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Genomic Study Finds Yersinia Pestis Has Plagued Humans for More Than 5,000 Years

NEW YORK (GenomeWeb) – The plague-causing Yersinia pestis has been infecting people longer than previously thought, according to a new genomic analysis of Bronze Age human teeth.

Researchers led by Eske Willerslev from the University of Copenhagen sequenced Y. pestis DNA isolated from those teeth, which are between 2,800 and 5,000 years old. In a study published today in Cell, they said that that those ancient bacterial genomes were basal to all known Y. pestis lineages, and estimated the origin of the Y. pestis lineage to some 5,783 years ago, about 3,300 years earlier than previous estimates.

"We found that the Y. pestis lineage originated and was widespread much earlier than previously thought, and we narrowed the time window as to when it developed," Willerslev said in a statement. "This study changes our view of when and how plague influenced human populations and opens new avenues for studying the evolution of diseases."

Three human plague epidemics have been recorded — the Plague of Justinian in the sixth century AD; the Black Death in Europe in the mid-1300s; and the Third Pandemic, which emerged in China in the mid-19th century — and have shaped human populations. The Black Death, for instance, likely killed between 30 percent and 50 percent of Europe's population.

Earlier outbreaks like the Plague of Athens some 2,500 years ago and the Antonine Plague in the second century may have also have occurred, though there is no solid evidence to tie them to Y. pestis.

As part of their work, Willerslev and his colleagues searched through about 89 billion raw DNA reads obtained from the teeth of 101 Bronze Age people who lived in Europe or Asia and discovered that seven of the individuals had sequences that resembled those of Y. pestis.

Through additional sequencing, the researchers assembled the Y. pestis genomes to an average depth ranging between 0.14X and 29.5X. They also recovered the sequences of the three bacterial plasmids, pCD1, pMT1, and pPCP1.

Willerslev and his colleagues argued that these sequences were authentic Y. pestis. In addition to using standard precautions for working with such ancient DNA, they reported Y. pestis sequences in only eight of the 101 samples, indicating there wasn't a ubiquitous contaminant in their lab. Also, among other indicators, the reads were highly fragmented with signs of C-T deamination, and the pathogen and host DNA molecules appear to be of similar ages.

They mapped all the reads to both the Y. pestis and then Y. pseudotuberculosis reference genomes. Seven of the samples matched Y. pestis, they noted, while an eighth did not.

To create a phylogenetic tree, the researchers mapped the two high coverage ancient Y. pestis strains — RISE505 and RISE509 — as well as Y. similis, Y. pseudotuberculosis, and Y. pestis strains to the Y. pseudotuberculosis reference genome. Using RAxML, they constructed a Maximum Likelihood phylogeny.

This tree, Willerslev and his colleagues reported, shows Y. pestis to be a monophyletic group within Y. pseudotuberculosis. They noted that the ancient strains RISE505 and RISE509 clustered together within the Y. pestis clade and fell basal to all other known Y. pestis strains.

In addition, they estimated the most recent common ancestor of Y. pestis and Y. pseudotuberculosis to have lived some 54,735 years ago, about twice as old as previous assessments. They also estimated the most recent common ancestor of all Y. pestis strains to have lived 5,783 years ago, also much older than previously thought.

These two high-coverage ancient Y. pestis genomes, the researchers noted, included most known plague virulence genes. They did, though, lack the Yersinia murine toxin (ymt) gene located in the pMT1 plasmid that encodes a phospholipase that protects the pathogen while in the flea gut.

Among the lower-coverage samples, they also found this gene to be missing in all but the youngest sample. The gene, they added, is present in all other published Y. pestis strains, both modern and ancient, except for three that lack the plasmid in its entirety. The researchers also noted that transposase regions flank the missing region.

This suggested to Willerslev and his colleagues that there was a late and rapid spread of the ymt gene and that the gene was acquired through horizontal gene transfer. They further placed the timing of the acquisition of this gene to between 1686 BC and 951 BC.

Six of the seven ancient plague genomes, the researchers reported, harbored the plasminogen activator gene pla on the pPCRP1 plasmid, which is essential for the development of bubonic and pneumonic plague as it facilitates deep tissue invasion. The one in which they didn't find it had the lowest depth of coverage.

The most ancient plague strains also appeared to have the pla protein isoleucine to threonine mutation that is needed for bubonic, though not pneumonic, plague.

However, RISE505 and RISE509 appear to have a functional flagellin regulator gene — a gene that is affected by a frameshift mutation in most known plague strains that prevents them from expressing flagellin. Flagellin, the researchers noted, is a potent initiator of the mammalian innate immune system.

The younger of the ancient plague strains, though, have partial loss of their flagella system, suggesting to the researchers that there was selective pressure on ancestral Y. pestis as it emerged as a mammalian pathogen.

Together, this indicated to Willerslev and his colleagues that the ancient Y. pestis strains could only lead to pneumonic and septicemic plague, not bubonic plague, and Y. pestis didn't become a flea-borne mammalian pathogen until the beginning of the first millennium BC.

"The underlying evolutionary mechanisms that facilitated the evolution of Y. pestis are still present today, and learning from this will help us understand how future pathogens may arise or develop increased virulence," co-first study author Simon Rasmussen from Technical University of Denmark added. "Additionally, our study changes the historical understanding of this extremely important human pathogen and makes it possible that other so-called plagues, such as the Plague of Athens and the Antonine Plague, could have been caused by Y. pestis."