Plague Genome: The Evolution Of a Pathogen

Plague has earned a place in history books as the Black Death of medieval Europe, and in novels, from Albert Camus' classic The Plague, to the more recent Year of Wonders.1,2 A different medium for telling the tale of the plague bacterium Yersinia pestis is its genome, recently sequenced by researchers at the Sanger Centre, the London School of Hygiene and Tropical Medicine, the Royal London School of Medicine and Dentistry, and the Imperial College of Science, Technology and Medicine.3 In addition to identifying 4,012 genes, comprising 84 percent of the sequence, the work reveals an unusually dynamic genome, with genes added, moved, and silenced into pseudogenehood. "This flexibility in its genome has made Yersinia pestis a finely tuned pathogen that is adaptable to new routes of transmission to humans. This is like watching evolution in action," says Brendan Wren, professor of microbial pathogenesis at the London School of Hygiene and Tropical Medicine. Clues in the genome support the hypothesis that Y. pestis evolved, 1,500 to 20,000 years ago, from the less fearsome human enteric pathogen Y. pseudotuberculosis. In 1999, researchers from the Max Planck Institute for Molecular Genetics and the Pasteur Institute compared six genes among 36 strains of Y. pestis and 13 of Y. pseudotuberculosis. The near genetic identity of the two types of bacteria suggested that the plague microbe veered onto its own evolutionary pathway.4 Ironically, University of Chicago professor of history William H. McNeill guessed the relationship in 1976, but got it backwards, suggesting that Y. pseudotuberculosis is a "mutant form of" Y. pestis.5 Plague Pathology Plague in humans takes two forms, representing two transmission routes. The more common bubonic plague passes from fleas to humans, usually via black rats. Today the disease remains endemic in squirrels and chipmunks and other small mammals in many parts of the world, rarely affecting humans. Jack Dixon, the Minor J. Coon professor of biological chemistry at the University of Michigan in Ann Arbor, explains how that can happen: "When infected rats and squirrels get sick and die, their fleas seek warm bodies for blood, which they digest. A flea bites an infected rat and takes in the bacillus, which multiplies so effectively that it obstructs the flea gut. The flea starves, going into a feeding frenzy and looking for another source. It bites a person or some other animal." A single fleabite delivers about 24,000 bacteria. Once in a human body, the bacteria head for the lymph nodes, where they proliferate. After an incubation period of two to six days, fever and headache begin, and then, about eight hours later appear the intensely painful swellings near the lymph nodes, called buboes, that gave the condition its name. The person goes into shock as the bacteria release a toxin that causes circulatory collapse. Widespread organ failure leads to death within days. On a molecular level, the bacteria cut off the immune response by injecting proteins into macrophages with a syringe of sorts. "Some of the proteins block macrophages from engulfing, and other proteins cut the lines of communication between the macrophages and the T and B cells. Bacterial proteins also block the release of tumor necrosis factor and other cytokines, stopping the immune response to amplification of the bacteria," Dixon says. His work focuses on YopJ, a type of "Yersinia outer protein" that disables macrophages.6 The Yops are vital for pathogenesis-mutants cannot infect. Pneumonic plague, the second guise of Yersinia pestis, is a quicker killer. If a person with bubonic plague survives long enough, the bacteria flood the bloodstream and end up in the lungs. "Then people aspirate them out into the air, as small droplets of sputum and blood. Lethality by pneumonic plague is much greater than by a flea bite," says Dixon. And it spreads fast. No longer must the bacteria arrive via rat and flea-a sneeze will do the trick. A person can die from the high fever, chills, and severe pneumonia of pneumonic plague within a day. Antibiotics can treat plague, and vaccines exist but offer temporary protection. Y. pestis survives in rodent colonies, jumping to human populations in epidemic proportions only when a confluence of circumstances, such as crowding and poor sanitation, force rats into contact with people. Today only about 18 cases occur in the United States per year, according to the Centers for Disease Control, usually in rural areas where rats wander indoors. Worldwide, 1,000 to 2,000 cases are reported each year. Genes Added, Lost, and Moved The genome sequence of Y. pestis reveals three types of changes, when compared to forebear Y. pseudotuberculosis. A key change is the acquisition of genes, many on two of its three plasmids, that enabled the immediate ancestor of the plague bacterium to hitch rides on fleas and rats. "Scrutiny of the genome sequence allowed us to identify 23 regions where the organism has gained key genes that allowed it to infect fleas and rodents," says Wren. A gene very similar to one from baculovirus, for example, enables the bacterium to penetrate the insect gut lining. The fact that the gene is sandwiched between transposons suggests that it "jumped" into the genome, a relic of horizontal gene transfer. Y. pestis has also lost genes, mutating into silence some 149 genes that provided forebear pseudotuberculosis its preference for the human gut. Of these pseudogenes, 58 have undergone frameshift mutations, 32 deletions, and the rest have nonsense mutations-all changes that prevent the genes from being expressed. "The loss of its enteric lifestyle is reflected in the vestigial 149 pseudogenes that Y. pestis has lost by natural selection. These are the genes I am most interested in, as they give us an Aladdin's cave of riches in terms of genes potentially involved in gastroenteritis," Wren says. The third type of change reflects a generalized instability of the Y. pestis genome, revealed in a skewing of the location and directionality of GC repeats compared to other microbial genomes. The fact that some of the repeats are turned around indicates movement of pieces of the genome. "The significance of the anomalies is that they indicate recent disruption of the pattern, by inversion, insertion, or translocation of tracts of DNA," says Wren. Adds Julian Parkhill, principal investigator of the Sanger Centre group, "The most surprising aspect was the large and rapid chromosomal rearrangements, with hundreds of kilobases of DNA inverting. This has not been seen on this scale in any other sequenced organism. In terms of the acquisition and loss of genes, this is a common theme that runs through much of prokaryote biology." Wren agrees that the biggest surprise was the fluidity of the genome, and he adds that the genome information can have some practical implications. "This basic information may be useful as a model for infectious disease outbreaks, and may be useful in terms of disease surveillance and, in the longer term, for predicting outbreaks of disease." Ricki Lewis (rickilewis@nasw.org) is a contributing editor for The Scientist. References 1. A. Camus, The Plague, New York: Random House, 1948. 2, G. Brooks, Year of Wonders, New York: Viking Penguin, 2001. 3. J. Parkhill et al., "Genome sequence of Yersinia pestis, the causative agent of plague," Nature, 413: 523-7, Oct. 4, 2001. 4. M. Achtman et al., "Yersinia pestis is a recently emerged clone of Y. pseudotuberculosis." Proceedings of the National Academy of Sciences, 96:14043-8, 1999. 5. W.H. McNeill, Plagues and Peoples, New York: Anchor Books, 1976. 6. K. Orth et al., "Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like protein protease," Science, 290:1594-9, 2000. Plague: An Ancient Scourge and Bioweapon Three plagues of plague killed more than 200 million people in the Justinian pandemic from the 6th through 8th centuries, the Black Death that peaked from 1347-1350 in Europe and Eurasia, and the modern pandemic, which began in South China in 1894. Historians think that pockets of plague existed in underground rodent colonies through Asia, and were usually contained when the disease would occasionally spring up among humans because the people had learned the value of quarantine. The disease jumped to epidemic status when Mongol invaders spread the contagion from the Himalayan foothills across the grasslands of Eurasia in the 12th and 13th centuries. The official start of the Black Death was 1346, when the army of a Mongol prince in Crimea fell ill, and, because of their travels, spread the disease all over. The bubonic form went pneumonic, and a third of the population of Europe ultimately perished. The 1894 near-pandemic is attributed to replacement of sailing ships with steamships. The sailing ships took so long to make their journeys that epidemics on board would burn themselves out by the time the destination was reached. But the steamships were larger and could sustain a larger rat population that survived the shorter journey to the New World. Flea-infected rats were thus delivered from Canton and Hong Kong at the turn of the 20th century, but quarantine measures eventually brought this plague under control. The first record of plague being spread intentionally dates from a 14th century Tartan army catapulting plague-ridden bodies over the city walls of Kaffa in the Crimea. After World War II, the Soviets discovered evidence of plague as a bioweapon in a Japanese facility in Manchuria. The Japanese at first had tried to drop bacterial bombs, but they found that the microbes did not survive the explosions. So instead, they developed porcelain containers that harbored fleas, dropping billions of the infected insects over rural China, killing thousands of people. The Japanese also reportedly infected U.S. and British prisoners of war. So impressed were the Soviets with the Japanese facility that they used it as a model for their own bioweapon program, called Biopreparat.1 Thanks to its history, plague was a prime candidate. "The weaponized, pneumonic form would spread from person to person particularly effectively. It would be undetected for a short time, then spread. Most weaponized kinds need to be spread by aerosols," says Jack Dixon, the Minor J. Coon professor of biological chemistry at the University of Michigan in Ann Arbor. As if pneumonic plague is not horrific enough, the Soviets genetically modified the bacterium to produce a myelin toxin that caused paralysis, according to Ken Alibek,2 who was part of the program but defected to the United States. The Soviet Union collapsed before this new deadlier plague could be pursued. In this new age of bioterrorism, the ability to engineer ever-more-lethal pathogens leads to the question of whether publishing genome sequence information can be subverted to create more virulent bioweapons. "This is a touchy and difficult question. Society has paid a price for opening Pandora's box, but in the long term, the open exchange of scientific information for the public good far outweighs prices we've had to pay. If we do the alternative, close everything down, many more lives will be lost to the lack of advances in biomedical research," says Bruno Sobral, director of the Virginia Bioinformatics Institute in Blacksburg. -Ricki Lewis References 1. Ken Alibek. Biohazard, New York: Delta Trade Paperbacks, 1999. 2. T. Hollon, "Ken Alibek: For the biodefense," The Scientist, 14[8]:18, April 17, 2000. Supplemental Materials Circular representation of the Y. pestis genome

Plague Genome: The Evolution Of a Pathogen

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