LONDON — New drugs to treat the tropical disease African trypanosomiasis — also known as sleeping sickness — could follow the latest insights into how the trypanosome parasite moves.
Researchers in Manchester, U.K., have inactivated a gene in the trypanosome whose protein product is a key component of the parasite's flagellum. They used antisense technology to switch off the gene and found they had produced mutants which were paralyzed.
Trypanosomiasis is caused by protozoan parasites of the genus Trypanosoma. In humans, the disease causes malaise, lassitude, headache, joint pains and swollen tissues initially, progressing to mental deterioration, coma and death as the parasites invade the central nervous system. Between 25,000 and 50,000 people are infected each year, in a belt of countries across sub-Saharan Africa.
The disease can be treated with drugs, but some strains of the parasite are resistant and the drugs have serious, sometimes fatal, side effects.
Trypanosomiasis is also of enormous agricultural importance. The disease, known as Nagana in cattle, kills between 3 million and 5 million cattle a year in Africa.
The trypanosome lives in the bloodstream of infected humans and cattle as an extracellular parasite. It is transmitted by the bite of the tsetse fly.
When the fly bites an infected person or animal, trypanosomes enter its gut along with the meal of blood. After a complex life cycle within the fly, the parasites migrate from the gut to the salivary gland, where they differentiate into a form capable of infecting the next host. When the fly bites, it injects anticoagulant into the wound — along with some trypanosomes.
Paralyzing Parasites May Prevent Disease
The parasites' need to travel from the fly's gut to its salivary glands may prove to be its Achilles' heel.
Philippe Bastin, research associate at the School of Biological Sciences of the University of Manchester, and colleagues have shown that the paraflagellar rod of the trypanosome's flagellum is crucial to its ability to move. They report their findings in a letter to Nature, Feb. 5, 1998, titled "Paraflagellar rod is vital for trypanosome motility."
Bastin and his coworkers, whose work is funded by the London-based Wellcome Trust, took the gene coding for a protein which is found in the paraflagellar rod and manufactured antisense DNA to match it, which they inserted into the parasite's genome.
When both the native gene and the antisense version began to manufacture RNA in preparation for protein production, the two RNAs produced — which are mirror images of each other — stuck together. As a result, protein manufacture came to a halt.
One mutant produced by this technique was apparently normal — except that it was paralyzed. While trypanosomes in laboratory culture normally swim constantly, the paralyzed mutants sedimented to the bottom of the well.
Bastin told BioWorld International, "We saw that in these mutants, the paraflagellar rod was not there any more and we knew then that we had hit the correct target with the antisense technique. Secondly, when we looked down the microscope at the trypanosomes in culture, we saw that they had lost their motility function and this was a very striking phenotype."
Parasitologists suspect that the trypanosomes' ability to move is essential to their survival in their hosts. Bastin added, "The flagellum is likely to be very important when the trypanosomes in the fly's gut have to migrate to the salivary glands — this is a long way to travel for a parasite which is only about 25 micrometers long. It has also been suggested that constant motility is important in order for the trypanosomes to evade capture by white blood cells in their mammalian hosts."
The team's next step is to check how the paralyzed mutant fares when taken in by a tsetse fly and when used to infect mice. Bastin said, "These mutants offer us a very good model to study the function of motility in the life cycle in real terms. We assume that they will fail to survive in the fly. They may be able to survive in its gut, but presumably they will be in deep trouble once they need to move to the salivary gland. If they are unable to do this, the life cycle will be interrupted and this means we should be able to stop the transmission of the disease."
The ball would then be in the pharmacologists' court. "It may be possible to produce a new drug acting against the paraflagellar rod," Bastin observed. "Neither humans nor cattle have a paraflagellar rod, so you would not expect such a drug to have serious side effects such as are produced by those currently used to treat the disease."
Commenting on the finding, Deborah Smith, reader in molecular parasitology at Imperial College of Science, Technology and Medicine, in London, said: "This report contains two findings of considerable interest. One is that Bastin and colleagues have been able to switch off the gene using antisense regulation. Others have tried to use this method to modulate gene expression in trypanosomes but they have not succeeded.
"Secondly, the phenotype of the mutant is very interesting and suggests that the paraflagellar rod may indeed provide a new target for drug therapy against this disease."
But the million-dollar question, she added, was whether loss of motility does indeed affect the viability of the parasite. "It may make them less fit to survive, but it may have no effect in terms of the virulence of the parasite. The next step is the crucial one."