Localized microbially induced inflammation influences distant healthy tissues in the human oral cavity.

Variation in human immune response to the same bacterial or viral pathogen is well established in the literature. Variation in immune response to microbial challenge has also been observed within the human oral cavity. Our recent study focused on characterizing observed variations in microbially induced gingival inflammation-resulting in three distinct clinical Inflammatory Responder Types (IRTs): High-IRT, Low-IRT, and Slow-IRT. Here, we applied a high-resolution temporal multiomic analysis during microbially induced inflammation in order to characterize the effects of localized oral inflammation on distant healthy tissues in young healthy adults. Our results highlight a nonlocalized subclinical effect with alterations in proinflammatory host mediators and an ecological shift toward dysbiosis within the subgingival microbiome in an IRT-dependent manner-despite maintained oral hygiene. Our results provide mechanistic insight into how healthy tissues within humans are influenced by distant localized inflammation and may ultimately become susceptible to disease.

Human variation in gingival inflammation.

Oral commensal bacteria actively participate with gingival tissue to maintain healthy neutrophil surveillance and normal tissue and bone turnover processes. Disruption of this homeostatic host-bacteria relationship occurs during experimental gingivitis studies where it has been clearly established that increases in the bacterial burden increase gingival inflammation. Here, we show that experimental gingivitis resulted in three unique clinical inflammatory phenotypes (high, low, and slow) and reveal that interleukin-1ß, a reported major gingivitis-associated inflammatory mediator, was not associated with clinical gingival inflammation in the slow response group. In addition, significantly higher levels of Streptococcus spp. were also unique to this group. The low clinical response group was characterized by low concentrations of host mediators, despite similar bacterial accumulation and compositional characteristics as the high clinical response group. Neutrophil and bone activation modulators were down-regulated in all response groups, revealing novel tissue and bone protective responses during gingival inflammation. These alterations in chemokine and microbial composition responses during experimental gingivitis reveal a previously uncharacterized variation in the human host response to a disruption in gingival homeostasis. Understanding this human variation in gingival inflammation may facilitate the identification of periodontitis-susceptible individuals. Overall, this study underscores the variability in host responses in the human population arising from variations in host immune profiles (low responders) and microbial community maturation (slow responders) that may impact clinical outcomes in terms of destructive inflammation.

Transcriptome of Epibiont Saccharibacteria Nanosynbacter lyticus Strain TM7x During the Establishment of Symbiosis.

Saccharibacteria Nanosynbacter lyticus strain TM7x is a member of the broadly distributed candidate phylum radiation. These bacteria have ultrasmall cell sizes, have reduced genomes, and live as epibionts on the surfaces of other bacteria. The mechanisms by which they establish and maintain this relationship are not yet fully understood. The transcriptomes of the epibiont TM7x and its host bacteria Schaalia odontolytica strain XH001 were captured across the establishment of symbiosis during both the initial interaction and stable symbiosis. The results showed a dynamic interaction with large shifts in gene expression for both species between the initial encounter and stable symbiosis, notably in transporter genes. During stable symbiosis, the host XH001 showed higher gene expression for peptidoglycan biosynthesis, mannosylation, cell cycle and stress-related genes, whereas it showed lower expression of chromosomal partitioning genes. This was consistent with the elongated cell shape seen in XH001 infected with TM7x and our discovery that infection resulted in thickened cell walls. Within TM7x, increased pili, type IV effector genes, and arginine catabolism/biosynthesis gene expression during stable symbiosis implied a key role for these functions in the interaction. Consistent with its survival and persistence in the human microbiome as an obligate epibiont with reduced de novo biosynthetic capacities, TM7x also showed higher levels of energy production and peptidoglycan biosynthesis, but lower expression of stress-related genes, during stable symbiosis. These results imply that TM7x and its host bacteria keep a delicate balance in order to sustain an episymbiotic lifestyle. IMPORTANCE Nanosynbacter lyticus type strain TM7x is the first cultivated member of the Saccharibacteria and the candidate phyla radiation (CPR). It was discovered to be ultrasmall in cell size with a highly reduced genome that establishes an obligate epibiotic relationship with its host bacterium. The CPR is a large, monophyletic radiation of bacteria with reduced genomes that includes Saccharibacteria. The vast majority of the CPR have yet to be cultivated, and our insights into these unique organisms to date have been derived from only a few Saccharibacteria species. Being obligate parasites, it is unknown how these ultrasmall Saccharibacteria, which are missing many de novo biosynthetic pathways, are maintained at a high prevalence within the human microbiome as well as in the environment.

Ultrasmall epibiont Nanosynbacter lyticus strain TM7x and host bacteria transcriptional activity after initial host parasitism.

Background: Oral Saccharibacteria Nanosynbacter lyticus strain TM7× lives as an ultrasmall epibiont on the surface of its host, Schaalia odontolytica strain XH001. Establishing this interaction is a poorly understood multi-step process. The recovery phase marks a shift in the TM7×/host interaction, switching from the early killing phase, with extensive host cell death, to a stable symbiosis phase where the host and epibiont can grow together. Results: Transcriptomes of TM7× and host, XH001, were captured during the recovery phase and compared to uninfected host and the early host/epibiont interaction (initial encounter). XH001 showed increased expression for rhamnose cell wall components and for the precursor to peptidoglycan while TM7× showed increases in the peptidoglycan pathway. Transporter expression was generally increased for both organisms during recovery compared to the initial encounter, though, XH001 showed lower amino acid transporter expression. Consistent with host parasitism, XH001 showed increased expression of various stress-related genes during recovery while TM7× showed reduced stress. TM7× displayed higher expression of type IV pili, consistent with increased attachment to new hosts. Conclusion: As TM7× is a member of the broadly distributed Candidate Phyla Radiation with small genomes lacking numerous biosynthetic pathways, this study provides further insights into how these epibionts interact and modulate their host bacteria.

Identifying essential genes in Schaalia odontolytica using a highly-saturated transposon library.

The unique epibiotic-parasitic relationship between Nanosynbacter lyticus type strain TM7x, a member of the newly identified Candidate Phyla Radiation, now referred to as Patescibacteria, and its basibiont, Schaalia odontolytica strain XH001 (formerly Actinomyces odontolyticus), require more powerful genetic tools for deeper understanding of the genetic underpinnings that mediate their obligate relationship. Previous studies have mainly characterized the genomic landscape of XH001 during or post TM7x infection through comparative genomic or transcriptomic analyses followed by phenotypic analysis. Comprehensive genetic dissection of the pair is currently cumbersome due to the lack of robust genetic tools in TM7x. However, basic genetic tools are available for XH001 and this study expands the current genetic toolset by developing high-throughput transposon insertion sequencing (Tn-seq). Tn-seq was employed to screen for essential genes in XH001 under laboratory conditions. A highly saturated Tn-seq library was generated with nearly 660,000 unique insertion mutations, averaging one insertion every 2-3 nucleotides. 203 genes, 10.5% of the XH001 genome, were identified as putatively essential.

Effects of a stabilized stannous fluoride dentifrice on clinical, immunomodulatory, and microbial outcomes in a human experimental gingivitis model.

BACKGROUND: Stannous fluoride dentifrice is well established for its beneficial clinical effects. In this study, we evaluated the effects of stannous fluoride on inflammation and oral microbiome. METHODS: In this randomized, parallel-arm, double-blind, controlled clinical trial, we compared clinical resolution of experimental gingivitis by evaluating bleeding on probing, gingival index, and plaque index between stannous fluoride stabilized with zinc phosphate (test) and sodium fluoride (control) dentifrices. Further, these groups were compared for oral neutrophil counts, systemic priming of neutrophils, gingival crevicular fluid (GCF) expression of inflammatory markers, and the oral microbiome. RESULTS: We found significant reduction in bleeding on probing in the test group compared to the control group in experimental gingivitis when participants used the test dentifrice prior to induction of experimental gingivitis. The test group also showed significant reductions in GCF levels of inflammatory markers (matrix metalloproteinase 8 [MMP8], receptor activator of nuclear factor kappa-Β ligand [RANKL]), oral polymorphonuclear neutrophil (PMN) counts, and systemic neutrophil priming (CD11b expression) during experimental gingivitis. Further, significant reductions in the gram-negative genera Porphyromonas, Tannerella, and Treponema were noted in the test group. CONCLUSION: The stannous fluoride stabilized with zinc phosphate dentifrice formulation demonstrated clinical reduction in gingival inflammation and a beneficial effect on microbiome and immune markers. This intervention should be explored as a preventive aid in the progression of plaque-induced gingivitis to periodontitis.

Draft Genome Sequences of Nine "Candidatus Nanosynbacter sp. HMT-352" Strains Cultured from the Human Oral Cavity.

Here, we report draft genome sequences for nine strains of "Candidatus Nanosynbacter sp. HMT-352." These strains and their sequences were used to interrogate strain-level variations in host range, gene content, and growth dynamics among the phylum "Candidatus Saccharibacteria."

Strain-Level Variation and Diverse Host Bacterial Responses in Episymbiotic Saccharibacteria.

Saccharibacteria (TM7), which are obligate episymbionts growing on the surface of host bacteria, may play an important role in oral disease, such as periodontitis (1, 2). As TM7 is a newly cultured lineage of bacteria, its research is limited by the small number of isolated representatives relative to the number of TM7 genomes assembled from culture-independent studies (3-5). A comprehensive view of both TM7 taxa and TM7 strain-level variations remains opaque. In this study, we expanded our previously developed TM7 baiting method into using many host bacteria in parallel, which allowed us to obtain 37 TM7 strains from the human oral cavity. These strains were further classified into low-enrichment (LE, n = 24) and high-enrichment (HE, n = 13) groups based on their proficiency at propagating on host bacteria. Of the 13 HE strains, 10 belong to "Candidatus Nanosynbacter sp." strain HMT-352 (human microbial taxon) (6), enabling us to explore both the phenotypic and genomic strain variations within a single TM7 species. We show that TM7 HMT-352 strains exhibit a diverse host range and varied growth dynamics during the establishment of their episymbiotic relationship with host bacteria. Furthermore, despite HMT-352 strains sharing a majority of their genes, we identified several gene clusters that may play a pivotal role in host affinity. More importantly, our comparative analyses also provide TM7 gene candidates associated with strain-level phenotypic variation that may be important for episymbiotic interactions with host bacteria. IMPORTANCE Candidate phylum radiation (CPR) bacteria comprise a poorly understood phylum that is estimated to encompass ∼26% of all diversity of domain bacteria. Among CPR bacteria, the Saccharibacteria lineage (TM7) is of particular interest, as it is found in high abundance in the mammal microbiome and has been associated with oral disease. While many CPR genomes, TM7 included, have been acquired through culture-independent methods, only a small number of representatives have been isolated. Such isolated representatives, however, shed light on the physiology, pathogenesis, and episymbiotic interactions of TM7. Combined with genomic analyses, experiments involving isolated representatives can distinguish phylogenetic to phenotypic discrepancies and better identify genes of importance. In this study, we utilized multiple host bacteria in parallel to isolate TM7 bacteria and examined strain-level variation in TM7 to reveal key genes that may drive TM7-host interactions. Our findings accentuate that broad phylogenetic characterization of CPR is the next step in understanding these bacteria.

Modified SHI medium supports growth of a disease-state subgingival polymicrobial community in vitro.

Developing a laboratory model of oral polymicrobial communities is essential for in vitro studies of the transition from healthy to diseased oral plaque. SHI medium is an enriched growth medium capable of supporting in vitro biofilms with similar diversity to healthy supragingival inocula; however, this medium does not maintain the diversity of gram-negative bacteria more associated with subgingival plaque. Here, we systematically modified SHI medium components to investigate the impacts of varying nutrients and develop a medium capable of supporting a specific disease-state subgingival community. A diseased subgingival plaque sample was inoculated in SHI medium with increasing concentrations of sucrose (0%, 0.1%, 0.5%), fetal bovine serum (FBS) (0%, 10%, 20%, 30%, 50%), and mucin (0.1, 2.5, 8.0 g/L) and grown for 48 hrs, then the 16S rRNA profiles of the resulting biofilms were examined. In total, these conditions were able to capture 89 of the 119 species and 43 of the 51 genera found in the subgingival inoculum. Interestingly, biofilms grown in high sucrose media, although dominated by acidogenic Firmicutes with a low final pH, contained several uncultured taxa from the genus Treponema, information that may aid culturing these periodontitis-associated fastidious organisms. Biofilms grown in a modified medium (here named subSHI-v1 medium) with 0.1% sucrose and 10% FBS had a high diversity closest to the inoculum and maintained greater proportions of many gram-negative species of interest from the subgingival periodontal pocket (including members of the genera Prevotella and Treponema, and the Candidate Phyla Radiation phylum Saccharibacteria), and therefore best represented the disease community.

Acquisition and Adaptation of Ultra-small Parasitic Reduced Genome Bacteria to Mammalian Hosts.

The first cultivated representative of the enigmatic phylum Saccharibacteria (formerly TM7) was isolated from humans and revealed an ultra-small cell size (200-300 nm), a reduced genome with limited biosynthetic capabilities, and a unique parasitic lifestyle. TM7x was the only cultivated member of the candidate phyla radiation (CPR), estimated to encompass 26% of the domain Bacteria. Here we report on divergent genomes from major lineages across the Saccharibacteria phylum in humans and mammals, as well as from ancient dental calculus. These lineages are present at high prevalence within hosts. Direct imaging reveals that all groups are ultra-small in size, likely feeding off commensal bacteria. Analyses suggest that multiple acquisition events in the past led to the current wide diversity, with convergent evolution of key functions allowing Saccharibacteria from the environment to adapt to mammals. Ultra-small, parasitic CPR bacteria represent a relatively unexplored paradigm of prokaryotic interactions within mammalian microbiomes.

Rapamycin rejuvenates oral health in aging mice.

Periodontal disease is an age-associated disorder clinically defined by periodontal bone loss, inflammation of the specialized tissues that surround and support the tooth, and microbiome dysbiosis. Currently, there is no therapy for reversing periodontal disease, and treatment is generally restricted to preventive measures or tooth extraction. The FDA-approved drug rapamycin slows aging and extends lifespan in multiple organisms, including mice. Here, we demonstrate that short-term treatment with rapamycin rejuvenates the aged oral cavity of elderly mice, including regeneration of periodontal bone, attenuation of gingival and periodontal bone inflammation, and revertive shift of the oral microbiome toward a more youthful composition. This provides a geroscience strategy to potentially rejuvenate oral health and reverse periodontal disease in the elderly.

Age is the single greatest risk factor for many human diseases, including cancer, heart disease, and dementia. This is because, as the body ages, it becomes less able to repair itself. One way to prevent age-related disease and extend lifespan, at least in laboratory animals, is to use a drug called rapamycin. Mice treated with rapamycin live longer, have stronger hearts, and respond better to vaccination. But, despite these promising observations, the use of rapamycin as an anti-aging treatment is still under investigation. One open question is what age-related diseases rapamycin can help to prevent or treat. In the United States, more than 60% of adults over the age of 65 have gum disease. These people are also more likely to have other age-related diseases, like heart disease or Alzheimer's. This association between gum problems and other age-related diseases prompted An et al. to ask whether it might be possible to treat gum disease by targeting aging. To find out whether rapamycin could improve gum health, An et al. performed three-dimensional CT scans on mice as they aged to measure the bone around the teeth. Some of mice were treated with rapamycin, while the rest received a placebo. The mice that received the placebo started to show signs of gum disease as they aged, including inflammation and loss of bone around the teeth. The types of bacteria in their mouths also changed as they aged. Treating mice with rapamycin not only delayed the onset of these symptoms, but actually reversed them. After eight-weeks of the drug, the older mice had lost less bone and showed fewer signs of inflammation. There was also a shift in their mouth bacteria, restoring the balance of species back to those found in younger mice. Rapamycin is already approved for use in people, so a clinical trial could reveal whether it has the same effects on gum health in humans as it does in mice. But there are still unanswered questions about how rapamycin affects the mouth as it ages. These include how the drug works at a molecular level, and how long the changes to gum health persist after treatment stops.