Repurposing Metformin for periodontal disease management as a form of oral-systemic preventive medicine.

BACKGROUND: Despite the improvements in treatment over the last decades, periodontal disease (PD) affects millions of people around the world and the only treatment available is based on controlling microbial load. Diabetes is known to increase the risk of PD establishment and progression, and recently, glucose metabolism modulation by pharmaceutical or dietarian means has been emphasised as a significant modulator of non-communicable disease development. METHODS: The impact of pharmaceutically controlling glucose metabolism in non-diabetic animals and humans (REBEC, UTN code: U1111-1276-1942) was investigated by repurposing Metformin, as a mean to manage periodontal disease and its associated systemic risk factors. RESULTS: We found that glucose metabolism control via use of Metformin aimed at PD management resulted in significant prevention of bone loss during induced periodontal disease and age-related bone loss in vivo. Metformin also influenced the bacterial species present in the oral environment and impacted the metabolic epithelial and stromal responses to bacterial dysbiosis at a single cell level. Systemically, Metformin controlled blood glucose levels and age-related weight gain when used long-term. Translationally, our pilot randomized control trial indicated that systemic Metformin was safe to use in non-diabetic patients and affected the periodontal tissues. During the medication window, patients showed stable levels of systemic blood glucose, lower circulating hsCRP and lower insulin levels after periodontal treatment when compared to placebo. Finally, patients treated with Metformin had improved periodontal parameters when compared to placebo treated patients. CONCLUSION: This is the first study to demonstrate that systemic interventions using Metformin in non-diabetic individuals aimed at PD prevention have oral-systemic effects constituting a possible novel form of preventive medicine for oral-systemic disease management.

Microbial transitions from health to disease.

Recent advances in our understanding of the microbial populations that colonize the human mouth, their acquisition, interdependency, and coevolution with the host, bring a different perspective to the mechanisms underpinning the maintenance of periodontal health and the development of disease. In this work we suggest that our knowledge map of the etiology of periodontal health and disease can be viewed as a broad, highly connected, and integrated system that spans the entire spectrum of microbe/host/clinical interactions. The overall concept of present Periodontology 2000, that the microbial biofilm can be considered a human tissue of bacteriological origin, is entirely consistent with this integrated system view. The health-associated community structure of microbial biofilms can be considered a system that is normally resilient to perturbation. Equally, there is evidence to suggest that the dysbiotic community structure in disease may share similar resilience properties. In both instances, the resilience may be governed by the precise makeup of the acquired microbiome and by the genetics of the host. Understanding the mechanisms that enable the resistance to change of healthy and dysbiotic microbial populations may be important in the development of approaches to prevent the progression of disease and to restore health in diseased individuals.

Hemin availability induces coordinated DNA methylation and gene expression changes in Porphyromonas gingivalis.

Periodontal disease is a chronic inflammatory disease in which the oral pathogen Porphyromonas gingivalis plays an important role. Porphyromonas gingivalis expresses virulence determinants in response to higher hemin concentrations, but the underlying regulatory processes remain unclear. Bacterial DNA methylation has the potential to fulfil this mechanistic role. We characterized the methylome of P. gingivalis, and compared its variation to transcriptome changes in response to hemin availability. Porphyromonas gingivalis W50 was grown in chemostat continuous culture with excess or limited hemin, prior to whole-methylome and transcriptome profiling using Nanopore and Illumina RNA-Seq. DNA methylation was quantified for Dam/Dcm motifs and all-context N6-methyladenine (6mA) and 5-methylcytosine (5mC). Of all 1,992 genes analyzed, 161 and 268 were respectively over- and under-expressed with excess hemin. Notably, we detected differential DNA methylation signatures for the Dam "GATC" motif and both all-context 6mA and 5mC in response to hemin availability. Joint analyses identified a subset of coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify altered methylation and expression responses to hemin availability in P. gingivalis, with insights into mechanisms regulating its virulence in periodontal disease. IMPORTANCE DNA methylation has important roles in bacteria, including in the regulation of transcription. Porphyromonas gingivalis, an oral pathogen in periodontitis, exhibits well-established gene expression changes in response to hemin availability. However, the regulatory processes underlying these effects remain unknown. We profiled the novel P. gingivalis epigenome, and assessed epigenetic and transcriptome variation under limited and excess hemin conditions. As expected, multiple gene expression changes were detected in response to limited and excess hemin that reflect health and disease, respectively. Notably, we also detected differential DNA methylation signatures for the Dam "GATC" motif and both all-context 6mA and 5mC in response to hemin. Joint analyses identified coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify novel regulatory processes underlying the mechanism of hemin regulated gene expression in P. gingivalis, with phenotypic impacts on its virulence in periodontal disease.

Molecular basis for avirulence of spontaneous variants of Porphyromonas gingivalis: Genomic analysis of strains W50, BE1 and BR1.

The periodontal pathogen Porphyromonas gingivalis is genetically heterogeneous. However, the spontaneous generation of phenotypically different sub-strains has also been reported. McKee et al. (1988) cultured P. gingivalis W50 in a chemostat during investigations into the growth and properties of this bacterium. Cell viability on blood agar plates revealed two types of non-pigmenting variants, W50 beige (BE1), and W50 brown (BR1), in samples grown in a high-hemin medium after day 7, and the population of these variants increased to approximately 25% of the total counts by day 21. W50, BE1 and BR1 had phenotypic alterations in pigmentation, reduced protease activity and haemagglutination and susceptibility to complement killing. Furthermore, the variants exhibited significant attenuation in a mouse model of virulence. Other investigators showed that in BE1, the predominant extracellular Arg-gingipain was RgpB, and no reaction with an A-lipopolysaccharide-specific MAb 1B5 (Collinson et al., 1998; Slaney et al., 2006). In order to determine the genetic basis for these phenotypic properties, we performed hybrid DNA sequence long reads using Oxford Nanopore and the short paired-end DNA sequence reads of Illumina HiSeq platforms to generate closed circular genomes of the parent and variants. Comparative analysis indicated loss of intact kgp in the 20 kb region of the hagA-kgp locus in the two variants BE1 and BR1. Deletions in hagA led to smaller open reading frames in the variants, and BR1 had incurred a major chromosomal DNA inversion. Additional minor changes to the genomes of both variants were also observed. Given the importance of Kgp and HagA to protease activity and haemagglutination, respectively, in this bacterium, genomic changes at this locus may account for most of the phenotypic alterations of the variants. The homologous and repetitive nature of hagA and kgp and the features at the inverted junctions are indicative of specific and stable homologous recombination events, which may underlie the genetic heterogeneity of this species.