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

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, through large-scale metagenomic analysis of archeological samples, we discovered bacterial species related to modern-day Clostridium tetani, which produces tetanus neurotoxin (TeNT) and causes the disease tetanus. We were able to assemble 38 draft genomes from distinct human archeological samples spanning five continents from as early as ~4000 BCE. They display hallmarks of ancient DNA damage to variable degrees. While 20 fall into known C. tetani clades, phylogenetic analysis revealed novel C. tetani lineages, as well as a novel Clostridium species (“clade X”) closely related to C. tetani. Within these genomes, we found 15 novel TeNT variants, including a unique lineage of TeNT found exclusively in ancient samples from South America. We experimentally produced and tested a TeNT variant selected from a ~6000-year-old Chilean mummy sample and found that it induced tetanus muscle paralysis in mice with extreme potency comparable to modern TeNT. Our work provides the first identification of neurotoxigenic C. tetani in ancient DNA, discovery of a new Clostridium species unique to ancient human samples, and a novel variant of tetanus neurotoxin that retains functional activity and ability to cause disease in mammals.