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Genome from the Time of Cholera

02/05/2014
Jeffrey M. Perkel, PhD

It has long been suspected that pathogens and their hosts significantly influence each other's evolution. Now, methods of amplifying and sequencing ancient DNA have made researchers better able to explore this question. Read more...


Some 165 years ago in Philadelphia, a man, likely of West African descent, succumbed to cholera. That wasn’t particularly unusual; the world at the time was in the grip of the second of seven cholera pandemics in recorded history.

Intestine sample preserved at the Mütter Museum, credit: McMaster University

But then something unusual did happen. Rather than simply burying the victim, Dr. John Neill, a Philadelphia physician affiliated with the Southeast Cholera Hospital, removed a section of the dead man's intestine and preserved it in a jar filled with alcohol. Neill presented that sample, along with several others to the College of Physicians in Philadelphia, to investigate the anatomy of a cholera victim’s intestines and as “a groundwork for any future investigation.”

Fast-forward to 2014. Researchers at McMaster University in Hamilton, Ontario have become specialists in reconstructing ancient DNA. In 2011, they were able to extract enough genetic material from the teeth of fourteenth century victims of the Black Death in London to reconstruct the genome of Yersinia pestis, the bacteria thought to be responsible for that pandemic.

Hendrik Poinar, Associate Professor of Evolutionary Genetics and Canada Research Chair at the McMaster Ancient DNA Centre, who codirected the Y. pestis team, is a “molecular archeologist.” He studies “the evolutionary dynamics of infectious diseases,” deciphering “the tempo and mode of the evolution of these bugs into humans.”

The best way to do that, he said, is to focus on the past, to decode ancient pathogens and compare them to their modern counterparts to see how they have changed over time. In this way, using what Poinar calls a “time machine”—the tools and techniques of modern molecular biology—he can begin to understand how pathogens evolve, where pandemics come from, and what, if anything, can be done to stop them.

Now Poinar wanted to take a crack at cholera. But Vibrio cholerae, the pathogen that causes that disease, is a tougher nut to crack than Y. pestis, because unlike the plague bacterium, V. cholerae is found not in the blood but in the gastrointestinal tract, and thus leaves no trace in the bones.

“It’s sort of a dead end for us to study, at least genetically,” Poinar said.

Unless, of course, his team could find an intact section of preserved intestinal material from somebody who died of cholera.

A Biological Treasure, Rediscovered

Hendrik Poinar, credit: McMaster University

Enter the Mütter Museum in Philadelphia, which now held possession of Dr. Neill’s samples. In a search of the museum’s collections, the curators rediscovered the biological treasures and informed Poinar and his team.

“We were like, ‘holy cow, that’s phenomenal. Is there any way we could sample a small bit of those?’” Poinar said.

That job fell to graduate student Alison Devault, who headed down to Philadelphia. There, she and collaborator Joseph Tien, an assistant professor of mathematics at Ohio State University who models infectious disease dynamics, excised a 2-cm × 2-cm piece from a sample described only as “Intestine. Cholera. Yellow. Presented by Dr. John Neill,” and returned the bulk of the specimen to storage. The extracted nucleic acid was horribly fragmented, containing pieces that averaged just 35 base pairs or so, and chemically damaged. Still, it was enough to sequence.

“The great advantage of this particular sample … was that it was actually kept in alcohol, which is a wonderful thing, because most of the time those remains are preserved in paraffin or formalin that make it difficult for DNA to be recovered,” said Johannes Krause, Professor of Archaeogenetics and Paleogenetics at the Institute for Archaeological Sciences at Eberhard Karls Universität Tübingen.

The resulting DNA, though, wasn’t all from V. cholerae. There also, obviously, were human genomic sequences, a lot of bacteria, and even substantial amounts of bovine nucleic acid, said Devault, who speculated that the jar may originally have held a piece of cow tissue. So, she purified the specific DNA sequences she was interested in using a DNA microarray carrying oligos complementary to the complete Vibrio genome, as well as human mitochondrial DNA, and sequenced and assembled what came out. The resulting sequence was sufficient to reconstruct the V. cholerae genome to 15×, and also establish that the victim likely was a male from “sub-Saharan western Africa.”

Comparison of the bacterial sequences to known Vibrio strains from other pandemics fills in a key gap in epidemiologists’ understanding of cholera pandemics since the 1800s, Devault said.

To this point, she explained, researchers only had sequenced strains causing the sixth and seventh pandemics, which occurred in the early-to-mid twentieth century. The sixth pandemic was caused by the so-called “classical” biotype, whereas the seventh was caused by “El Tor,” which today is the predominant human cholera pathogen. Indeed, outside of laboratory strains, the classical biotype essentially has disappeared from the earth, said John Mekalanos, Professor and Chair of Microbiology and Immunology at Harvard Medical School. “You can’t go anywhere in the world today … and isolate classical Vibrio cholerae anymore,” he said.

Researchers had hypothesized that all previous cholera pandemics also were caused by the classical biotype, but they weren’t sure. Given that both the second and the sixth pandemics derive from the classical biotype, that seems more likely—though the possibility remains that the untested pandemics may have been caused by another strain, Devault admitted.

“We have clearly some sort of continuity between ancient classical and modern classical [strains],” she said. That isn’t to say that the 1849 strain directly caused later pandemics. Instead, she suspects all six pandemics essentially bubbled up from the same pool. “They’re breaking off from the same group probably.”

The results also suggested that the ancestor of both the classical and El Tor biotypes emerged in India several thousand years ago, coinciding with changes in climate and trade, and that both strains coexisted for a long time—though precise dating is complicated by the organism’s high rate of genetic recombination. “The roots of both the El Tor and the classical [biotypes] are very deep, much deeper than some estimates have been in terms of emergence of these things,” Poinar said.

The results were published in the New England Journal of Medicine in January. [1]

A Classic

Alison Devault examines a preserved cholera-infected intestine, credit: McMaster University

The study, said Mekalanos, is “a classic.” It fills in a key gap in epidemiologists’ understanding of cholera pandemics through the nineteenth century. But more to the point, he said, it suggests that a cholera vaccine could work.

The elimination of the classical biotype by El Tor in the mid-twentieth century suggested to some researchers that each cholera outbreak may have been caused by phylogenetically distinct subspecies—in other words, that the disease reemerged over and over again from harmless Vibrio species de novo. Instead, the new study implies that for 150 years, cholera pandemics were caused by reinfection by the same pathogen before it was supplanted by another more fit strain.

“It really says that you can’t just envision that the classical was a freak strain that was dominating disease for a few decades in the early 1900s or something,” Mekalanos explained. “It was a very successful human pathogen for a long, long time. The fact that it got eliminated coincidentally by another strain might just argue that if we could eliminate cholera from people, we would eliminate cholera as a threat on this planet.”

And that, he said, suggests that a cholera vaccine could be effective because repeated pandemics are not arising anew from environmental (and harmless) Vibrio strains; instead, the infectious strains are specially adapted to cause disease.

Deciphering Justinian’s Plague

Shortly after the cholera study appeared, Poinar and Devault’s team published another report in which they sequenced the plague bacteria from the devastating Plague of Justinian in the sixth century AD. Using the same techniques that were worked out in the cholera study, the researchers extracted, enriched, and sequenced DNA from the teeth of plague victims from a sixth century cemetery in Bavaria. [2]

Comparison of the resulting genome to modern and Black Death Y. pestis sequences suggests a different sort of dynamics from cholera, said Poinar. The Justinian plague killed perhaps 100 million people and then essentially disappeared from the earth. When the Black Death strain arose, it arose anew, claimed a comparable number of victims and then survived, so to speak, as the ancestor of plague strains found today.

“So you have two horrifically successful, if you will, pandemics that have two very different outcomes,” Poinar said. “One is a dead end, and the other one goes on to seed the world with Yersinia strains which are responsible for the third pandemic. And the reason we have plague in the US today is because the Black Death was so successful basically at increasing the dissemination of Yersinia pestis around the globe.”

According to Devault, both studies relied on sequence enrichment strategies. Sequencing is simply still too expensive to decipher everything in such a complex sample and then reassemble the genome bioinformatically. But enrichment, she said, is a biased approach, as it requires researchers to know or guess a priori what pathogen they’re looking for—something that is not always obvious. Plus, the current strategy ignores the possibility of coinfecting pathogens, which likely contribute to the lethality of many pandemics.

Devault is working on a microarray-based enrichment tool that can detect DNA from a wide range of bacteria in order to help researchers determine overall pathogen load in a more unbiased fashion.

“The technology is just getting to the point where it’s really feasible to look at things on this scale,” said Devault, who plans to continue with molecular archeology as a postdoc. “When I started my PhD in 2008, we had a completely different idea of how we were going to get the cholera data. We were going to do it with PCR, and we were going to look at much smaller sets of genes. But then as the technology progressed towards next-gen sequencing, and then when we did the original Black Death paper, we were like, oh, let’s do it that way.”

Given the changes Devault has seen even in her short research career, it’s hard to guess how such studies will be performed in the future. But if Poinar has his wish, museum specimens will play an ever-larger role. “These archival collections, as dead as they may be to administrators who are seeking additional room for expanding departments, are treasure troves for studying the past. A lot of universities have been forced to get rid of them, burn them, throw them out. I hope this paper will act as a savior for all those collections, which house tremendous mysteries.”

References

[1] A.M. Devault et al., “Second-pandemic strain of Vibrio cholerae from the Philadelphia Cholera Outbreak of 1849,” New England J Med, Jan. 8, 2014.

[2] D.M. Wagner et al., “Yersinia pestis and the Plague of Justinian 541–543 AD: A genomic analysis,” Lancet Infectious Disease, 2014.