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, to our knowledge, 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) 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 direct investigation of common diseases into the human evolutionary past.

Figure 1. Taxonomic and phylogenetic characterization of ancient dental calculus

a, Relative proportion of bacterial, archaeal, eukaryotic, and viral DNA in ancient calculus estimated from assembled whole metagenome shotgun sequences of two individuals. b, Phylogenetic tree of the 100 most abundant OTUs in ancient dental calculus samples of four pooled individuals. Evidence for the presence and abundance of each microbial OTU is represented by colored, size-scaled circles for each targeted 16S rRNA region (V3, V5, V6), shotgun 16S rRNA sequences, and other genes and proteins assigned to that OTU. OTUs for which no reference genome exists or for which insufficient proteome data has been validated for inclusion in the protein search databases are marked with a gray square, as no hits could ever be matched to that OTU. Relative phylum abundance (normalized mean of all genetic data generated from 16S sequences) is represented by a column chart showing phyla represented in top 100 OTUs (color), remaining phyla (dark gray), and unidentified OTUs (light gray).

Figure 2

Genomic coverage plot for the periodontal pathogen Tannerella forsythia, with details of gene and protein coverage of the virulence factor TF2663/tfsB, from medieval human dental calculus (G12). a, Plot of the T. forsythia genome with depth of coverage (0 to 30-fold shown) in red and identity in purple. Gene locations of major virulence factors present (black) or absent (gray) in the assembly are indicated in the outer ring. Notably absent are two transposon-related tra pathogenicity islands containing putative tetracycline resistance genes in the T. forsythia ATCC 43037 reference strain. Proteins identified by MS/MS are indicated in blue by an asterisk (*) at the corresponding gene locus. b, Enlarged view of forward (blue) and reverse (red) DNA reads mapped to gene TF2663, which encodes the surface layer B protein, tfsB. c, Enlarged view of peptide coverage for tfsB, a species-diagnostic virulence factor involved in haemagglutination, adherance, and host tissue invasion. d, Detail of a representative unique tfsB peptide (arrowhead) with corresponding spectrum.

Figure 3. Metaproteomic comparison of human proteins in modern and ancient dental samples

a, Functional characterization of human proteins identified in modern dental calculus (two individuals), ancient dental calculus (four individuals), and ancient tooth roots (four individuals). b, Venn diagram of shared human proteins by sample type. c, STRING network representation of human proteins identified in ancient dental calculus; nodes are labeled by gene name and colored in accordance with functional categories and connections (gray) to predicted functional partners. The network is set to medium confidence (0.4) for all active prediction methods. d, Gene ontology categories with significant enrichment (p < 0.001, FDR corrected) in ancient dental calculus for biological process (black) and molecular function (white) relate to proteinase regulation, substrate binding, and innate immune function. Enrichment calculated relative to the human genome using the STRING-embedded AmiGO term enrichment tool.

Figure 4. Genetic, microfossil, zooarchaeological, and stable isotopic evidence for medieval human diet at Dalheim

Neighbor joining trees for Genbank sequences aligning to putative dietary sheep (a), crucifer (b), and pig/boar (c) sequences. Trees include accessions with alignment scores >45, except for (c), which is limited to the top 250 alignments; highly significant alignments (E-value < 1e-30) are starred (*). BLAST top hits for each dietary sequence are highlighted. Maximum fraction of mismatched bases is 0.75 for tree generation, and distance was calculated using a Jukes-Cantor substitution model. d, One sequence aligned to two accessions of bread wheat only. e-h, Microfossils recovered from ancient human dental calculus yielded morphological matches to animal collagen fibers (e), a monocot phytolith (f), and starch grains of the grass tribe Triticeae (g) and the legume family Fabaceae (h). i, C and N stable isotopic values of human bone collagen (black circles) fall within two standard deviations (boxes) of those measured for other Central European populations and are consistent with a diet of mixed C₃ terrestrial plant and animal resources. j-k, Recovered food waste includes skeletal material from Sus sp. (j) and Caprinae (k).

Figure 5. Evidence of microscopic and biomolecular preservation of ancient dental calculus

a, Labio-lingual section of a mandibular incisor with dental calculus (arrow) on the labial crown surface; both dentine and cementum within the tooth root show extensive evidence of postmortem alteration. b, EDS visualization of Ca (red) and Si (green) shows Si is restricted to the surface except for one biogenic Si inclusion (arrow). c, Detail of dental calculus which exhibits a layered structure suggesting outward-downward incremental growth. d, Detail of a stained section showing Gram-negative (red) and Gram-positive (blue) bacteria. e, Detail of Hoechst stained section showing abundant in situ dsDNA. f, g, The calculus matrix contains numerous lacunae of microorganisms (arrows), some of which are mineralized (arrow-heads). h, Proportions of human and bacterial proteins identified in ancient and modern samples. i, DNA extraction yields from modern (M) and ancient dental calculus, dentine, and alveolar bone samples. j, Comparison of microbial communities in ancient dental samples (squares) to those in diverse publicly available samples (circles). 16S rRNA data was generated by shotgun sequencing (S) and targeted amplification and sequencing of hypervariable regions (V3, V5, V6), followed by OTU clustering. Ancient metagenomes (n=38) were plotted within a network where distance scales with OTU community similarity. Modern metagenomes with >20% shared community are shown connected by black lines; ancient metagenomes with >20% shared community are connected by green lines. A total of 315 Genbank studies were recruited to the network. Ancient metagenomes segregate into two distinct groups: ancient dental calculus samples cluster tightly together, are connected by thick lines, and show similarity to modern metagenomes of primarily human and oral origin; ancient dentine and abscessed bone tissue samples form a more diffuse cluster and recruit primarily soil and environmental metagenomes. Carious dentine forms an intermediate cluster that shares OTUs with both human-associated and environmental sources.