[Effects of pleiotropic regulator catabolite control protein A in prompting the biofilm formation of cariogenic Streptococcus mutans].

Objective: To explore the effects of pleiotropic regulator, catabolite control protein A (CcpA), on the biofilm formation of Streptococcus mutans (Sm). Methods: The CcpA knockout strain ΔccpA and complementation strain ΔccpA/pDL278-ccpA were constructed. The cell morphologies and growth curves of UA159 and ΔccpA cells were detected. The pH meter and acid tolerance assay were conducted to evaluate the effect of CcpA on the acid-producing and acid resistance capacity of Sm, respectively. Crystal violet assay (CVS), anthrone-sulfuric method, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were conducted to evaluate the characteristics of Sm biofilms. Moreover, real-time fluorescence quantitative PCR (RT-qPCR) and electrophoretic mobility shift assay (EMSA) were employed to explore the molecular mechanism of CcpA in prompting the biofilm formation at transcriptional level. Results: Growth curve analysis indicated that ΔccpA demonstrated slightly reduced growth rate compared with UA159, while the final yield showed no significant difference. SEM observation showed ΔccpA bacteria formed loose biofilm architectures and exhibited a reduced capacity in synthesizing extracellular matrix, and these findings were further confirmed by CVS that ΔccpA (0.55±0.11) exhibited decreased biomass relative to UA159 (0.82±0.01) (P<0.01), while no significant difference between ΔccpA/pDL278-ccpA and UA159 (P>0.05). CLSM observation exhibited a reduced capacity to synthesize exopolysaccharides of ΔccpA strain, and these findings were further confirmed by data of anthrone-sulfuric method that ΔccpA exhibited decreased water-soluble glucans (0.10±0.01) and water-insoluble glucans (0.08±0.00) relative to UA159 (0.14±0.02, 0.20±0.03) (all P<0.01), while there was no significant difference between ΔccpA/pDL278-ccpA and UA159 (all P>0.05). RT-qPCR showed the expression levels of gtfB/C/D genes were markedly downregulated in ∆ccpA strain with different carbon resources (all P<0.05). Specifically, the promoter regions of gtfB and gtfC were directly regulated by CcpA. Conclusions: In summary, CcpA can trigger transcription of biofilm-formation genes via direct binding. Our study supports a role for CcpA as a crucial regulator that exploit the nuanced control of Sm biofilm formation.