Scientists reveal new insights into the plague pathogen

Among other skeletons found during an excavation in a cemetery in Sejet, Denmark, provided genetic samples for new plague research from Kiel University. Credit: Unit for Anthropology, ADBOU, SDU

A group of researchers from Kiel University and MPI-EB have identified genetic factors as the pathogen Yersinia pestis acquired during its recent development. This discovery improves our understanding of the emergence of the modern plague pandemic in the 19th century.

The origins of the plague can be traced back to the Neolithic, with the earliest evidence of the causative bacterium Yersinia pestis found in human bones that are approximately 5000 years old. Two notable instances in the history of plague include the late antique Justinian plague of the sixth century and the infamous Black Death that struck in the late Middle Ages.

Both of these devastating outbreaks were demonstrably caused by Y. pestis and, according to estimates, it resulted in the death of up to half the population in certain regions of Europe. While there were several smaller, regionally confined outbreaks across different continents over the centuries, a third significant plague pandemic occurred from the mid-19th to early 20th centuries.

At first it mainly affected Asia, with a focus on India, and subsequently spread globally. With around 15 million confirmed deaths, it is one of the deadliest pandemics in human history. Plague continues to occur regionally today and is almost always fatal if not treated quickly with antibiotics.

Dr. Joanna Bonczarowska and her colleagues discovered that a genetic element newly acquired before the modern pandemic is associated with the virulence of Y. pestis. Credit: Małgorzata Znarowska

Through thousands of years, Y. pestis have evolved into numerous strains, via both acquisition and loss of genes. Researchers worldwide are studying the development of Y. pestis to find out more about the causes of historical pandemics and the dangers that the plague continues to pose. In particular, they investigate the pathogen’s genetic characteristics, which are responsible for transmission, geographical distribution and disease severity, among other things.

In a new study, a research team from Kiel University and the Max Planck Institute for Evolutionary Biology in Plön (MPI-EB) investigated ancient and modern Y. pestis genomes ranging from the Neolithic to the modern pandemic. The researchers led by Dr. Daniel Unterweger, research group leader at MPI-EB and Kiel University, and professors Almut Nebel and Ben Krause-Kyora from the Institute of Clinical Molecular Biology (IKMB) at Kiel University found that Y. pestis must have acquired a new genetic element, known as the YpfΦ prophage, between the Middle Ages and the modern pandemic, which is related to the pathogen’s virulence, i.e. its disease-causing effect.

The prophage produces a protein that strongly resembles certain toxins from other pathogens, for example the cholera pathogen. The researchers, who among other things contribute to the Kiel Evolution Center (KEC) at Kiel University, have recently published their results together with colleagues from the University of Southern Denmark in Odense (SDU) in the journal Proceedings of the Royal Society B: Biological Sciences.

New genetic elements increased the virulence of the pathogen

The research team from Kiel has obtained the genetic samples thanks to a collaboration with the Department of Forensic Medicine at SDU, which administers skeletal material from various Danish museums. In this specific case, the researchers examined the skeletal remains of 42 people who were buried in two Danish parish cemeteries between the 11th and 16th centuries. The genetic information contained in the samples was sequenced and the Y. pestis genes contained therein were compared to other published genomes dating to the Neolithic, medieval and modern periods.

“Previous research has shown that, early in its development, the pathogen lacked the genetic makeup required for efficient transmission via the flea, which is typical of today’s bubonic plague. During its development, Y. pestis acquired a remarkable level of virulence, which contributed to the later outbreaks of some of the deadliest pandemics in human history,” says Dr. Joanna Bonczarowska, first author of the paper, who carried out this research as part of her PhD at IKMB with support from the International Max-Planck-Research School for Evolutionary Biology (IMPRS).

In the aDNA laboratory at Kiel University, so-called DNA libraries are prepared for sequencing. Credit: Jan Steffen, Kiel University

“In our study, we show that everyone knew Y. pestis strains before the 19th century lacked a certain genetic element, the YpfΦ prophage”, says Bonczarowska, who now works as a postdoctoral researcher at the IKMB, where she is funded by the Cluster of Excellence “Precision Medicine in Chronic Inflammation” (PMI). The prophage was probably taken up from the environment through lateral gene transfer. This genetic information affects the pathogen’s virulence, i.e. the severity of the disease resulting from an infection. Y. pestis strains that have the prophage were shown to require a significantly lower lethal dose compared to those without YpfΦ. This inclusion of new genetic elements could thus provide an advantage for Y. pestis during the modern plague pandemic.’

How did the increased virulence since the Middle Ages arise?

The mechanisms by which the prophage contributes to the increased virulence of the modern plague pathogen have not yet been investigated in detail. Previous studies suggest that such new genetic information may help the pathogen to infect body tissues far away from the original site of infection. In their search for such a mechanism, the Kiel researchers examined all proteins encoded by the new DNA is about. They discovered that one of these proteins is very similar to a toxin known from other pathogens.

A tooth is drilled in the laboratory to obtain powder for DNA extraction. Credit: Jan Steffen, Kiel University

“This protein is similar in structure to zonula occludens toxin (ZOT), which facilitates the exchange of harmful substances between infected cells and has a harmful effect on the mucosa and epithelium. This connection was first discovered in the cholera pathogen, where it causes the typical gastroenteritis- symptoms”, explains Bonczarowska. The Kiel researchers therefore want to investigate this ZOT-like protein in Y. pestis more closely in the future, as it provides a plausible explanation for the increased virulence of the plague pathogen in the present and the recent past.

Further research into the evolution of the plague and other pathogens

Such a rapid development of Y. pestis contributing to the pandemic threat it continues to pose. “Acquisition of new genetic elements can lead to new symptoms of infection. These misleading disease signs can make it difficult to diagnose plague in time and thus delay rapid treatment, which is crucial for survival,” emphasizes Unterweger. “In addition, some strains of the plague pathogen already show resistance to various antibiotics, which further contributes to the great potential danger of this disease,” continues Unterweger.

An important aspect of the work is also the newly discovered parallels to other bacteria species, as genetic elements very similar to YpfΦ, were also found in other bacteria. These results provide clues to their future evolution towards increased virulence.

Overall, the research results emphasize that there is much knowledge to be gained for modern science and medical application in the study of historical disease development using aDNA, which goes back hundreds or even thousands of years. “Understanding how the pathogen was able to increase its virulence in the past, sometimes by evolution, will help us detect new forms of the disease and prevent new pandemics in the future,” sums up Krause-Kyora.

Reference: “Old Yersinia pestis genomes lack the virulence-associated YpfΦ prophage present in modern pandemic strains” by Joanna H. Bonczarowska, Julian Susat, Ben Krause-Kyora, Dorthe Dangvard Pedersen, Jesper Boldsen, Lars Agersnap Larsen, Lone Seeberg, Almut Nebel and Daniel Unterweger, July 19, 2023, Proceedings of the Royal Society B: Biological Sciences.
DOI: 10.1098/rspb.2023.0622