Construction Of Comd Gene Mutant Of Streptococcus Mutans And Virulence Investigation

BackgroundStreptococcus mutans (S.m) is recognized as an important cariogenic bacteria, the main virulence factors including acid-production, acid-resistance, adhesin, the production of dextran, and the formation of dental plaque structure plays an essential role in caries formation.Quorum sensing is a special signaling mechanism between bacteria, the nature of which is that the bacteria identify the signal from the outside world and change it into gene activation or other cellular responses. Quorum sensing signaling system plays an important role in biofilm formation, competence development, the expression of other virulence factors of Streptococcus mutans. Streptococcus mutans is a gram positive bacteria, which have a Lux-S and AI-2-mediated quorum sensing system and a signal peptide-mediated two-component quorum sensing system, the former control signals communication between different species, the latter control signal exchange within bacterial species.Com competent genes family is one of the important two-component signaling system, with comD gene encoding a dimerization transmembrane receptor which can sense specific stimuli outside and change it into the intracellular signaling. ComD gene has been demonstrated to have relation to bacteria transformation, the acid tolerance and induction, bacteriocin generation and the biofilm formation, while few intensive studies were about the correlation between comD gene and the major caries related virulence factors of Streptococcus mutans.ObjectiveTo construct comD gene mutant of S.m UA159, which is preparing for the further of the effect of comD gene on cariogenic virulence factors. To investigate the correlation of Quorum sensing signal system and cariogenic virulence factors in S.m on the gene level, and to provide a theoretical evidence to study the significance of the regulation of quorum sensing signaling system in the prevention and treatment of dental caries .Method1 Construction of comD gene mutant of S.m UA159Long flanking homology polymerase chain reaction(LFH- PCR) was introduced to generate a gene disruption construct consisting of erm cassette with long flanking homology regions to the target gene. Then S.m UA159 was transformed directly with this PCR product. The comD-deletion mutant was obtained on the BHI agar containing erythromycin and identified by morphology observation, biochemical characteristic test,PCR, RT-PCR and sequencing.2 Comparing the differences of cariogenic virulence between S.mUA159 and comD mutant2.1 To analyse biofilm of S.mUA159 and comD mutant by SEM: Biofilms were developed on the human enamel surface, a BHI medium supplemented with final concentrations 2% sucrose was used as the biofilm medium, After the cultures were incubated at 37℃anaerobicly for 6h, 12h, 24h, then observe the biofilm structure at three different time points by SEM.2.2 To analyse mRNA expression levels of polysaccharide metabolism genes (gtfB, gtfD, gtfC, ftf) of S.mUA159 and comD mutant by Fluorescent quantitative polymerase chain rection(FQ-PCR)2.3 To detect the activity of lactate dehydrogenase( LDH) which is associated with acid production of S.mUA159 and comD mutantResult1 Study of morphological and biochemical confirmed the transformant was S.mutans. RT-PCR, PCR and sequencing showed that comD gene was completely replaced by erm genes, S.m UA159 comD defective strain was successfully constructed.2 Observation of the biofilm structure at three time points showed that : there were visible biofilm formation after 6h,12h,24h in both two strains, while the comD defects strains had larger pore in the biofilm ,and the structure was relatively looser than reference strain.3 As the LFH-PCR results showd, there was significant difference(P <0.001) of mRNA expression levels of four polysaccharide metabolism related genes between the reference strains, and the defect strain had a reduced expression of gtfB, gtfD, gtfC, ftf.4 The LDH detection showed that the defect strain had a decreased LDH activity levels, and the differences were significant (P <0.05) between the reference strain and comD defective strains.Conclusion1 LFH-PCR is an effective functional genomics method in construction of S.m mutant ,which can completely replace the target gene.2 With the defect of comD gene, mRNA expression levels decreased in polysaccharide metabolism gene ( gtfB, gtfD, gtfC, ftf ), and so as to the LDH activity which is closely related to acid production, the formation of plaque biofilm also has structural defects. So the research results show that the comD gene plays a part in the regulation of important cariogenic virulence factors of S.m UA159 .