type, which indicated that Gcp protein might contribute to the S. mutans biofilm formation in some degree.Biofilm structures of the gcp mutant and wildtype strains on enamel chips were observed by scanning electron microscopy (SEM). Upon inspection, gcp mutant has a noticeable difference in biofilm structure from that of the wild type. The wild type biofilms have a thick and confluent appearance, with cells regularly arranged. However, the gcp mutant biofilms are very thin and seem much more heterogeneous, with large gaps and more long-chains in the biofilm. This phenomenon suggested the important role of Gcp protein in S. mutans biofilm structure.To investigate the adhesion properties of gcp mutant and wildtype strains, the bacteria cells were labeled with a fluorescent indicator (BCECF/AM) and incubated with saliva-coated hydroxyapatite (SHA). Bacteria adhered to the SHA were measured using a spectrofluorometer. The result showed a significantly lower adherence percentage in the gcp mutant group (19.7%±0.91) as compared with the wild type strains (29.5%±2.9). After the labeled wild type bacteria were co-incubated with anti-Gcp antibody and SHA, the adherence percentage was decreased compared to the non-antibody controls (P <0.05). The test indicated that GGDEF domain played a role in the S. mutans adhesion to SHA .