By David N. Leff

For some years now, sleeping sickness has again been ravaging sub-Saharan Africa with a major epidemic outbreak.

"Through Sudan, Uganda and especially Zaire," observed Yale University epidemiologist Serap Aksoy, "it's estimated to be as bad, as powerful, as the AIDS epidemic."

Two co-conspiratorial organisms team up to spread this death-dealing scourge. One is a brownish, half-inch blood-sucking insect, the tsetse fly (Glossina morsitans morsitans). It delivers to its victims, both man and beast, a microscopic protozoan parasite, Trypanosoma brucei rhodiense, which can kill its human target within one agonizing year.

"The parasite begins to replicate in the blood," Aksoy recounted, "and goes to parasitemia [setting up housekeeping in the circulation]. The immune system of the host can clear its first peak [of replication], but because T. rhodiense is able to switch its surface coat antigens — probably the most fascinating antigenic variation that we know of — a second peak begins to come up in the blood, and the host can eventually clear that peak as well. But the parasite can code for practically an endless number of these coat genes, so eventually the host just dies.

"Especially," she said, "when the parasite crosses the blood-brain barriers, then it gets into the final stage of the disease. That's why it's called the sleeping sickness, where you see people comatose."

The tsetse fly holds the smoking gun that shoots T. rhodiense into its victim's bloodstream in the first place. Among biting insects, it's known as a pool feeder, a term explained by Yale parasitologist Michael Cappello.

"Pool feeders," he said, "are the biting insects that actually create a hematoma [pool of blood]. The way the tsetse feeds, it has a long-nosed, horny proboscis with a very serrated edge So, it will break the skin repeatedly over a small area, sawing in and out. The serrated blade will lacerate lots of tissue and blood vessels. As it does this, the fly is actually injecting parasites in its saliva, which contains a thrombin inhibitor and a potent blocker of platelet aggregation, apyrase. Those two molecules probably work in conjunction to inhibit the blood's clot-forming response locally.

"So you get bleeding into a confined area, and blood pools underneath the skin," Cappello said. "Then, the fly sucks that blood. Tsetse flies feed fairly quickly; they get in and get out in a matter of seconds, often before the host can detect that they're there."

Aksoy and Cappello are respectively senior author and lead author of a paper in the current Proceedings of the National Academy of Sciences (PNAS), published Nov. 24, 1998. It bears the title, "Tsetse thrombin inhibitor: Bloodmeal-induced expression of an anticoagulant in salivary glands and gut tissue of Glossina morsitans morsitans."

"It presents the unique aspect of our work," Aksoy told BioWorld Today. "People always thought that these anticoagulant molecules were expressed only in the fly's salivary glands, to keep the blood uncoagulated while feeding. But we have shown that it's also expressed in the gut, so that, in addition to this anticoagulation, it may have a role in the insect's digestion as well. Whether this affects the transmission of the parasite, by keeping the blood bolus-free, is a question we're addressing."

Long-Held View Short-Changed Anticoagulant's Uses

Cappello added an historical footnote to this general assumption that the tsetse secretes anticoagulants in its saliva in order to facilitate taking a blood meal from their mammalian host: "It's very interesting to know," he told BioWorld Today, "that some observations in the early part of this century reported in fact that tsetses with their salivary glands dissected out could still feed quite well. But they died within a day or so, with clots found throughout their alimentary canal." This neglected finding suggested to Cappello that these anticoagulants "may have less to do with the taking of a blood meal than what to do with managing the ingested blood."

The co-authors have named their tsetse thrombin inhibitor TTI, for short. In fact, it's a very short molecule, only 32 amino acids long. "It appears to represent," their paper reports, "a unique class of naturally occurring protease inhibitors" of unprecedented clot-busting potency.

Cappello recalled how the team identified and isolated the molecule:

"First, I purified the protein from approximately 1,000 salivary glands, dissected from Yale University's tsetse fly colony," he said. "Salivary glands are actually large relative to size of fly. The paired set of glands run the length of the insect. Then I made soluble extracts from the glands, and was able to get a substantial amount of protein sequence. At that point, Serap Aksoy's lab used that protein sequence to design PCR primers. So she was able to pull out the TTI sequence from a salivary-gland-specific cDNA library.

"When we looked at the expression of TTI following a blood meal," Cappello said, "what we found was that TTI was expressed not just in the salivary glands but also in the gut of the fly. In fact, it was up-regulated there in response to a blood meal, which really, I think, speaks to my initial point about the biological role of this protein. Meaning it would suggest that a primary function of TTI is to keep the blood meal liquid once it's in the fly's gut. To characterize the recombinant protein, we expressed it in vivo in E coli, and then we had the molecule chemically synthesized, and tested both versions for anticoagulant activity."

Disappointingly, neither lab-made TTI was as active a clot-buster as the native tsetse anticoagulant.

Cappello suspects this "has to do with post-translational modifications that can only occur in a eukaryotic system. That is, expressing eukaryotic genes in E. coli, which is a prokaryote, may be the problem."

The co-authors have now expressed their cDNA TTI in a eukaryotic baculovirus system, which utilizes insect vectors. "Although we haven't yet quantified the material, our preliminary data suggest that TTI expressed in a baculovirus system is comparable in activity to the native protein."

Cappello said Yale has filed a patent application to cover the thrombin inhibitor, "and I hope [the university] is actively seeking industrial partners who might be interested in developing it further."

Aksoy concluded: "We've been characterizing the TTI from different species of fly, to see if we can find the active domain, the binding site for example. We may even chop the molecule smaller, eventually." *