Last year, I reviewed a PLoS Pathogens paper that found European Black Plague victims from the mid 14th century were infected with more than one clone of Yersinia pestis. While the Y. pestis-specific sequences amplified from several skeletal samples from various countries were evidence of the bacterium as the etiological agent, questions still remained about the virulence of the outbreak. What allowed that ancient strain of Y. pestis to cause such widespread death? Another group of researchers decided to further analyze the causative agent of the Black Plague by enriching for and sequencing one of the extrachromasomal plasmids present in the bacterial genome: the 9.6kb virulence-associated pPCP1 plasmid.
The recently published research in the Proceedings of the National Association of Science USA chose to use samples from East Smithfield (ES) cemetery in London, England, a burial ground that was established during the medieval Black Death pandemic of 1348–1350 to cope with the influx of the dead. This information is recorded in historical record and reinforced by archeological evidence (the presence of coins dated to the 1340s).
Using these skeletal remains as established victims of the Black Plague, the molecular analysis began with DNA extraction. Human DNA samples from St. Nicholas Shambles (SNS), a cemetery in London that predated the Black Plague, were used as negative controls, and a sample of ancient cave bear DNA served as a cross-contamination control during mitochondrial DNA (mtDNA) studies to ensure there was no contamination by modern human DNA. Furthermore, DNA extraction from 53 skeletal samples and 46 teeth occurred at two different locations: McMaster University in Hamilton, Ontario, Canada, and Max Planck Institute in Leipzig, Germany.
The McMaster University samples were screened using quantitative PCR, looking for a 52bp Y. pestis-specific gene, plasminogen activator (pla), located on the pPCP1 virulence-associated plasmid. The pla-positive samples were further screened for the chaperone protein for the fraction 1 antigen gene (caf1M) located on the pMT1 plasmid, a larger plasmid at lower copy number in Y. pestis. None of the negative control samples yielded any Y. pestis sequences.
One difficulty in dealing with ancient DNA is the high incidence of transitions that can mask a true mutation or polymorphism. A transition is an alteration in the nucleotide sequences that changes a purine to another purine or a pyrimidine to another pyrimidine. Using the five human samples positive for both pla and caf1M, researchers amplified the pla gene by multiplex PCR and the gene sequenced with at least 4X coverage over 78% of the region. The researchers distinguished polymorphisms from DNA damage by comparing the resulting sequence with pla sequences generated from two independent amplifications of the same ancient DNA library, and using a third amplification of the same library to resolve any nonmatches. Additional PCR primer pairs were used to place the Y. pestis strain on two of the three branches of the phylogenetic tree. Only one product amplified, suggesting placement in branch 2, the Medievalis biovar.
The researchers at the Max Planck Institute in Leipzig, Germany, focused on enriching for Y. pestis pPCP1 and human mtDNA then further analyzing the sequences. DNA was isolated from five Y. pestis-postive ES samples, based on pla copy number, and used to create libraries. Five SNS (Y. pestis-negative) samples and one ancient cave bear sample were also chosen for DNA isolation and library creation. Immobilized sequences from the human mitochondrial genome and modern Y. pestis pPCP1 sequence were used for enrichment of those sequences in the ancient DNA samples followed by amplification of the resulting enriched samples. All ten ancient human samples had unique mtDNA genomes, indicating there was no cross contamination between samples. Two of the ES samples were extracted twice, yielding identical sequences and haplotypes. The mtDNA consensus sequences for each sample were compared against a worldwide dataset of 311 mtDNAs and the Cambridge reference sequence, and found up 17 positions considered unique or rare. These variants also had hallmarks of DNA degradation, suggesting they were truly ancient human DNA.
Interestingly, all the libraries contained sequences that matched the Y. pestis pPCP1 reference genome including the blank, cave bear DNA and preBlack Death samples. The fragments in these nonES samples all mapped to the same position on the plasmid: 3,000–4,200. This region’s sequence was similar to vector sequences used to express recombinant enzymes and were eliminated from further analysis. Libraries from the five ES samples had up to 6,435 unique fragments that mapped to the pPCP1 plasmid, and because the researchers assumed that all people from the same grave in London suffered from the same strain of plague, the libraries were pooled to construct a consensus sequence covering 99% of the plasmid with at least 2X coverage. AT-rich regions had less coverage than GC-rich ones, reflecting a bias found in other ancient human DNA shotgun sequencing studies (reference in Schuenemann et al.). Furthermore, the same DNA nucleotide substitution patterns found in the ancient mtDNA were also found in the pPCP1 sequence (e.g., C to T at the 3´ ends), and the plasmid fragments were smaller than the contaminating vector sequence, more evidence that this was sequence of ancient DNA. Comparing the pPCP1 sequence from the ES samples to modern pPCP1 sequences found identity with 11 of the 14 Y. pestis strains but did not place this strain any one branch of the phylogenetic tree.
What can be concluded from this study? Y. pestis-specific genes and the virulence-associated plasmid pPCP1 were isolated from teeth that originated from a Black Death cemetery. Because the cemetery can be precisely dated, its occupants are known to have died from the Black Death, and Y. pestis is a blood-borne pathogen and would likely be found in the pulp of teeth, we can conclude that the people that were buried in the East Smithfield cemetery were infected with Y. pestis. These victims died from a possible Medievalis biovar Y. pestis strain that contained a virulence-associated plasmid that does not differ much from the plasmid carried by many modern strains. Sequencing the full length of a nearly 10kb plasmid from ancient DNA is an important achievement because of the high degree of fragmentation in ancient DNA. However, for the Y. pestis virulence-associated plasmid pPCP1, the sequence similarity with modern strains leaves researchers wondering what differences existed in the Black Plague strain that make it so devastating to the human population.
Schuenemann VJ, Bos K, Dewitte S, Schmedes S, Jamieson J, Mittnik A, Forrest S, Coombes BK, Wood JW, Earn DJ, White W, Krause J, & Poinar HN (2011). From the Cover: Targeted enrichment of ancient pathogens yielding the pPCP1 plasmid of Yersinia pestis from victims of the Black Death. Proceedings of the National Academy of Sciences of the United States of America, 108 (38) PMID: 21876176
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