Small RNA SmsR1 modulates acidogenicity and cariogenic virulence by affecting protein acetylation in Streptococcus mutans.

Post-transcriptional regulation by small RNAs and post-translational modifications (PTM) such as lysine acetylation play fundamental roles in physiological circuits, offering rapid responses to environmental signals with low energy consumption. Yet, the interplay between these regulatory systems remains underexplored. Here, we unveil the cross-talk between sRNAs and lysine acetylation in Streptococcus mutans, a primary cariogenic pathogen known for its potent acidogenic virulence. Through systematic overexpression of sRNAs in S. mutans, we identified sRNA SmsR1 as a critical player in modulating acidogenicity, a key cariogenic virulence feature in S. mutans. Furthermore, combined with the analysis of predicted target mRNA and transcriptome results, potential target genes were identified and experimentally verified. A direct interaction between SmsR1 and 5'-UTR region of pdhC gene was determined by in vitro binding assays. Importantly, we found that overexpression of SmsR1 reduced the expression of pdhC mRNA and increased the intracellular concentration of acetyl-CoA, resulting in global changes in protein acetylation levels. This was verified by acetyl-proteomics in S. mutans, along with an increase in acetylation level and decreased activity of LDH. Our study unravels a novel regulatory paradigm where sRNA bridges post-transcriptional regulation with post-translational modification, underscoring bacterial adeptness in fine-tuning responses to environmental stress.

SMU_1361c regulates the oxidative stress response of Streptococcus mutans.

Dental caries is the most common chronic infectious disease around the world and disproportionately affects the marginalized socioeconomic group. Streptococcus mutans, considered a primary etiological agent of caries, depends on the coordinated physiological response to tolerate the oxidative stress generated by commensal species within dental plaque, which is a critical aspect of its pathogenicity. Here, we identified and characterized a novel tetracycline repressor family regulator, SMU_1361c, which appears to be acquired by the bacteria via horizontal gene transfer. Surprisingly, smu_1361c functions as a negative transcriptional regulator to regulate gene expression outside its operon and is involved in the oxidative stress response of S. mutans. The smu_1361c overexpression strain UA159/pDL278-1361c was more susceptible to oxidative stress and less competitive against hydrogen peroxide generated by commensal species Streptococcus gordonii and Streptococcus sanguinis. Transcriptomics analysis revealed that smu_1361c overexpression resulted in the significant downregulation of 22 genes, mainly belonging to three gene clusters responsible for the oxidative stress response. The conversed DNA binding motif of SMU_1361c was determined by electrophoretic mobility shift and DNase I footprinting assay with purified SMU_1361c protein; therefore, smu_1361c is directly involved in gene transcription related to the oxidative stress response. Crucially, our finding provides a new understanding of how S. mutans deals with the oxidative stress that is required for pathogenesis and will facilitate the development of new and improved therapeutic approaches for dental caries.IMPORTANCEStreptococcus mutans is the major organism associated with the development of dental caries, which globally is the most common chronic disease. To persist and survive in biofilms, S. mutans must compete with commensal species that occupy the same ecological niche. Here, we uncover a novel molecular mechanism of how tetracycline repressor family regulator smu_1361c is involved in the oxidative stress response through transcriptomics analysis, electrophoretic mobility shift assay, and DNase I footprinting assay. Furthermore, we demonstrated that smu_1361c mediates S. mutans sensitivity to oxidative stress and competitiveness with commensal streptococci. Therefore, this study has revealed a previously unknown regulation between smu_1361c and genes outside its operon and demonstrated the importance of smu_1361c in the oxidative stress response and the fitness of S. mutans within the plaque biofilms, which can be exploited as a new therapy to modulate ecological homeostasis and prevent dental caries.

Molecular and regulatory mechanisms of oxidative stress adaptation in Streptococcus mutans.

Dental caries is a chronic progressive disease, which destructs dental hard tissues under the influence of multiple factors, mainly bacteria. Streptococcus mutans is the main cariogenic bacteria. However, its cariogenic virulence is affected by environmental stress such as oxidative stress, nutrient deficiency, and low pH to some extent. Oxidative stress is one of the main stresses that S. mutans faces in oral cavity. But there are a variety of protective molecules to resist oxidative stress in S. mutans, including superoxide dismutase, nicotinamide adenine dinucleotide oxidase, Dps-like peroxide resistance protein, alkyl-hydrogen peroxide reductase, thioredoxin, glutamate-reducing protein system, and some metabolic substances. Additionally, some transcriptional regulatory factors (SloR, PerR, Rex, Spx, etc.) and two-component systems are also closely related to oxidative stress adaptation by modulating the expression of protective molecules. This review summarizes the research progress of protective molecules and regulatory mechanisms (mainly transcription factors) of oxidative stress adaptation of S. mutans.

Acetylation of Lactate Dehydrogenase Negatively Regulates the Acidogenicity of Streptococcus mutans.

Lysine acetylation, a ubiquitous and dynamic regulatory posttranslational modification (PTM), affects hundreds of proteins across all domains of life. In bacteria, lysine acetylation can be found in many essential pathways, and it is also crucial for bacterial virulence. However, the biological significance of lysine acetylation events to bacterial virulence factors remains poorly characterized. In Streptococcus mutans, the acetylome profiles help identify several lysine acetylation sites of lactate dehydrogenase (LDH), which catalyzes the conversion of pyruvate to lactic acid, causing the deterioration of teeth. We investigated the regulatory mechanism of LDH acetylation and characterized the effect of LDH acetylation on its function. We overexpressed the 15 Gcn5 N-acetyltransferases (GNAT) family members in S. mutans and showed that the acetyltransferase ActA impaired its acidogenicity by acetylating LDH. Additionally, enzymatic acetyltransferase reactions demonstrated that purified ActA could acetylate LDH in vitro, and 10 potential lysine acetylation sites of LDH were identified by mass spectrometry, 70% of which were also detected in vivo. We further demonstrated that the lysine acetylation of LDH inhibited its enzymatic activity, and a subsequent rat caries model showed that ActA impaired the cariogenicity of S. mutans. Collectively, we demonstrated that ActA, the first identified and characterized acetyltransferase in S. mutans, acetylated the LDH enzymatically and inhibited its enzymatic activity, thereby providing a starting point for the further analysis of the biological significance of lysine acetylation in the virulence of S. mutans. IMPORTANCE Lysine acetylation, a dynamic regulatory posttranslational modification, remains poorly characterized in bacteria. Hundreds of proteins have been identified to be acetylated in bacteria, with advances made in acetylome analyses. However, the regulatory mechanisms and functional significance of the majority of these acetylated proteins remain unclear. We analyzed the acetylome profiles of Streptococcus mutans and found that lactate dehydrogenase (LDH) contains several lysine acetylation sites. We also demonstrated that the acetyltransferase ActA, a member of the Gcn5 N-acetyltransferases (GNAT) family in S. mutans, acetylated LDH, inhibited its enzymatic ability to catalyze the conversion of pyruvate to lactic acid, and impaired its cariogenicity in a rat caries model. Therefore, LDH acetylation might be a potential target that can be exploited in the design of novel therapeutics to prevent dental caries.

Post-translational Modifications in Oral Bacteria and Their Functional Impact.

Oral bacteria colonize the oral cavity, surrounding complex and variable environments. Post-translational modifications (PTMs) are an efficient biochemical mechanism across all domains of life. Oral bacteria could depend on PTMs to quickly regulate their metabolic processes in the face of external stimuli. In recent years, thanks to advances in enrichment strategies, the number and variety of PTMs that have been identified and characterized in oral bacteria have increased. PTMs, covalently modified by diverse enzymes, occur in amino acid residues of the target substrate, altering the functions of proteins involved in different biological processes. For example, Ptk1 reciprocally phosphorylates Php1 on tyrosine residues 159 and 161, required for Porphyromonas gingivalis EPS production and community development with the antecedent oral biofilm constituent Streptococcus gordonii, and in turn Php1 dephosphorylates Ptk1 and rapidly causes the conversion of Ptk1 to a state of low tyrosine phosphorylation. Protein acetylation is also widespread in oral bacteria. In the acetylome of Streptococcus mutans, 973 acetylation sites were identified in 445 proteins, accounting for 22.7% of overall proteins involving virulence factors and pathogenic processes. Other PTMs in oral bacteria include serine or threonine glycosylation in Cnm involving intracerebral hemorrhage, arginine citrullination in peptidylarginine deiminases (PADs), leading to inflammation, lysine succinylation in P. gingivalis virulence factors (gingipains, fimbriae, RagB, and PorR), and cysteine glutathionylation in thioredoxin-like protein (Tlp) in response to oxidative stress in S. mutans. Here we review oral bacterial PTMs, focusing on acetylation, phosphorylation, glycosylation, citrullination, succinylation, and glutathionylation, and corresponding modifying enzymes. We describe different PTMs in association with some examples, discussing their potential role and function in oral bacteria physiological processes and regulatory networks. Identification and characterization of PTMs not only contribute to understanding their role in oral bacterial virulence, adaption, and resistance but will open new avenues to treat oral infectious diseases.

Acetylation of glucosyltransferases regulates Streptococcus mutans biofilm formation and virulence.

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.

Roux-en-Y Gastric Bypass Improved Insulin Resistance via Alteration of the Human Gut Microbiome and Alleviation of Endotoxemia.

BACKGROUND: Obesity is a main contributing factor for the development of glucose intolerance and type 2 diabetes mellitus (T2D). Roux-en-Y gastric bypass (RYGB) is believed to be one of the most effective treatments to reduce body weight and improve glucose metabolism. In this study, we sought to explore the underlying mechanisms of weight reduction and insulin resistance improvement after RYGB. METHODS: This was a prospective observational study using consecutive samples of 14 obese subjects undergoing bariatric surgery. Main assessments were serum indexes (blood metabolites, glucose-lipid regulating hormones, trimethylamine-N-oxide (TMAO), and lipopolysaccharide-binding protein (LBP), fecal short-chain fatty acids (SCFAs), and gut microbiota. Correlation analysis of the factors changed by RYGB was used to indicate the potential mechanism by which surgery improves insulin resistance. RESULTS: The subjects showed significant improvement on indices of obesity and insulin resistance and a correlated change of gut microbiota components at 1 month, 3 months, and 6 months post-RYGB operation. In particular, the abundance of a counterobese strain, Akkemansia muciniphila, had gradually increased with the postoperative time. Moreover, these changes were negatively correlated to serum levels of LBP and positively correlated to serum TMAO and fecal SCFAs. CONCLUSIONS: Our findings uncovered links between intestinal microbiota alterations, circulating endotoxemia, and insulin resistance. This suggests that the underlying mechanism of protection of the intestine by RYGB in obesity may be through changing the gut microbiota.

The Adc regulon mediates zinc homeostasis in Streptococcus mutans.

Zinc (Zn2+ ) is an essential divalent trace metal for living cells. Intracellular zinc homeostasis is critical to the survival and virulence of bacteria. Thus, the frequent fluctuations of salivary zinc, caused by the low physiological level and the frequent exogenous zinc introduction, present a serious challenge for bacteria colonizing the oral cavity. However, the regulation strategies to keep intracellular Zn2+ homeostasis in Streptococcus mutans, an important causative pathogen of dental caries, are unknown. Because zinc uptake is primarily mediated by an ATP-binding ABC transporter AdcABC in Streptococcus strains, we examined the function of AdcABC and transcription factor AdcR in S. mutans in this study. The results demonstrated that deletion of either adcA or adcCB gene impaired the growth but enhanced the extracellular polymeric matrix production in S. mutans, both of which could be relieved after excessive Zn2+ supplementation. Using RNA sequencing analysis, quantitative reverse transcription polymerase chain reaction examination, LacZ-reporter studies, and electrophoretic mobility shift assay, we showed that a MarR (multiple antibiotic resistance regulator) family transcription factor, AdcR, negatively regulates the expression of the genes adcR, adcC, adcB, and adcA by acting on the adcRCB and adcA promoters in response to Zn2+ concentration in their environmental niches. The deletion of adcR increases the sensitivity of S. mutans to excessive Zn2+ supply. Taken together, our findings suggest that Adc regulon, which consists of a Zn2+ uptake transporter AdcCBA and a Zn2+ -responsive repressor AdcR, plays a prominent role in the maintenance of intracellular zinc homeostasis of S. mutans.