The Relationship Between Aciduric Virulence Factor F-ATPase Expression In Biofilms And Caries

Dental caries can be related directly to acid production by bacteria in the dental plaque which is the initiating factor of the disease. The cariogenic bacteria exert virulence mainly in adherenc, acidogenicity and aciduricity three functions. While acidurance is essential prerequisite for cariogenesis, and the acid tolerances of dental plaque bacteria appear to depend on the activities of F-ATPase (membrane-bound proton-translocating ATPase, F0F1 ATPase, F-ATPase, H--ATPase, F-type ATPase), which is able to transport protons out of cell in association with ATP hydrolysis and thereby to maintain ApH across the cell membrane with the interior bacteria carrying out glycolysia to produce acid even in low pH value biofilms environment, as a result, the dental plaque pH value can plumment to 5.4-5.5, then the aqueous solubility of dental enamel increases exponentially that cannot be repaired during the normal acidification-demineralization-alkalinization- remineraliz-ation cycle, and cause the caries. Therefore, F-ATPase plays a key role in dental caries occurance. In general, dental plaque biofilm is the situ cariogenic environment for cariogenic bacteria, and the structure of bacteria in dental plaque biofilm is not the simple superposition. They construct a complex microbial community, biofilm structure and function of microbial physiological characteristic. Meanwhile, the organisms in biofilm exist mutual antagonism, interdependent and signal transduction with each other. They start a completely different genetic systems from the planktonic status and affect the expression of virulence and toxicological role of cariogenic bacteria,'so it is undoubted that only in the dental pique biofilms the pathogens can performance their cariogenic function, Therefore, observing the expression of F-ATPase in biofilm could more truly reflect the aciduric virulence of cariogenic bacteria in the development of dental caries mechanism.Objective:To analyze F-ATPase mRNA expression in artificial biofilm and clinical situ plaque with different caries-sensitive individuals, and evaluate F-ATPase activity, also the fluorescent expression of the F-ATPase operon in artificial biofilm with different pH.Methods:1. F-ATPase mRNA expression in different pH artificial biofilm:The strains S. mutans UA159,S.sanguis ATCC10556,S.sobrinus ATCC33478, A.naeslundii ATCC19246, A.viscosus ATCC12104, S.oralis ATCC10557, Lactobacillus ATCC4356 and Neisseria 100603 were used to construct the artificial biofilm. The strains growed in continuous culture for 24 hours and cultured in different pH medium for 3 hours, then biofilm total RNA were extracted to analyze F-ATPase mRNA expression by real-time PCR.2. F-ATPase mRNA expression in plaque from different caries-sensitive individuals:One test is based on adults population,61 plaque samples collected from the caries site with sever caries and 50 healthy controls from the intact enamel with caries free. Another test is based on old people,56 plaque samples pooled from the caries site with root caries and 41 healthy controls pooled from the health cementum. Real-time PCR also be used to analyze ATPase mRNA expression in the dental plaque, and ANOVA valuate the results.3. The F-ATPase activity expression in the artificial biofilm:Bacteria were collected from the artificial biofilm constructed like above method, and were prepared as permeability cell. F-ATPase activity in different pH condition was determined through detecting the releasing contents of phosphorus at different time, then ANOVA valuate the results.4. Pre-constructed F-ATPase promotor transformed into bacteria and Fluorescent recombinant expression vector expressed in artificial biofilm:Two shuttle vectors (PLFgfp and PLRred) were constructed previously, electronic transformed into S. mutans. Counting the colony on kan+ agar plate of the S. mutans cultured in different pH condition for 12 hours. The E.coli strain DH5αwhich contained PLFgfp and PLRred were selected to construct artificial biofilm with some other bacteria like above method. CLSM (confocal laser scanning microscope) assayed fluorescent expression.Results:1. F-ATPase mRNA expression in different pH bacterial biofilm was quite different. The expression level was the highest at initial pH value 5 or 5.5,which was approximately 2.8-fold than that at initial pH value 7. The second high level of mRNA expression is at the initial pH value 6, followed by initial pH value 6.5,4.5,7 in succession, and the lowest expression level achieved at the initial pH value 4. Compared to the neutral environment (pH 7), F-ATPase expression in the acid environment showed statistical significance(p<0.05).2. In dental plaque, F-ATPase expression in two caries groups (adults and old population)were much higher than in health groups (P<0.01); and the data in this study had shown that in adults caries group F-ATPase expression was higher than that in old people(P<0.01), but there was no significant difference between adults health group and old people health group(P>0.05). F-ATPase expression in the situ plaque biofilm from clinical individuals was much higher than that in vitro biofilm(P<0.01).3. F-ATPase activity in artificial oral biofilm at different pH value has different characteristics:on the first 10 min, F-ATPase activity decreased while the pH value increased. After continuous culturing for 3h, the activity at pH 5 and 5.5 was the highest, successively was pH 4.5,6.5,4, the lowest is pH 7. Compared to the neutral environment(pH 7), F-ATPase activity in the acid environment differences have statistical significance(P<0.05).4. Both of the two vectors respectively containing F-ATPase promoters and recA gene were separately transformed into the host cells S.mutans UA159,which could grow on the kan+ BHI agar medium. PCR result also proved the transformation succeed. After culturing in different pH condition, the colony quantity of the S.mutans contained PLFgfp was similar to F-ATPase expression level in different pH. From the CLSM images of artificial biofilm which contained the fluorescent recombinant expression vector found that the green fluorescent (F-ATPase promotor) expression at the initial pH value 5.5 was the highest, successively was initial pH 5,6,4.5,6.5,4, when at initial pH 7,the green fluorescent expression was the lowest, but the red fluorescent (recA promotor) expression was higher than the others, and the red fluorescent as the pH decreased to reduce, when at initial pH 4 was the lowest.Conclusion:External pH decrease causes an increase in F-ATPase expression and activity, when initial pH value at 5 to 5.5, the expression was the highest(pH 5 to 5.5 was the critical pH of enamel in imbalance between demineralizaion and remineralization which caused caries). In situ plaque biofilm with high-caries, the expression of F-ATPase is much higher than that without caries. It demonstrated that the cariogenic microorganisms in the dental plaque achieved acid tolerance and caused caries through F-ATPase gene and protein expression up-regulation in acid environment. This research provide valuable foundation for further clarification of the etiology, risk assessment and prevention of dental caries.