NEW YORK (GenomeWeb News) – A genetic study appearing online last night in the Proceedings of the National Academy of Sciences supports the notion that Yersinia pestis was the bacterial species behind the Black Death plague — but hints that the Y. pestis variant involved in the pandemic may have since disappeared.
Researchers from Germany, Canada, and the US screened DNA isolated from more than 100 medieval skeletal samples at burial sites in London using quantitative PCR before doing targeted enrichment, coupled with high-throughput sequencing, to sequence mitochondrial genomes and Y. pestis plasmids from 10 of the samples. They reported that the version of the Y. pestis pPCP1 plasmid they detected matches some still found in Y. pestis bacteria.
"That indicates that at least this part of the genetic information has barely changed in the past 600 years," co-corresponding author Johannes Krause, an archeology and human genetics researcher at the University of Tübingen, said in a statement. "Without a doubt, the plague pathogen known today as Y. pestis [was] also the cause of the plague in the Middle Ages."
Although the plasmid sequences correspond to those in some modern day strains of the bug, the researchers also identified some Y. pestis chromosomal sequences that seem to differ from those present in known versions of the species.
"Our data reveal that the Black Death in medieval Europe was caused by a variant of Y. pestis that may no longer exist," Krause and co-authors wrote, "and genetic data carried on its pPCP1 plasmid were not responsible for the purported epidemiological differences between ancient and modern forms of Y. pestis infections."
It's estimated that one-third of the European population died during the Black Death between 1347 and 1351. Y. pestis — which resembles the Y. pseudotuberculosis species but contains virulence plasmids, called pMT1 and pPCP1, that aren't found in that soil bacterium species — has been implicated in the pandemic.
But there has been some debate about whether the symptoms, timing, and epidemiology patterns documented in the Black Death outbreak jive with those seen in other waves of Y. pestis disease.
To determine whether Y. pestis could be detected in ancient samples from medieval plague victims, the researchers first isolated DNA from 53 bone and 46 tooth specimens originating at a burial site called East Smithfield in London that was used during the plague's peak between 1348 and 1350. For comparison purposes, they also isolated DNA from 10 pre-plague medieval samples from another burial site called St. Nicholas Shambles.
They then screened these samples for pPCP1, a Y. pestis plasmid that harbors several immunity- and virulence-associated genes, using a quantitative PCR assay that targeted the plasminogen activator gene pla, one of the pPCP1 virulence genes.
The researchers were able to amplify bits of the gene in nearly six percent of bone samples and 37 percent of the teeth samples from the plague burial site. PCR-based approaches showed that a subset of these samples also contained DNA from the less abundant pMT1 plasmid, they reported. On the other hand, Y. pestis DNA was not detected in the negative controls from the St. Nicholas Shambles burial site.
In their subsequent experiments, the team amplified and sequenced the pla gene and assayed sequences from the Y. pestis chromosome. They also showed that it was possible to enrich for specific bacterial and human sequences from their samples using hybridization to long-range PCR products.
"[T]argeted enrichment strategies in combination with high-throughput DNA sequencing allow for long stretches of ancient DNA to be reconstructed from a complex metagenomic background," they explained, "and it is clearly the way forward for ancient pathogen research."
Using this approach, the researchers targeted DNA representing the human mitochondrial sequences and the 9,600 base pPCP1 plasmid from skeletal samples from five of the individuals who died of plague and five negative controls from the other burial site.
They then sequenced pooled samples using the Illumina GAIIx, using characteristic nucleotide degradation patterns to distinguish between ancient DNA and present-day contaminants.
From the complete or nearly complete mitochondrial DNA genome sequences generated from the medieval samples, the team found that nine of the 10 individuals belonged to a known European mitochondrial haplogroup.
The team's analyses of the Y. pestis sequences in the samples, meanwhile, suggest that the plague-associated version of the pPCP1 plasmid matches some still circulating in Y. pestis today.
"The data presented here represent the oldest and longest assembled authentic sequences from an ancient pathogen," the study authors wrote, "and in turn, they suggest that the Black Death was caused by a Y. pestis variant that harbors a pPCP1 plasmid found in some modern isolates."
Given the conservation found between the ancient and modern versions of the plasmid, the researchers speculated that pPCP1 itself likely didn't contribute to the characteristic epidemiological profiles that distinguish Black Death from other Y. pestis pandemics. Instead, their initial sequence data suggests the plague strain may have harbored chromosomal DNA variations distinct from those previously reported for Y. pestis.
"[T]he medieval form of Y. pestis may harbor additional information regarding the organism's evolutionary history as a human pathogen," the researchers concluded. "This information may prove instrumental in identifying factors that influence the different epidemiology of ancient and modern forms of the disease."