Ancient Clostridium DNA and variants of tetanus neurotoxins associated with human archaeological remains

Abstract

The analysis of microbial genomes from human archaeological samples offers a historic snapshot of ancient pathogens and provides insights into the origins of modern infectious diseases. Here, we analyze metagenomic datasets from 38 human archaeological samples and identify bacterial genomic sequences related to modern-day Clostridium tetani, which produces the tetanus neurotoxin (TeNT) and causes the disease tetanus. These genomic assemblies had varying levels of completeness, and a subset of them displayed hallmarks of ancient DNA damage. Phylogenetic analyses revealed known C. tetani clades as well as potentially new Clostridium lineages closely related to C. tetani. The genomic assemblies encode 13 TeNT variants with unique substitution profiles, including a subgroup of TeNT variants found exclusively in ancient samples from South America. We experimentally tested a TeNT variant selected from an ancient Chilean mummy sample and found that it induced tetanus muscle paralysis in mice, with potency comparable to modern TeNT. Thus, our ancient DNA analysis identifies DNA from neurotoxigenic C. tetani in archaeological human samples, and a novel variant of TeNT that can cause disease in mammals.

Fig. 1. Petabase-scale screen of the NCBI sequence read archive reveals C. tetani-related genomes in ancient human archeological samples.

a General bioinformatic workflow starting with the analysis of 43,620 samples from the NCBI sequence read archive. Each sample is depicted according to its C. tetani k-mer abundance (y axis) versus the natural log of the overall dataset size in megabases (x axis). A threshold was used to distinguish samples with high detected C. tetani DNA content, and these data points are colored by sample origin: modern C. tetani genomes (red), non-human (light blue), modern human (blue), ancient human (black). The pie chart displays a breakdown of identified SRA samples with a high abundance of C. tetani DNA signatures. The 38 aDNA samples predicted to contain C. tetani DNA were further analyzed as shown in the bioinformatic pipeline on the right. b Top—density plot of the percentage identities of all BLAST local alignments detected between acBins and reference genomes including C. tetani, C. cochlearium, and other Clostridium spp. Bottom—density plot of the checkM results for the 38 acBins including estimated completeness, contamination, and strain heterogeneity levels. Completeness and contamination levels are percentage values. c MapDamage damage rates (5’ C → T misincorporation frequency) for acBins (n = 38 biologically independent samples) subdivided by UDG treatment [none (n = 27), partial (n = 5), and full (n = 6)]. Also shown are the damage rates for modern C. tetani genomes (n = 21 biologically independent samples). The boxplots depict the lower quartile, median, and upper quartile of the data, with whiskers extending to 1.5 times the interquartile range (IQR) above the third quartile or below the first quartile. d Damage plots for the top five acBins with the highest damage rates, and corresponding mtDNA damage plots. Shown is the frequency of C → T (red) and G → A (blue) misincorporations at the first and last 25 bases of sequence fragments. Increased misincorporation frequency at the edges of reads is characteristic of ancient DNA. Source data for (a–d) are provided as a Source Data file.

Fig. 2. Phylogenetic analysis reveals known and novel lineages of C. tetani in ancient DNA samples, as well as a previously unidentified Clostridium species (“X”).

a Dendrogram depicting relationships of acBins from ancient samples with modern C. tetani genomes. Novel branches are labeled “X” and “Y”, which are phylogenetically distinct from existing C. tetani genomes. Shown on the right of the dendrogram are metadata and statistics associated with each acBin including the estimated date of the associated archeological sample. All metadata can be found in Supplementary Data 2. b Geographic distribution of ancient DNA samples from which the 38 acBins were identified. Each sample is colored based on the acBin clustering pattern shown in (a). The global map was derived from the Natural Earth [https://www.naturalearthdata.com/] medium-scale data and plotted using the rnaturalearth and ggplot2 R packages. c SNP-based phylogenetic tree of a subset of acBins from lineage 1 and 2 showing high similarity and coverage to the C. tetani reference genome. See Supplementary Fig. 9 for more details. Source data for (a, c) are provided as a Source Data file.

Fig. 3. Comparative genomics of acBins versus modern C. tetani strains.

a Visualization of the chromosomal and plasmid multiple sequence alignment. Orthologous blocks are shown in black and the missing sequence is colored white. The reference gene locations are plotted above the alignments. b Gene neighborhoods surrounding the repA gene (left) and tent gene (right) in modern strains versus acBins. Selected unique differences identified in acBin gene neighborhoods are highlighted. The boxed region shows the assembled tent locus in two clade X acBins. Comparison reveals a putative deletion event in the clade X strains that has removed the majority of the tent gene along with five upstream genes, leaving behind conserved flanking regions. See Supplementary Fig. 18 for more information. c Per-clade coverage of the tent gene normalized to the coverage of repA. The data include n = 33 biologically independent samples, including acBins from clade 1 (n = 3), 1B (n = 1), 1 F (n = 1), 1H (n = 8), 2 (n = 9), X (n = 7), Y (n = 1), and acBins whose clade affiliation could not be determined (N.D., n = 3). The coverage was calculated as the average depth of coverage based on mapped reads to each gene. The boxplots depict the lower quartile, median, and upper quartile of the data, with whiskers extending to 1.5 times the interquartile range (IQR) above the third quartile or below the first quartile. See Supplementary Fig. 17 for the associated read pileups. Source data for (a–c) are provided as a Source Data file.

Fig. 4. Analysis and experimental testing of a novel TeNT lineage identified from ancient DNA.

a Maximum-likelihood phylogenetic tree of tent genes including novel tent sequences assembled from ancient DNA samples and a non-redundant set of tent sequences from existing strains in which duplicates have been removed (see “Methods” for details). The phylogeny has been subdivided into three subgroups. Sequences are labeled according to sample followed by their associated clade in the genome-based tree (Fig. 2a), except for the Barcelona-3031-Tooth sequence (*) as it fell below the coverage threshold. b Visualization of tent sequence variation, with vertical bars representing nucleotide substitutions found uniquely in tent sequences from ancient DNA samples. On the right, a barplot is shown that indicates the number of unique substitutions found in each sequence, highlighting the uniqueness of subgroup 3. c Structural model of TeNT/Chinchorro indicating all of its unique amino acid substitutions, which are not observed in modern TeNT sequences. Also shown is a segment of the translated alignment for a specific N-terminal region of the TeNT protein (residues 141–149, Uniprot ID P04958). This sub-alignment illustrates a segment containing a high density of unique amino acid substitutions, four of which are shared in TeNT/El-Yaral and TeNT/Chinchorro. d MapDamage analysis of the tent/Chinchorro gene, and associated C. tetani contigs and mtDNA from the Chinchorro-Mummy-Bone sample. e Cultured rat cortical neurons were exposed to full-length toxins in culture medium at the indicated concentration for 12 h. Cell lysates were analyzed by immunoblot, and the image shown is a representative of four independent experiments. WT TeNT (uniprot accession # P04958) and TeNT/Chinchorro (“ch”) showed similar levels of activity in cleaving VAMP2 in neurons. f, g Full-length toxins ligated by sortase reaction were injected into the gastrocnemius muscles of the right hind limb of mice. The extent of muscle rigidity was monitored and scored for 4 days (means ± s.e.; n = 3 per group, 9 total). TeNT/Chinchorro (“ch”) induced typical spastic paralysis and showed a potency similar to WT TeNT. Source data for (a, b, d, e, g) are provided as a Source Data file.