By David N. Leff

A serial killer that knocks off its victims slowly rather than quickly is doing itself a favor.

This multiple murderer is the globe-girdling, tropical parasite named Leishmania. The microscopic pathogen infects more than 10 million people in tropical countries.

Just as the Anopheles mosquito ferries malarial parasites to their victims' defenseless skin, so Leishmania rides to the scenes of its crimes in a similar insect partner, the sand fly. The Leishmania parasite's smoking gun chambers two bullets: One, L. major, causes severe, disfiguring skin lesions. The other, L. donovani, strikes inward from the skin bite to infect the body's visceral organs with death.

In a court of law, Leishmania's defense counsel would counter this prosecutorial indictment with a plea that the parasite often spares its victim's life by deploying low rather than high virulence. Here is how molecular microbiologist Stephen Beverley justifies this allegedly altruistic defense:

"The connection that we made - a hypothesis at this point, but a very attractive one - is that in fact the pathogens don't always want to be fully virulent. If they are too deadly, they kill their hosts very rapidly. That means they don't have a chance to be transmitted onto a fresh host.

"So ultimately, from the evolutionary microbiology point of view, a good pathogen doesn't kill its host too fast, or hurt it too bad."

Beverley is senior author of a research paper in today's Science, dated April 13, 2001. It's titled: "Regulation of differentiation to the infective stage of the protozoan parasite Leishmania major by tetrahydrobiopterin." He holds an endowed chair of Molecular Microbiology at Washington University School of Medicine, in St. Louis.

Evolution Slyly Softens Pathogen's Vindictiveness

"We reported two critical findings," Beverley told BioWorld Today. "One is that we identified the metabolic pathway controlling the ability of the Leishmania parasite to differentiate into the infective form that's carried by the sand flies. The other thing we found is that the parasite's gene encoding pteridine reductase, (PTR1), which is involved in this pathway, actually acts to limit the parasite's virulence, rather than to increase it. From an evolutionary point of view, that's quite interesting. Usually, when you find those genes and inactivate them, the pathogen becomes less virulent.

"In our case, we got not that result but the opposite outcome, where the parasites became hypervirulent - more deadly. That's unusual, because why would the pathogen have such a gene? We're used to thinking about genes that promote virulence, not hypervirulence."

Beverley compares Leishmania's life cycle to the metamorphosis of a caterpillar into a butterfly - but in spades.

"The parasite's transmogrification," he began, "starts with the sand fly. The fly bites an already-infected person, and picks up some parasites. These grow in the gut of the insect, in a replicating form called the promastigote. These come in with the blood meal that the fly picks up. At the same time the fly is digesting the blood meal and growing on it, the parasite is, too.

"Eventually," Beverley narrated, "that food is consumed, so the fly then gets hungry and buzzes off to bite somebody else. The parasite also senses that the meal is gone, and therefore stops proliferating. Instead, it differentiates into its metacyclic form. The replicating promastigote form is not very infective in an animal or person. But, once the pathogen stops dividing, it differentiates to this metacyclic stage, which is highly virulent.

"At this point," Beverley continued, "we have these metacyclical parasites in a hungry fly, which starts to bite. It not only takes on a blood meal, but it deposits some parasites into the host. The pathogens are able to resist the mammalian host - notably, the cell-killing complement system. Instead, the parasites are ingested by phagocytic cells - typically, macrophages.

"Once they get inside a macrophage," he went on, "they differentiate into another stage, the amastigote. It lives very happily in the macrophage's debris-scavenging phagolysosome. There the amastigotes sit inside the macrophage, replicating, and in the meantime inducing a lot of immune responses. These don't do a very good job of killing the parasite, but cause many unfortunate consequences to the host, which ultimately lead to the Leishmania disease. Eventually, the infected host will get bitten again, by a second sand fly. The ones the fly picks up this time are in the amastigote stage. The macrophage then bursts open, because it's not very happy in the gut of a sand fly. It releases the amastigote, which differentiates back to the promastigote stage - and completes the parasite's life cycle."

To reach their conclusions, Beverley and his co-authors conducted experiments on mouse models of Leishmania major infection. "At this point," he observed, "we were studying the human cutaneous disease caused by L. major. The mouse model is also pretty good for measuring the human visceral disease.

"We took parasites grown in culture," he recounted, "and inoculated them into the footpads of the mice. The parasites went on causing an infection, and we could monitor that very conveniently, because as the infection grew, we got infiltration of cells, and swelling of the lesion. The size of some footpads quadrupled, infected with hundreds of millions of parasites. We could monitor it by measuring the size of the foot."

The co-authors knocked out the pathogen's gene for pteridine reductase, which converts biopterin to tetrahydrobiopterin (H₄B) to see if the parasite survived. In fact, it did more than survive; it became more infectious. After about two weeks, mice injected with the mutant microbe had more than 50 times as many parasites in their bodies as mice injected with normal Leishmania.

Results showed that low H₄B caused high metacyclogenesis. Mutants lacking the PTR1 gene had low H₄B levels, remained infectious to mice, and induced hypervirulence - evidenced by large skin ulcerations. "Thus," the Science paper summed up, "the control of pteridine metabolism has relevance to the mechanism of Leishmania differentiation, and the limitation of virulence during evolution."

Beverley concluded: "This is the first pathway we have identified that controls the virulence of this deadly pathogen. Remarkably, it normally acts to limit parasitic virulence rather than increase it." n