The plague, of which Yersinia pestis is the bacterial pathogen, comes in two highly virulent versions bubonic and pneumonic. Bubonic plague inflames and swells the lymph nodes (buboes). It’s slow to progress, and treatable by antibiotics. The more lethal and contagious pneumonic plague, which attacks the lungs, can kill its victims within 48 hours of onset.
Some time between 1,500 and 20,000 years ago, bubonic plague gradually changed from an enteric microbe that causes mild human stomach illness due to contaminated food or water to the “Black Death,” which in the l4th century wasted one-fourth of Europe’s population. It brought with it a genetic souvenir of its one-time intestinal origins. Microbiologist B. Joseph Hinnebusch, a plague expert at the NIAID’s Rocky Mountain Laboratories in Hamilton, Mont., discovered “the gene that probably set the stage for that abrupt change of transmission route from food- and water-borne to being transmitted by a blood-feeding insect, the flea.” The wingless Oriental rat flea (Xenopsilla cheopsis) injects masses of Yersinia pestis bacteria into its flea bites.
Hinnebusch is lead author of a paper in today’s Science, dated April 26, 2002. It’s title: “Role of Yersinia murine toxin in survival of Yersinia pestis in the midgut of the flea vector.”
“This gene we report finding,” Hinnebusch told BioWorld Today, “encodes an enzyme called phospholipase D. These PLD enzymes break down phospholipids, which are very common in cellular membranes. The PLD gene resides on a Yersinia pestis plasmid, which is a small bit of circular DNA that replicates independently of the bacterial chromosome. This plasmid is unique to Y. pestis; it’s not found in its close genomic relatives. So apparently the bacterium has acquired this new plasmid, carrying the PLD gene.
“The PLD enzyme,” he continued, “has been assumed to be a virulence factor. It was known to be toxic for mice and rats, so was originally described as a toxin, and called Yersinia murine toxin Ymt. But it was completely innocuous to other animals only murine species showed toxicity. However, what we’re showing in our Science paper is that it’s really important biological role is not as a virulence factor.”
Knocked-Out Gene Gives Germ Knockout Blow
To gauge the role and importance of their new-found PLD gene in the lifestyle of Y. pestis, Hinnebusch and his team knocked the gene out in the bacterial genome. “What we did,” he recounted, “was create two defined mutations in this PLD or Ymt gene. One was a deletion mutation in which the entire gene was deleted from the Y. pestis plasmid. The second was a more specific mutation in the catalytic site of this expressed PLD enzyme. The two were kind of mutually supportive. But the deletion mutant was what we did first to show that this gene was important. And to narrow it down to its PLD enzyme function, we made the more specific mutation in that catalytic site.
“Then we fed fleas blood, containing either the wild-type bacterium or the mutant bacterium, and compared the ability of these bacteria to colonize the flea’s midgut. It turned out that the mutant strains did not survive in the flea; they just died out almost immediately within the first 24 hours. That result meant that this PLD gene is required by Y. pestis to infect its flea vector. The recent acquisition of that plasmid with this gene would have been a key evolutionary step in allowing plague to be transmitted by fleas.
“Now it’s known,” Hinnebusch observed, “that Yersinia pestis that is mutated or has lost its gene is still fully virulent. So this gene is not required for virulence in the mammal. The really striking phenotype occurs in the insect,” he went on, “and that’s what we report in this paper. That is, when we knock out this PLD gene, then Yersinia pestis is unable to colonize the gut of its flea vector. We don’t know the mechanism.”
Hinnesbusch summed up the weirdly complex colonization strategy the flea deploys. “The way plague is transmitted is that the flea takes up the bacteria in a blood meal when it feeds on an infected person. Then the bacteria multiply in the flea midgut to form large clumps of bacterial aggregates. These can actually block the flea’s foregut. When the constipated insect next tries to feed, it can’t pump blood into its stomach because this clump of bacteria is clogging its foregut. Those fleas do try to feed rather aggressively, and in that process they upchuck some of the bacteria into the bite wound. That’s basically how plague is transmitted to humans by fleas.
“It’s not unique to Y. pestis,” he pointed out. “A relatively innocuous microbe can pick up one or two genes from its neighbors, repackage itself and become much more pathogenic. This is a major way in which new diseases emerge. For instance, most E. coli are fairly innocuous, but one strain has picked up a virulent plasmid in E. coli. So now it has become a dangerous pathogen for humans.” He recalled those fast-food restaurants a decade ago, which caused an outbreak of food poisoning from raw or undercooked hamburger.
Running Down Flea’s Own Antibiotic
“But it appears that when the fleas digest their blood meals,” Hinnebusch went on, “one of the byproducts of digestion is a serum component that is toxic to the Gram-negative bacteria. That much we know. There is some antibacterial agent that is produced in the flea as the result of it just digesting its blood meal. And somehow, this PLD enzyme activity counteracts that toxicity. And that’s been enabling the bacterium to survive in the gut of the flea.
“Most organisms prefer not to be infected with bacteria. The flea is essentially deploying an antibacterial,” he observed. “The midgut of the flea does tend to be a hostile environment for bacteria. This may be a defense mechanism to keep the flea from being infected with Y. pestis that may actually harm the insect itself. That’s why it will be worth putting some effort into trying to identify that toxic antibacterial agent in the flea’s gut. We plan to find out.”