| Background: Streptococcus mutans(S.mutans)is considered to be the main pathogen causing human dental caries.Biofilm formation,acidogenesis and acid resistance play crucial roles in S.mutans pathogenicity.As Gram-positive bacterium,Lipoteichoic acid(LTA)is essential component of the cell wall and supposed to be main pathogenic factor.Previous researches show that the D-alanyl ester substituent of LTA modulates physiological and metabolic activities of bacteria.D-alanyl ester substituent is the result of Lipoteichoic acid D-alanylation process,and the enzymes required for this process are encoded by the dlt operon.It was demonstrated that D-alanylation of LTA was closely related to biofilm formation and acid resistance of S.mutans.However,related mechanisms and regulatory approaches are still not clear.Objectives: 1)In this research,dlt C gene mutation strain of S.mutans was constructed to explore the influence of D-alanylation of LTA on S.mutans biofilm formation,acidogenesis,acid resistance and interspecies competition.2)To investigate the regulation of D-alanylation of LTA in S.mutans involved two-component signal transduction system.Materials and methods: 1)SMUA159-△dlt C was constructed by homologous recombination.The content of D-alanine in SMUA159-△dlt C LTA was detected by the 2,4-dinitrophenylhydrazine the growth curve was observed by an automatic growth curve analyzer,and the bacterial morphology was observed by Gram staining;2)Auto-aggregation of SMUA159 and SMUA159-△dlt C were detected by spectrophotometric assay.Bacteria adhesion was observed by glass surface adherence assay.The surface charges of planktonic bacteria and biofilm were measured by cytochrome c.The young(6 h)and mature(24 h)biofilms of SMUA159 and SMUA159-△dlt C were formed and the content of water-soluble and water-insoluble extracellular polysaccharide within biofilms were measured with anthrone.The distribution of extracellular polysaccharides and the biofilm formation process were observed by fluorescent staining.Gene expression related biofilm formation and bacteria adhesion were detected by Real-time PCR.3)the terminal p H of SMUA159 and SMUA159-△dlt C culture in variable p H initial medium were measured to evaluate the acidogenesis of bacteria.CFU(Colony-Forming Units)was conducted to evaluate innate acid resistance and ATR(induced acid resistance response)in planktonic and biofilm state SMUA159 and SMUA159-△dlt C.4)Agar plates assay and conditioned medium assay were conducted to compare the inhibitory effects of SMUA159 and SMUA159-△dlt C metabolites on the oral commensal bacteria,Streptococcus sanguinis(S.sanguinis)and Streptococcus gordonii(S.gordonii).Dual-species biofilms(SMUA159+S.sanguinis、SMUA159+S.gordonii、SMUA159-△dlt C+S.sanguinis、SMUA159-△dlt C+S.gordonii)were formed on bovine enamel blocks placed in a vertical position in 24-well plates.The p H and the concentration of calcium were measured to evaluate demineralization of enamel causing by biofilms and the morphology of demineralized blocks were observed by SEM(Scanning electron microscope).The proportion of each strain in dual-species biofilms was measured by CFU.Mutacins synthesis and acid produced related genes were detected by Real-time PCR.5)Inactivation of Lia SR two-component signal transduction system of S.mutans by construction of SMUA159-△lia S and SMUA159-△lia R;The expression levels of dlt C gene in SMUA159-△lia S and SMUA159-△lia R within acidic environment and biofilm state were measured by Real-time PCR.Results: 1)S.mutans dlt C deletion strain,named SMUA159-△dlt C,was constructed successfully and dlt C gene was replaced by antibiotic gene which was confirm by DNA sequence.After the dlt C gene inactivation,the content of D-alanine in LTA of S.mutans decreased significantly(p <0.05).Growth curve showed that mutant had a slight decrease in growth rate and bacterial chain length increased.2)Deletion of dlt C gene resulted in defects in biofilm formation.Compared with parental three-dimensional mature biofilm,the biofilm formed by mutant was flat and gaps existed,in which cells lacked contact with each other and were scattered on the substratum as clusters.Compared with parental strain,the auto-aggregation of mutant strain enhanced significantly(p <0.05),while,the adhesion ability was decreased(p <0.05).The negative charge on the surface of bacteria increased significantly in both the planktonic and the biofilm state(p<0.05).The mutant was found to produce more water-soluble glucan(WSG)and less water-insoluble glucan(WIG)than parental strain during the biofilm formation process.In the early stage of the biofilm formation,the expression of gtf and gbp genes which related to the extracellular polysaccharide and surface adhesion proteins synthesis decreased significantly(p <0.05)compared to the parental strain.In the late stage,gtf D expression level was same as parental strain’s(p> 0.05).gtf C and gbp A were relatively high expression,and gtf B expression was still lower than the parental strain(p <0.05).3)the acidogenesis of SMUA159-△dlt C in acid broth compromised compared to the parental strain.Moreover,planktonic SMUA159-△dlt C showed more sensitive to acid while the ATR of SMUA159-△dlt C could still be induced.By contrast,SMUA159-△dlt C in biofilm showed same response to acid treatment with parental strain.4)Deletion of dlt C gene resulted in decrease of the metabolites of S.mutans which inhibit the growth of S.sanguinis and S. gordonii.The proportion of S.mutans significantly decreased in dual-species biofilms(p <0.05)and the demineralization effect on the enamel blocks was significantly weaken.The Real-time PCR results illustrated that the levels of m mutacins synthesis and acid produced related genes decreased significantly both in early and late stage of biofilm formation(p <0.05).5)S.mutans lia S deletion strain and S.mutans lia R deletion strain were constructed successfully,which was named SMUA159-△lia S and SMUA159-△lia R,separately.Deletion of lia S gene resulted in dlt C gene expression up regulation(p <0.05)in the acidic environment or biofilm compared to parental strain.Different form SMUA159-△lia S,deletion of lia R gene resulted in same dlt C gene expression with parental strain in the acidic environment(p> 0.05)while up regulation in biofilm(p <0.05).Conclusion: To summarize,D-alanylation of LTA plays a critical role in S.mutans cariogenic factors expression.1)D-alanylation of LTA can influence S.mutans biofilm formation by modulating bacteria auto-aggregation,adhesion,surface charge,and extracellular polysaccharides synthesis.2)D-alanylation of LTA can also affect the acidogenesis and acid sensitivity of planktonic S.mutans while shows no effect on S.mutans in biofilm.3)D-alanylation of LTA modulate the interspecies competition of S.mutans by regulating the produce mutacins and acid.4)In the acidic environment and biofilm state,Lia SR two-component signal transduction system shows negative regulatory effect on D-alanylation of LTA in acid resistance and biofilm formation of S.mutans.These findings provide a theoretical basis for further clarifying the cariogenic mechanism of S.mutans. |
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