Acetylation of glucosyltransferases regulates Streptococcus mutans biofilm formation and virulence

Author summaryLysine acetylation is a regulatory post-translational modification (PTM) important in physiological processes across all domains of life. Although it has been well studied and characterized in eukaryotes, new insights into the lysine acetylation in bacteria have gained momentum in recent years, and hundreds to thousands of protein acetylation processes have been identified in various bacteria with novel enrichment strategies. However, the specific mechanisms of regulating lysine acetylation and function are still poorly understood. Therefore, we screened for the KAT mediating Gtfs acetylation by constructing 15 strains of S. mutans that overexpressed the GCN5-related N-acetyltransferases (GNAT) family members. Eventually, we identified and characterized ActG, a GNAT family member, that could catalyze the acetylation of GtfB and GtfC in S. mutans by the enzymatic mechanism, inversely related to their enzymatic activities, subsequently affecting the water-insoluble EPS synthesis and biofilm formation. In addition, ActG impaired the cariogenicity of S. mutans in a rat caries model. Thus, this study provides significant insights into the effect of lysine acetylation on S. mutans virulence and pathogenicity by regulating target protein functions and relative physiological processes. Lysine acetylation is a frequently occurring post-translational modification (PTM), emerging as an important metabolic regulatory mechanism in prokaryotes. This process is achieved enzymatically by the protein acetyltransferase (KAT) to specifically transfer the acetyl group, or non-enzymatically by direct intermediates (acetyl phosphate or acetyl-CoA). Although lysine acetylation modification of glucosyltransferases (Gtfs), the important virulence factor in Streptococcus mutans, was reported in our previous study, the KAT has not been identified. Here, we believe that the KAT ActG can acetylate Gtfs in the enzymatic mechanism. By overexpressing 15 KATs in S. mutans, the synthesized water-insoluble extracellular polysaccharides (EPS) and biofilm biomass were measured, and KAT (actG) was identified. The in-frame deletion mutant of actG was constructed to validate the function of actG. The results showed that actG could negatively regulate the water-insoluble EPS synthesis and biofilm formation. We used mass spectrometry (MS) to identify GtfB and GtfC as the possible substrates of ActG. This was also demonstrated by in vitro acetylation assays, indicating that ActG could increase the acetylation levels of GtfB and GtfC enzymatically and decrease their activities. We further found that the expression level of actG in part explained the virulence differences in clinically isolated strains. Moreover, overexpression of actG in S. mutans attenuated its cariogenicity in the rat caries model. Taken together, our study demonstrated that the KAT ActG could induce the acetylation of GtfB and GtfC enzymatically in S. mutans, providing insights into the function of lysine acetylation in bacterial virulence and pathogenicity.

Automated summary

The study elucidated the important role of lysine acetylation in bacteria by identifying and characterizing ActG, a GNAT family member that catalyzes the acetylation of GtfB and GtfC in S. mutans, subsequently affecting EPS synthesis and biofilm formation, while also reducing cariogenicity in a rat caries model. This provides insight into the impact of lysine acetylation on bacterial virulence and pathogenicity by regulating protein functions and physiological processes.