Deletion ofgene modulates biofilm matrix and acid metabolism to attenuatecariogenicity.

Abstract

INTRODUCTION: is one of the key pathogenic bacteria in the occurrence and development of dental caries. Its complex virulence regulation network has become an important target in current ecological caries prevention research. This study explored howattenuatescariogenicity using standard strainUA159, high-cariogenic clinical strain593, and theirdeletion/complemented strains. METHODS: In this study, the clinical serotype C593 clinical strain isolated from caries-active patients (DMFT6), the593 dltD deletion strain (593-), and593-complementary strain (593-) were selected as the experimental strains. Rat caries model was constructed to detect the cariogenicity. Colony forming counting units (CFU) counting was used to detect the colonization ability. The adhesion ability and surface hydrophobicity of each strain were examined by tube attachment assay and microbial adhesion to hydrocarbons method. Biofilm of each strain was constructed., CFU counting and MTT staining were used to analyze thebiofilm formation. Laser confocal scanning microscope were used to observe the biofilm morphology, live/dead staining distribution. Anthrone-sulfuric acid assay, laser confocal scanning microscope, SYTOX probe assay and BCA protein kit assay were used to detect the extracellular polysaccharide content, extracellular polysaccharide distribution, eDNA content and extracellular protein content of the biofilm. Acid production was examined by detecting the pH of the biofilm supernatant. Potassium iodide assay and lactate dehydrogenase detection kit assay were used to examine intracellular polysaccharides and lactate dehydrogenase activity. CFU counting was used to detect the adaptive acid tolerance ability. Laurdan fluorescent probe was used to examine the cell membranes fluidity under the acidic condition. The expression of genes related to biofilm formation and acid tolerance was detected by RTqPCR. RESULTS: ,deletion significantly reduced fissure and proximal caries severity (P<0.05), with strain-specific colonization differences.,deletion strains showed decreased biofilm viable cells (P<0.05), metabolic activity (P<0.01), and water-insoluble polysaccharides (P<0.01), associated with downregulatedandexpression (P<0.05), increased autolysis, and extracellular DNA (P<0.01). Acidogenicity and acid tolerance were impaired, associated with downregulated,, andexpression (P<0.05). DISCUSSION: These findings confirmed thatdeletion attenuatescariogenicity by disrupting biofilm EPS and acid metabolism, supportingas a potential target for caries prevention.