The Role Of C-di-GMP Signaling Pathway In The Formation Of Dental Caries Caused By Streptococcus Mutans

Dental caries is a common oral infectious disease that affect people's health. One of the dorminant pathogenic microorganisms cause human dental caries is Streptococcus mutans ( S. mutans), which attaches on the surface of the teeth, produce polysaccharide and acid, and survive acid environment. With the contribution of GGDEF, S. mutans generates c-di-GMP, which has been reported mediating biofilm formation and virulent factors expression in Staphlococcus Aureus, Vibrio Cholerae, Pseudomonus Aurugenosa, and it also stimulated innate immunity which limited the infection of the bacteriae mentioned above. It is one of key regulatory factors inbacterium exist and metabolism,however, the research in S. mutans is still unknown. In this study, we focus on the effect of exogenous c-di-GMP on S. mutans induced dental caries and its mechanism,which might theoretically indicate a new way in caries prevention.Section I:exogenous c-di-GMP's effect on dental caries caused by S MutansBiofilm Formation:48 hours after exposed to exogenous c-di-GMP with different dosage, biofilm formed in 96 well plates by S Mutans were stained and dissolved by ethanol/acetone, the OD measured by spectrophotometry presents the amount of biofilm in each well. 200μM c-di-GMP and the saline with the same volume incubated with S Mutans on the fresh tooth slices, the biofilm on the slices were investigated with scanning electronic microscope. Result shown that c-di-GMP decreased the biofilm formation and the bacteriae disorganized on the tooth slices with less extracellular matrix, which implies that 200uM c-di-GMP inhibit S Mutans's capability to form biofilm.Acid generation and acid resistance: Exogenous c-di-GMP and saline added into variable pH bacteria medium with UA159 S Mutans strain, the change of pH values (△pH)at the end of culture were compared;after cultured in pH=5 medium for 2 hours, the number of live bacteriea presents the ability of acid resistance. Results: after treat with c-di-GMP, S. Mutans's capability of acid generation and acid resistance decreased.Extracellular polysaccharide formation: S. Mutans strain UA159 cultured in anaerobic environment 24 hour after exogenous c-di-GMP and saline added into culture system, S. Mutans were broken by distilled water (x2 times) and the supernatant (supernatant I) of 3 centrifugations were collected and combined as water-soluble extracellular polysaccharide. The pellets were added with same volume 0.5 mol/L NaOH, centrifuged, collected supernatant, and repeated 3 times. Combine the supernatant (supernatant II) as water-insoluble polysaccharide. 1.5ml from supernatant I and II (respectively) plus 3 volumes of ethanol, 4℃overnight, collect and centrifuge and discard the upper water phase. Pellets dissolved in distilled water and 0.1mol/L NaOH with same volume. Anthrone method measured the content of glucan in the water-soluble and–insoluble supernatant,OD620,the standard curve was made with glucose and calculate the water-soluble and–insoluble glucan generation. With the result shown that exogenous c-di-GMP treatment significantly decreased the formation of water-insoluble glucan. Attached to the surface of HA coated with saliva: fluorescence- BCECF/AM labeled UA159 strain put on SHA coated with saliva mimic oral teeth surface, exogenous c-di-GMP and saline co-cultured with UA159 for 30 minutes, fluorescence measured presents the bacteria attached on SHA (485nm→535nm). Results: c-di-GMP treatment decreased the bacteria attachment.Token together, exogenous c-di-GMP inhibits S Mutans'capacity to form dental caries. So it is possible that exogenous c-di-GMP could be used as anti-caries agent, either alone or with other methods.Section II: Exogenous c-di-GMP Prevent Caries Formation in Animals Dental Caries were made on the teeth of 20 day age SD rat infected with S Mutans, and fed with caries inducing food 2000 #. Exogenous c-di-GMP, NaF solution and 0.9% NaCl applied to the teeth of the rats, the bacteria number and scores of dental caries were recorded. Results shown that the food and water consume are same among different treatments, which enabled same number of dental caries on the teeth. No red plague, swelling appeared during the treatment, which implies that exogenous c-di-GMP has no toxic effect in the oral cavity. Exogenous c-di-GMP treatment group downgrade the scores of dental caries on occlusal surface and smooth surface, but no difference has been found on the number of bacterial plagues, implies that c-di-GMP could be used as anti-caries agent.Section III: S Mutans with gcp gene inactivation, strain construction, identification and its caries inducing charactersTake pMD19T-gcp as template, PCR amplify gcp sequence (873bp) , after connected to T vector, the gene frame inserted into suicide vector pVA8912. Enzymatic analysis found the product of PCR product had the same size as the inserted piece, which implies the construct of pVA8912/gcp is successful. Thereby pVA8912/gcp transformed into UA159 and positive clone selected. The selected clone was then verified by PCR and enzymatic analysis on the extracted genomic DNA of the positive clone, with the result shown that the construction of S. Mutans with gcp gene inactivation strain was successful.S. Mutans with gcp gene inactivation strain was culture in anaerobic environment, the growth curve based on values of OD 600 presents the bacterial growth condition. Results shown that no difference between S. Mutans with gcp gene inactivation strain and wildtype bacteria was found, which implies that inactivation of gcp gene did not change the growth of S Mutans.S. Mutans with gcp gene inactivation strain and wildtype bacteria were plated into 96 well plates and cultured in anaerobic environment for 48 hours, the bacteria attached were stained with crystal violet, ethanol/acetone dissolve the precipitated stain and the spectrophotometric values of OD575 represent the amount of biofilm formation, with the results shown that S. Mutans with gcp gene inactivation strain formed much less biofilm as compared with wildtype. This implies that gcp gene has relationship with S. Mutans'capacity to form biofilm.Fluorescence labeled S. Mutans with gcp gene inactivation strain and wildtype bacteria were put on SHA coated with saliva,the fluorescence on SHA represents the number of bacteria attached. S. Mutans with gcp gene inactivation strain (45.33%±6.35%)attached less (P <0.05)than wildtype bacteria(64.05%±6.73%), which implies that gcp gene could inhibit the SHA surface attachment by S Mutans. ConclusionIn our study, c-di-GMP signaling pathway has been found existed in S. Mutans and mediated its capacity of the biofilm formation and surface attachment. Exogenous c-di-GMP reduced S. Mutans'ability of biofilm formation, acid generation, acid resistance and surface attachment ex vivo. In brief, our research provided molecular mechanism for explanation how S Mutans'cause dental caries and experimental evidences for developing new anti-caries method.