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.

Treponema denticola is a Gram-negative, anaerobic spirochete associated with periodontal disease. It navigates highly viscous environments using periplasmic flagella located between the inner and outer membranes. The flagellum is composed of three homologous flagellin proteins-FlaB1, FlaB2, and FlaB3-encoded by the flaB1, flaB2, and flaB3 genes, respectively, and is enwrapped by the sheath protein FlaA, encoded by the flaA gene. To investigate the roles of the three FlaB flagellins in cell morphology and motility, we constructed mutants lacking different combinations of the flaB genes, including double and triple deletions. The deletion of one or two flaB genes did not affect the transcription or protein expression of the remaining flaB gene(s). Normal flagellar filaments were observed when at least one FlaB was expressed but were absent in the complete flaB deletion mutant, which also exhibited an elongated cell shape. The flagella had no significant impact on bacterial growth. In liquid medium, most mutants exhibited rotational movement comparable to the parent strain, although some showed an increased population of cells with higher rotation rates. However, in a medium containing 0.5% agar, deletion of any two or three flaB genes significantly reduced motility. These findings suggest that the expression of at least two FlaB flagellins is required for the potent movement of T. denticola.

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.

As first responders, neutrophils are a vital component of the host defense against oral pathogens, and their function is critical in preventing the progression of periodontal diseases. Streptococcus gordonii, a generally commensal oral bacterium, has been implicated in the pathogenesis of diseases by operating as a pathobiont with Porphyromonas gingivalis in periodontitis, and as an independent pathogen in infective endocarditis. Although the pathogenicity of S. gordonii is variable, its role in modulating, as well as responding to, host neutrophils remain, poorly understood. This study focuses on neutrophil activation, migration, and bactericidal activity towards S. gordonii. Our results found S. gordonii induced significant upregulation of surface markers CD63 and CD66 on neutrophils, a phenotypic change reminiscent of an oral neutrophil, and was enhanced by pre-activation of neutrophils by lipopolysaccharide (LPS) or the oral pathogen P. gingivalis. Co-incubations with P. gingivalis also led to a decreased ability of neutrophils to kill the normally commensal S. gordonii, though not other commensals with opportunistic pathogen potential, including Escherichia coli or Staphylococcus aureus. This increase in survival correlated with changes in phagosomal maturation, a decrease in cytoplasmic and phagosomal-associated granules, and increased IL-1β production. These results suggest oral streptococci may significantly contribute to oral neutrophil phenotypes associated with health, but introduction of oral pathogens can exacerbate a neutrophil shift and contribute to the persistence of S. gordonii, and its ability to contribute to the pathogenesis of periodontal disease.

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.

Streptococcus mutans is a key cariogenic pathogen of dental caries due to its strong ability to synthesize extracellular glucans and form biofilms. Glucosyltransferases, encoded by gtfB/C/D genes in S. mutans, are responsible for producing biofilm exopolysaccharides (EPS) and are considered to be critical virulence factors. Previous studies have highlighted the roles of various regulatory factors of gtf genes in S. mutans. Here, we investigated the role of the global transcriptional regulator CcpA encoded by ccpA in regulating the EPS synthesis and biofilm formation of S. mutans. A ccpA in-frame deletion strain was observed to develop shiny, round colonies and longer cell length. In addition, the deletion of ccpA resulted in impaired growth, diminished synthesis of EPS, and reduced biofilm formation. Transcriptome analysis revealed that differentially expressed genes in the ccpA deletion strain were significantly enriched in pathways of carbohydrate transport and metabolism, in which the expressions of gtfB and gtfC were downregulated markedly. Electrophoretic mobility shift assays confirmed that CcpA directly binds to the promoter sequences of gtfB and gtfC, with a higher affinity for gtfC. Moreover, the expression level of ccpA in part explained differences in the ability to synthesize sufficient EPS and form stable biofilm in clinically isolated strains. These findings highlight that CcpA plays a crucial role in the EPS production and biofilm formation of S. mutans through directly binding to the promoter regions of gtfB and gtfC. This study provides novel insights into the pathogenic mechanisms of S. mutans and potential strategies for the prevention and treatment of dental caries.

The morphological and viscoelastic properties of blood neutrophils and neutrophils isolated from the oral cavity of the same donor were compared using scanning ion conductance microscopy. It was found that cell morphology, including morphometric parameters (height, diameter, and cell volume), did not differ significantly. However, the elasticity of the membrane-cytoskeletal complex was primarily determined by the cell compartment and its functional state (e.g., migration or adhesion) rather than the neutrophils' ecological niche (blood or oral cavity). The ability of blood neutrophils and oral neutrophils to produce reactive oxygen species (ROS) was assessed using luminol-dependent chemiluminescence. Neutrophils isolated from the oral cavity demonstrated a significantly higher ROS-producing activity compared to blood neutrophils from the same donor. Additionally, oral neutrophils exhibited high variability in ROS production levels, even within the same donor on different days of collection. When comparing the stimulation of ROS production in blood and oral neutrophils exposed to S. aureus 2879M, the cells displayed opposing responses: blood neutrophils were stimulated by Staphylococci, whereas oral neutrophils were inhibited. This response persisted when cells were stimulated by Staphylococci preincubated with saliva. The sequential addition of unfiltered saliva to neutrophils and Staphylococci enhanced this trend, while filtered oral fluid attenuated it. For the first time, it was established that saliva without bacteria (filtered through bacterial filters) suppresses the production of ROS by both blood neutrophils and oral neutrophils, most likely indicating an anti-inflammatory effect of saliva.

Porphyromonas gingivalis is a key pathogen in periodontitis, with secreted proteases as major virulence factors. We developed a screening method to generate and identify P. gingivalis mutants with elevated protease activity. Mutations were induced using the mutagens 2,6-diaminopurine (2,6-DAP) or ethyl methanesulfonate (EMS), and the mutagenized cells were subsequently plated on casein agar. During colony growth, the medium became opaque due to partial casein precipitation, whereas colonies with higher protease activity produced clear halos through casein degradation. Colonies that formed halos earlier than the wild type were selected for further analysis. Liquid culture assays of the supernatants identified four strains with enhanced protease activity, of which two were 2,6-DAP-derived and two were EMS-derived. Whole-genome sequencing revealed that the two 2,6-DAP-derived strains carried mutations in iron transport-related genes (foeA and tonB, respectively), likely increasing protease levels through iron limitation-induced upregulation of rgpA. The two EMS-derived strains contained multiple mutations, including one in rgpA, a major protease gene. The N-terminal region of RgpA, which contains the protease motif, harbored the G450D mutation in one strain and the C600Y mutation in the other. These results demonstrate that our method efficiently generates P. gingivalis mutants with protease gene alterations that increase enzymatic activity. This approach provides a useful tool for studying protease function and virulence mechanisms in this pathogen, and for identifying genes that affect protease secretion.

Bacteria produce membrane vesicles (MVs) in response to environmental stress and genetic changes. Previous studies have shown that MVs can trigger inflammatory responses and may serve as important mediators of host-microbe interactions. Given the dynamic nature of the oral microbiome, bacteria such as Streptococcus mutans are frequently exposed to environmental fluctuations that could alter MV production. The objective of this study was to investigate whether inducing stress conditions would affect MV production and morphology in S. mutans, a prominent oral pathogen. Cultures were subjected to different pH conditions to mimic environmentally relevant stress. MVs were isolated and purified in order to characterize and assess changes in yield, size, and cargo. Our findings show that acidic stress significantly increased MV production while reducing average MV size. We also observed significant differences in MV content when compared to control conditions. These changes may reflect bacterial adaptation strategies and could influence how MVs interact with host immune systems. Overall, this study highlights the potential for environmental stress to reshape MV-mediated communication in the oral microbiome and provides a foundation for exploring how such changes may contribute to inflammation and oral disease.

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.