A tetanus bacillus from a 6,000-year-old mummy caused paralysis in a mouse

(ORDO NEWS) — Analysis of the genetic database of the National Center for Biotechnology Information allowed scientists to discover fragments of Clostridia genomes sequenced along with the DNA of ancient people.

They managed to collect 24 tetanus bacillus genomes, and also discovered two previously unknown Clostridium species.

In addition, the scientists showed that a variant of the tent gene encoding the production of tetanus neurotoxin, which had previously been sequenced from a sample of a 6,000-year-old South American Chinchorro mummy, could cause paralysis in mice.

A preprint of this work is available on the bioRxiv service.

Tetanus bacillus (Clostridium tetani) is a species of Gram-positive bacteria belonging to the genus Clostridium, which also includes the causative agents of gangrene (C. perfringens), pseudomembranous colitis (C. difficile) and botulism (C. botulinum).

As the name implies, the tetanus bacillus is the causative agent of tetanus, an acute infectious disease accompanied by damage to the nervous system, skeletal muscle tension and generalized convulsions (more about the disease in our text “Tetanus attacked me”).

Man has been confronted with this extremely dangerous pathogen since antiquity, as evidenced by information from written sources: the writings of Hippocrates (about 380 BC) and the ancient Egyptian papyrus of Edwin Smith (about 1550 BC).

This is not surprising, since the bacterium is very widespread, and soil is its main natural source. When it enters the body through a wound, the pathogen begins to produce tetanus toxin, which consists of two fractions: tetanospasmin (a neurotoxin for which the tent gene is responsible) and tetanolysin.

A tetanus bacillus from a 6 000 year old mummy caused paralysis in a mouse 2
Geographic origin of ancient specimens

Harold Hodgins of the University of Waterloo, together with colleagues from Denmark, Canada, the United States and Japan, explored the genetic database of the National Center for Biotechnology Information using the newly developed STAT (Sequence Taxonomic Analysis Tool).

In this database, they found 136 examples of sequences characteristic of tetanus bacillus. Moreover, 76 of them were contained in samples of ancient DNA extracted from 38 human teeth and bones.

Among them were, in particular, the teeth of the indigenous inhabitants of the Canary Islands (7th-11th centuries AD), the teeth of a resident of prehistoric Japan during the Jomon period (about 1044 BC), ancient Egyptian mummies (about 1879 BC – 53 AD ), as well as mummies of the South American Chinchorro culture (circa 3889 BC).

The scientists noted that the 24 assembled genomes were 96.4–99.7 percent identical to the reference tetanus bacillus genome. According to the researchers, this allows them to be considered one species.

Phylogenetic analysis showed that one accession each belongs to clades 1B and 1F, and nine accessions each belong to clades 1H and 2. The remaining four genomes are also grouped within clade 1, but outside the previously identified lineages.

However, not all collected genomes fell within the scope of tetanus bacillus variability. Examination of eight variants showed that they belong to a previously unknown species, which scientists tentatively named Clostridium sp. X. It is noteworthy that seven of these eight specimens come from Europe.

They were sequenced along with the ancient DNA of people who lived around 2253 BC – 1787 AD. Moreover, among them were victims of the plague, as well as a person who suffered from tuberculosis.

Another variant, read when analyzing the DNA of a man from the Canary Islands, who lived around 935 AD, the researchers attributed to a previously unknown species Clostridium sp. Y.

Scientists paid special attention to the tent gene, which is responsible for the production of tetanus neurotoxin. The nucleotide sequence of this gene differed most from the reference in a sample extracted from the bone of a mummy about six thousand years old, belonging to the Chinchorro culture.

Experiments have shown that, despite numerous substitutions, this gene encodes an active tetanus neurotoxin, which, when ingested, caused paralysis in mice.

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