Preliminary Study Of LuxS Of Streptococcus Mutans-significant Effect On Pathogenic Ability In Dental Caries

Oral micro-ecological niche is composed of many kinds of microorganism, which inhabit, grow and multiply, compete and inhibit with each other by the Quorum Sensing in the oral cavity. When bacteria in the micro-ecological environment reach a certain concentration, they can yield extracellular regulative chemical signals. With those signaling molecules accumulating to a certain amount, the expression of target genes will be escalated or inhibited. Moreover, signaling molecules can regulate some functional genes which are closely related with biofilm formation, bacteriocin synthesis, spore production, acid secretion, virulence reaction and so on, thereby improving the viability of bacteria. Streptococcus Mutans, one of the most important pathogen in dental caries development, can also regulate themselves through Quorum Sensing according to the change in the surrounding environment, to survive and thrive. luxS dependant Quorum Sensing in S. mutans can play a crucial role in pathogenic ability in dental caries. In our study, we set out the study of relation of luxS gene in S. mutans and their resistance acid killing and forming biofilm.1,Constructing the luxS mutans by knocking out luxS gene in S. mutans:Kanamicin resistance gene and the upstream and downstream of luxS were cloned via PCR, respectively through plasmid pEGFP-N1 and DNA of Streptococcus mutans as template, then were ligated into Multiple Cloning Site(MCS) of vector pMD19-T in certain order and transformed into E. coli Competent Cells, finally transformants were selected for resistance to Kanamicin and Ampicillin. pMD-19TUKD was extracted from E. coli transformants harvested and transformed into S. mutans UA159 to obtain the luxS mutans by homologous recombination and double crossover, furthermore, the luxS mutans were examined via PCR and V. harveyi bioluminescence assay.2,Acid tolerance assay of the luxS mutans:S.mutans strains and the luxS mutans were inoculated in BHI, and were acidified by maintaining in 0.1 M pH 3.0 glycine buffer. Their Acid tolerance was examined by gradient dilution and plate count way. Furthermore, S.mutans strains and the luxS mutans were adaptively grown in the pH 5.0 BHI medium for 2h, then were acidified by the above way. Result: luxS mutant strain and S. mutans UA159 survival rate both show a degression in glycine buffer, pH 3.0 accompanying time prolonged, and luxS mutant strain show a lower degression. Compared to grown in BHI medium, pH 7.0, the luxS mutant strain and strain UA159 preadapted acid killing for 2 hours in pH 5.0 BHI medium both displayed a prolong lag phase in glycine buffer, pH 3.03,Assay of biofilm on the surface of teeth:Adult human teeth were cut into slices which were put in BHI containing 1% sucrose medium, then S.mutans UA159 and the luxS mutans were inoculated into the medium, and observed biofilm formation on the surface of teeth slices after 2h, 1d, 3d. S.mutans UA159 and the luxS mutans were inoculated into BHI containing 0.5% glucose medium in one day, then observed biofilm formation on the surface of teeth slices. Result: Compared to the wild-type strain UA159, the luxS mutant maintained in BHI containing 1.0% sucrose medium displayed an apparent defect in the biofilm formation in 2h, 1d and 3d by scanning electron microscopy. Biofilm formation by the luxS mutant in BHI supplemented with 0.5% glucose was not greatly attenuated compared to that of S.mutans UA159. Hence, result suggested that the luxS genes was possible to regulate sucrose dependent biofilm formation.