# Deletion of dltD gene modulates biofilm matrix and acid metabolism to attenuate Streptococcus mutans cariogenicity

## TL;DR

Deleting the dltD gene in Streptococcus mutans reduces its ability to cause tooth decay by affecting biofilm and acid production.

## Contribution

This study identifies dltD as a novel target for preventing dental caries by modulating biofilm and acid metabolism in S. mutans.

## Key findings

- dltD deletion reduced caries severity and colonization in a rat model.
- dltD deletion impaired biofilm formation and acid production in vitro.
- dltD deletion altered gene expression related to biofilm and acid tolerance.

## Abstract

Streptococcus mutans (SM) 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 how dltD attenuates SM cariogenicity using standard strain SMUA159, high-cariogenic clinical strain SM593, and their dltD deletion/complemented strains.

In this study, the clinical serotype C SM593 clinical strain isolated from caries-active patients (DMFT6), the SM593 dltD deletion strain (SM593-dltD), and SM593-dltD complementary strain (SM593-dltD-c) 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 in vivo. 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 in vitro., CFU counting and MTT staining were used to analyze the SM biofilm 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.

In vivo, dltD deletion significantly reduced fissure and proximal caries severity (P<0.05), with strain-specific colonization differences. In vitro, dltD deletion strains showed decreased biofilm viable cells (P<0.05), metabolic activity (P<0.01), and water-insoluble polysaccharides (P<0.01), associated with downregulated gtfB and gtfC expression (P<0.05), increased autolysis, and extracellular DNA (P<0.01). Acidogenicity and acid tolerance were impaired, associated with downregulated dexA, fabM, and atpD expression (P<0.05).

These findings confirmed that dltD deletion attenuates SM cariogenicity by disrupting biofilm EPS and acid metabolism, supporting dltD as a potential target for caries prevention.