Molecular Oral Microbiology最新文献

Periodontal bacterial pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) accelerate inflammatory osteoclastogenesis, resulting in alveolar bone loss. The core PAMP and DAMP prototype molecules are periodontal bacterium Porphyromonas gingivalis-derived virulence lipids, for example, phosphoglycerol dihydroceramide (PGDHC) and lipopolysaccharide (LPS Pg), and the host non-histone alarmin high mobility group box protein-1 (HMGB1), respectively. Although it was reported that extracellularly released HMGB1 is critical for the promotion of sepsis inflammation in response to non-periodontal bacterial LPS, our understanding of the crosstalk between HMGB1 and P. gingivalis-derived virulence lipids remains limited. Therefore, we used Hmgb1^fl/fl LysM-Cre⁺ mice with ablated HMGB1 mRNA and littermate Hmgb1^fl/fl LysM-Cre^- controls. We observed limited Hmgb1^fl/fl LysM-Cre⁺ osteoclastogenesis compared to Hmgb1^fl/fl in response to RANKL in vitro. Furthermore, recombinant HMGB1 protein restored osteoclast formation in Hmgb1^fl/fl LysM-Cre⁺ cells, indicating the pivotal role of extracellular HMGB1 in osteoclastogenesis in vitro. Using bulk RNA-sequencing, we identified the diminished osteoclastogenesis in Hmgb1^fl/fl LysM-Cre⁺ cells are linked to accelerated expression of canonical osteoclast-suppressing interferon genes. We surprisingly detected that PGDHC and LPS Pg accelerate osteoclastogenesis in Hmgb1^fl/fl LysM-Cre⁺ cells in vitro. Using bulk RNA-sequencing and real-time PCR assays, we confirmed that PGDHC diminishes the expression patterns of different interferon-inducible guanylate-binding proteins (GBP 3, 4, 5, 9). At the same time, LPS Pg accelerates the expression of osteoclast-promoting matrix metalloproteases (MMP 8 and 12) mRNAs. The results suggest that the RANKL-primed osteoclastogenesis accelerated by P. gingivalis-derived virulence lipids is mediated by different MMP or GBP signaling pathways independently from canonical HMGB1 signaling.

Oral infectious diseases, particularly inflammatory periodontal lesions, exert a substantial impact on healthcare systems and economies, as acknowledged by the World Health Organization. The prevailing consensus attributes the onset of oral infectious diseases to dysbiosis within the intricate oral microbiome. In this context, Fretibacterium-a strictly anaerobic genus whose representative species, Fretibacterium fastidiosum, was classified in 2013 as the third human oral species within the Synergistetes phylum-has garnered attention for its progressive enrichment in periodontitis and distinct abundance profiles in health versus disease. This review synthesizes current knowledge on Fretibacterium's role in periodontal disease, dental caries, endodontic infections, and peri-implantitis, with emphasis on its virulence mechanisms. Then further explore its clinical associations with systemic conditions (e.g., diabetes) and evaluate conventional and emerging therapeutic strategies. By providing evidence-based insights, this work aims to guide clinical management and future research directions.

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.

Over the years, humanity has accumulated knowledge about the pathogens of infectious diseases and the ability of the human body to resist external aggression. In the last century, it became clear that the normal microflora of the human body can be used as an ally to resist a whole range of diseases. However, the intestinal microflora is the main object of modern complex studies. This review focuses on the microflora of the oral cavity. It describes the main microbiological composition of the microflora, including the most important bacterial species, fungi, and viruses. The main factors influencing the emergence of balance in the system "human oral cavity-microorganisms" are considered as well as environmental features that affect the formation of the species composition. The main functions performed by the oral microflora are described. Possible mechanisms for correcting initial dysbiotic disorders are also considered, including probiotics, bacteriophages, gases and thermotherapy, photobiomodulation, and diet correction.