Pathogens and host immunity in the ancient human oral cavity

Abstract

Calcified dental plaque (dental calculus) preserves for millennia and entraps biomolecules from all domains of life and viruses. We report the first high-resolution taxonomic and protein functional characterization of the ancient oral microbiome and demonstrate that the oral cavity has long served as a reservoir for bacteria implicated in both local and systemic disease. We characterize: (i) the ancient oral microbiome in a diseased state, (ii) 40 opportunistic pathogens, (iii) the first evidence of ancient human-associated putative antibiotic resistance genes, (iv) a genome reconstruction of the periodontal pathogen Tannerella forsythia, (v) 239 bacterial and 43 human proteins, allowing confirmation of a long-term association between host immune factors, “red-complex” pathogens, and periodontal disease, and (vi) DNA sequences matching dietary sources. Directly datable and nearly ubiquitous, dental calculus permits the simultaneous investigation of pathogen activity, host immunity, and diet, thereby extending the direct investigation of common diseases into the human evolutionary past.

Successful enrichment and recovery of whole mitochondrial genomes from ancient human dental calculus

“…The reasons for this are unclear, but may be related to differences in the cell types contributing to the human DNA component of dental calculus. For example, neutrophils, an abundant blood leukocyte involved in host innate immune response to dental plaque and calculus formation (Warinner et al 2014b), contain 10–15 times fewer mtDNA genome copies compared to peripheral blood mononuclear cells (PBMC) (Maianski et al 2004). However, during immune response, neutrophils utilize mtDNA as a structural molecule to bind cytotoxic proteins into neutrophil extracellular traps (NETs), which are then released onto advancing plaque biofilms (Yousefi et al 2009).…”

“…For dentine samples, the digestion buffer solution was refreshed after 48 hours and the two supernatants were combined for subsequent analyses. After digestion, DNA was extracted by a previously described phenol-chloroform separation protocol and purified by silica adsorption (Qiagen MinElute PCR Purification kit) (Warinner et al 2014b) using a modified protocol to increase binding buffer volume (Dabney et al 2013a). DNA was eluted into 30 μl of EB buffer and 1 μl of each extract was quantified using a Qubit High Sensitivity dsDNA assay (Life Technologies) (Table 1).…”

“…The subsequent application of shotgun metagenomics using Next-Generation Sequencing (NGS) technology further confirmed the presence of both mitochondrial and nuclear DNA within archaeological dental calculus, and moreover provided an estimate of its overall relative abundance: approximately 0.5% of all identifiable DNA sequences in one deeply sequenced sample (Warinner et al 2014b). Given that dental calculus is the richest known source of aDNA in the archaeological record, with reported DNA yields routinely exceeding 100 ng/mg in well-preserved samples (Warinner et al 2015a), dental calculus represents a potentially important source of ancient human DNA.…”

“…Calcification occurs when calcium phosphate ions present in saliva and gingival crevicular fluid (GCF) precipitate within dental plaque deposits, forming sequential mineralized layers corresponding to discrete plaque calcification events through time (Schroeder 1969; White 1991). The biological content of dental calculus is primarily microbial in origin (Warinner et al 2014b), and its parent material, dental plaque, is estimated to contain >200,000,000 microbial cells per milligram (Socransky and Haffajee 2005). In addition to microbes (bacteria and archaea), dental calculus also contains trace amounts of dietary (plant and animal) biomolecules and microfossils, viral DNA (primarily bacteriophages, or bacteria-infecting viruses), and human DNA and proteins (Warinner et al 2014a; Warinner et al 2014b).…”

“…The biological content of dental calculus is primarily microbial in origin (Warinner et al 2014b), and its parent material, dental plaque, is estimated to contain >200,000,000 microbial cells per milligram (Socransky and Haffajee 2005). In addition to microbes (bacteria and archaea), dental calculus also contains trace amounts of dietary (plant and animal) biomolecules and microfossils, viral DNA (primarily bacteriophages, or bacteria-infecting viruses), and human DNA and proteins (Warinner et al 2014a; Warinner et al 2014b). …”