Molecular Oral Microbiology最新文献

Tyrosine phosphorylation/dephosphorylation post-translational modification (PTM) of proteins in bacteria can control their function and location. PTM of transcriptional regulators and DNA-binding proteins, as well as components of their signaling pathways, can impact gene expression. However, little is known regarding the global impact of tyrosine phosphatases on the bacterial transcriptome. In this study, we performed RNA-Seq of Porphyromonas gingivalis wild type (WT) along with strains Δltp1 and Δphp1 with mutations in the genes encoding the two major tyrosine phosphatases, Ltp1 and Php1, respectively. Moreover, these strains were tested in vitro and in vivo (mouse abscess) conditions. Both the Δltp1 and the Δphp1 mutants exhibited little transcriptional difference to the parental strain when cultured in vitro. In vivo, comparison of the Δphp1 mutant to the WT showed a number of differentially regulated genes (DEGs) associated with transporter systems. In vivo DEGs in Δltp1 included one of the efflux ABC transporter systems also regulated in the Δphp1 mutant; however, the primary biological process populated by DEGs in Δltp1 involved genome stability. Comparison of the WT strain between the in vitro and in vivo condition indicated that DNA metabolic processes, including recombination and transposition, were significantly upregulated in vivo. Hence, a major role of Ltp1 phosphatase activity at the transcriptional level may be control of adaptation to in vivo conditions. Additionally, both Ltp1 and Php1 have common functions in the control of the expression of genes encoding transporter systems.

Lysine succinylation (Ksuc) is a novel post-translational modification (PTM), which regulates biological functions in bacteria. Streptococcus mutans has been identified as a predominant cariogenic pathogen responsible for the initiation and progression of dental caries. However, lysine succinylation in S. mutans has not yet been investigated. In this study, a global lysine succinylome was analyzed to examine Ksuc in S. mutans. Overall, 2250 succinylated sites in 580 proteins were identified. Quantitative analysis demonstrated that Ksuc substrates were substantially altered in the biofilm growth state compared with the planktonic growth state. These differentially succinylated proteins were distributed across various cellular components and involved in crucial biological pathways, including translation, ribosomal structure, and biogenesis. Furthermore, lysine acetylation and succinylation extensively overlapped in S. mutans, and these bimodified proteins were associated with biofilm formation, glycolysis, and pyruvate metabolism. These results provided a foundation to further investigate the role of Ksuc in S. mutans pathogenicity and expand our understanding of Ksuc functions in bacterial physiology and virulence.

The collagen-binding adhesin Cnm is a known virulence factor of Streptococcus mutans. It is present in specific serotypes (mostly e, f, and k strains) of S. mutans and belongs to the LPXTG family of cell wall-anchored surface adhesins. Here, we report the crystal structure of the collagen-binding N₂ domain of S. mutans Cnm. Using the Staphylococcus aureus collagen-binding protein Cna, which shares high sequence and structural homology with Cnm, we modeled collagen binding to S. mutans Cnm. The comparative analysis identified three conserved collagen-binding residues (Y176, F192, N194) and four equivalent residues that are different in their composition (D224, T226, S232, M276). This study also discovered the multifunctional attributes of this protein, where Cnm-FL, Cnm-N_12, and the individual domains of Cnm-N₁ and Cnm-N₂ adhere with high affinity to the scavenger receptor cysteine-rich (SRCR) domains of glycoprotein 340 (Gp340). Protein-protein docking of Cnm-N₂ and SRCR₁ showed the possibility of a shared binding site at the collagen-binding interface of Cnm-N₂. Furthermore, competition experiments using collagen and SRCR₁₂₃ with Cnm-N₂, Cnm-N_12, and Cnm-FL constructs confirmed that collagen and SRCR₁ share a binding site. Subsequent alanine substitution mutagenesis of the predicted collagen-binding residues validated our modeling results, confirming that Y176 and F192 are important residues for collagen and SRCR/Gp340 binding.

Streptococcus mutans is considered the main pathogen causing dental caries and has a strong ability to establish biofilms and respond to environmental stimuli, which are essential for its survival and cariogenicity. Fourteen two-component signal transduction systems (TCSs) in S. mutans have been reported to regulate a broad range of physiological processes such as bacterial biofilm formation, acid resistance, competence development, and toxic oxygen metabolite resistance. These systems collectively contribute to the cariogenicity of S. mutans by coordinating adaptive responses to environmental challenges. Among them, the VicRK system has been one of the most extensively studied, with epidemiological evidence linking vicK mutations to increased caries risk in children. Other TCSs, such as ComDE, LiaRS, CiaRH, and the orphan response regulator GcrR, also contribute to cariogenicity regulation. The present review summarizes the regulatory roles of TCSs in virulence traits of S. mutans, with an emphasis on those involved in biofilm formation, which highlights their potential as therapeutic targets to prevent dental caries through biofilm inhibition.

Hypothiocyanite (OSCN^-/HOSCN) is an antimicrobial molecule found at high concentrations in saliva. HOSCN is thought to differentially affect oral streptococci, since noncariogenic streptococci (e.g. Streptococcus sanguinis) possess HOSCN reductase activity that cariogenic streptococci (e.g. Streptococcus mutans) lack. However, the enzyme responsible for this activity and the effects of HOSCN and HOSCN reductase activity on biofilm formation by oral streptococci have not been previously established. In this work, we developed an artificial saliva medium for growth of oral streptococci with minimal redox-active components, called Defined Recipe Optimized Oral Liquid (DROOL), and used it to characterize the HOSCN responses of S. sanguinis and S. mutans. We identified a homolog of the Streptococcus pneumoniae Har protein in S. sanguinis as HOSCN reductase. S. mutans wild-type and S. sanguinis ∆har mutants were more sensitive to inhibition by physiological concentrations of HOSCN in DROOL than wild-type S. sanguinis when grown planktonically. S. mutans biofilm formation and glucan production were strongly decreased by HOSCN treatment, suggesting HOSCN inhibits S. mutans exopolysaccharide production. Collectively, our data demonstrate the specific ability of HOSCN to inhibit functions of cariogenic but not noncariogenic oral streptococci and show that Har is responsible for mediating this difference.

Oral spirochetes are among the small group of keystone pathogens contributing to dysregulation of periodontal tissue homeostasis, leading to breakdown of the tissue and bone supporting the teeth in periodontal disease. Of the more than 60 oral Treponema species and phylotypes, Treponema denticola is one of the few that can be grown in culture and the only one in which genetic manipulation is practicable. T. denticola is thus a model organism for studying spirochete behavior, metabolism, and interactions with other microbes and host tissues that are relevant to oral diseases. We recently demonstrated enhanced transformation efficiency using a synthetic shuttle plasmid resistant to T. denticola restriction-modification systems. Here, we report further optimization of the shuttle plasmid system by minimizing its size and by characterizing an array of promoter-gene constructs for plasmid-based genetic complementation, including the first inducible system for controlled expression of potentially toxic plasmid-encoded genes in Treponema. Our results highlight the importance of precise pairing of promoters and genes of interest for obtaining biologically optimal protein expression. This work expands the utility of the T. denticola shuttle plasmid system and will facilitate future studies in the analysis of Treponema physiology and behavior. Rigorous genetic analysis in oral spirochetes has been hampered by the limited utility of available versions of the Escherichia coli-T. denticola shuttle plasmid system. We report expanded characterization, refinement, and minimization of the shuttle plasmid, including relative activity of diverse promoters and the first inducible expression system described for T. denticola. We show that careful customization of the shuttle plasmid for specific applications is crucial for obtaining successful results.

Although the species is extensively studied, limited data are available on antiphage defense systems (APDSs) in Streptococcus mutans. The present study aimed to explore the diversity and the occurrence of APDSs and to search for prophages in the genomes of clinical isolates of S. mutans using bioinformatics tools. Forty-four clinical isolates of S. mutans were obtained from saliva samples of people with Parkinson's disease. Genomic DNA was extracted, sequenced using Illumina MiSeq technology, and analyzed for the presence of defense systems using DefenseFinder and PADLOC. CRISPR-Cas systems were characterized using CRISPRCasFinder, and prophages were detected by the PhiSpy pipeline from RAST. AcrFinder and AcrHub were used to identify anti-CRISPR proteins. Each strain harbored between 6 and 12 APDS, with restriction-modification systems being the most prevalent, followed by the MazEF toxin-antitoxin system and CRISPR-Cas systems. Type II-C CRISPR-Cas systems were not identified here in S. mutans. Novel variations in type II-A signature protein Cas9 were identified, allowing their classification into four distinct groups. Variability in direct repeat sequences within the same CRISPR array was also observed, and 80% of the spacers were classified as targeting "dark matter". A unique prophage, phi_37bPJ2, was detected, showing high similarity with previously described phages. The AcrIIA5 protein encoded by phi_37bPJ2 was conserved and suggested to remain functionally active. This study reveals the diversity of APDSs in S. mutans and the limited presence of prophages. The findings provide a foundation for future research on the evolutionary dynamics of these systems and their role in S. mutans adaptation to phage pressure.

Tolerance refers to a hyporesponsiveness toward repeated stimulations with bacteria and their virulence factors, which might exist in the development of periodontitis. To identify the roles of tolerance induced by Porphyromonas gingivalis (P. gingivalis) in periodontitis, an original tolerized mice model was established by high-dose of oral P. gingivalis inoculation following a primary infection. The alveolar bone loss of maxillae was detected by Micro-CT. The infiltration of neutrophils and macrophages, and macrophage polarization were detected by IHC and flow cytometry, respectively. Residual P. gingivalis in subgingival plaque with and without macrophage/neutrophil depletion was measured by real-time PCR. Moreover, a real-time PCR chip and bioinformatic analysis were then employed to explore the cytokine expression profiles in gingivae. The abundance of TNF-α, Toll-like receptor 2 (TLR2), and TLR4 were further verified by western blot. In comparison with the non-tolerance group, TNF-α protein levels, alveolar bone loss, and the infiltration of neutrophils and macrophages in the tolerance group were significantly suppressed (p < 0.05), while the quantities of residual P. gingivalis in subgingival plaque were increased (p < 0.05). Moreover, the depletion of macrophages by liposomal clodronate weakened the inhibitory effect of tolerance, as evidenced by the lack of differences in the quantities of residual bacteria between the tolerance and non-tolerance groups (p > 0.05). Macrophages in gingivae of tolerized mice were more likely to polarize into M2 type. In addition, the expressions of cytokines related to neutrophil and macrophage infiltration and recruitment and the protein levels of TLR2 and TLR4 were decreased in tolerized mice (p < 0.05). Tolerance induced by repeated P. gingivalis stimulations suppressed inflammatory responses in periodontal tissues, and the established periodontal tolerance model provided a reliable tool for the further study on periodontal tolerance in vivo.

Epithelial-mesenchymal transition (EMT) is a fundamental biological process where epithelial cells lose their polarity and adhesion properties, acquiring mesenchymal characteristics such as enhanced migratory ability and invasiveness. Cells undergoing EMT exhibit enhanced motility, aggressiveness, and stemness, contributing to a pro-tumor environment that facilitates malignant metastasis in cancer. Numerous studies have suggested that oral microbes facilitate carcinogenesis through EMT. Oral microbes can directly initiate EMT by adhering to mucosal layers and provoking the disintegration of intercellular adhesion among epithelial cells, thereby modifying cell polarity and downstream signaling pathways. Indirectly, the microbial metabolites and associated compounds can affect the dynamics of EMT. This review summarizes the mechanisms by which oral microbes regulate EMT and thus contribute significantly to cancer. Elucidating the mechanisms underlying the increased plasticity of cancer cells induced by the oral microbiota will facilitate the development of novel targeted therapeutic strategies.

With the growing threat of antimicrobial resistance (AMR), antivirulence strategies present a promising alternative to traditional antibiotics, particularly in dentistry. Dental caries, a chronic biofilm-associated disease primarily driven by the AMR pathogen Streptococcus mutans, results in enamel demineralization and significant oral health challenges. This study explores the anticariogenic mechanism of marine-derived cyclo(l-leucyl-l-prolyl) (CLP), a biomolecule known to inhibit key virulence factors of S. mutans UA159. LC-MS/MS proteomic analysis revealed 30 and 71 significantly regulated proteins following 12 and 24 h of CLP treatment, respectively. Protein-protein interaction and gene ontology analyses demonstrated that CLP downregulates critical virulence proteins related to d-alanylation of lipoteichoic acid (LTA), glucan synthesis, acid production and acid tolerance, while upregulating proteins associated with translation, DNA repair and protein metabolism. KEGG pathway analysis highlighted the involvement of downregulated proteins in key metabolic pathways, including d-alanine metabolism, starch and sucrose metabolism, glycolysis and branched-chain amino acid metabolism. Given the pivotal role of d-alanine metabolism in modulating interconnected virulence pathways, a comparative analysis of in vitro virulence assays-including cell adherence, biofilm formation, acid production and cell surface charge-alongside proteomic data signify that CLP specifically targets the d-alanylation of LTA. This hypothesis was further validated by LTA and d-alanine quantification assays, which confirmed a significant reduction in d-alanine content within LTA after CLP treatment, leading to a marked attenuation of S. mutans cariogenic virulence. Additionally, qPCR and molecular docking analyses corroborated that CLP disrupts S. mutans virulence by interfering with the d-alanylation of LTA. These findings highlight CLP's potential as a novel therapeutic agent for combating dental cariogenesis by targeting S. mutans virulence, offering a promising avenue for the development of advanced anticariogenic therapies.