Hanover, NH – Dartmouth Medical School geneticists in the Department of Microbiology and Immunology have discovered how to weaken a common human parasite to prevent disease in an animal model after infection by the normal parasite. The work, reported in the February 21 issue of Nature, opens new avenues for the development of vaccines and other treatments for diseases such as toxoplasmosis caused by a diverse, but related group of protozoan parasites.
Barbara A. Fox, a research associate, and David J. Bzik, PhD, associate professor of microbiology and immunology, found that inactivating a single enzyme in a key biochemical pathway prevented Toxoplasma gondii from causing disease.
The T. gondi parasite, most commonly spread through undercooked meat (and occasionally through cats), causes toxoplasmosis, which generally poses no problems in infected people, but can be life threatening in immunocompromised patients and cause severe birth defects in newborns from primary infections during pregnancy. T. gondii belongs to a family of parasites that include the human pathogens Cryptosporidium parvum, also a danger for immunocompromised patients, and Plasmodium falciparum, a cause of virulent malaria that kills more than 2 million children world-wide each year.
Fox and Bzik have devised a mutant T. gondii strain that causes no disease and, more importantly, provides protection against the normal parasite. "Because of the extraordinary ability of this mutant parasite to infect the animal host without apparent ill effects, this mutant could be used as a prototype vaccine strain," said Bzik.
"This parasite is amenable to further genetic manipulation; it has an amazing ability to elicit a strong immune response that is likely to be beneficial for certain vaccines targeted against other challenging infectious diseases or cancer," added Fox.
"Parasites have been around a long time and have become very proficient at stealing things from their host, if they can. This parasite seemed to rely on just one pathway for obtaining some of the key components of its genetic material. Most other organisms have retained two functional pathways: they can make components new (de novo), or they can salvage them from their nutrient environment, " Bzik explained.
The investigators determined that the parasite relied on a sole pathway. They showed that knocking out a single enzyme of the de novo pathway blocked T. gondii from making the essential pyrimidine components of parasite DNA and RNA. This in turn destroyed the parasite’s ability to replicate and survive in an animal host, neutralizing its virulence.
Pyrimidines comprise two of the four DNA and RNA units (nucleobases). "If we destroyed the ability of the parasite to make its pyrimidines de novo, we had no idea whether we could add pyrimidines to the parasite growth medium and actually grow the type of parasite mutant we were seeking. We simply had to test this experimentally," said Bzik.
Using a novel genetic approach, Bzik and Fox knocked out the first and key regulatory enzyme, carbamoyl phosphate synthetase II, in the de novo pyrimidine pathway. Inactivating "this enzyme made the parasite completely dependent on the added pyrimidine nucleobase uracil for its growth in the laboratory. These results suggest that pyrimidines are limited in the host cell, explaining why these parasites have retained the ability to synthesize these essential (genetic) precursors," said Fox.
In an animal model of toxoplasmosis, Bzik continued, "We were expecting to observe a modest difference between the virulence of the mutant compared to its highly virulent parent. Instead, the mutant did not cause any disease in a mouse model."
One dose of the parental type parasite will kill a mouse. Yet millions of the new mutant parasites inoculated into mice do no harm; all mice survive without any sign of disease. "More surprisingly, this mutant parasite was equally avirulent in mice with severe immune deficiency," said Fox, "It is the only known T. gondii parasite strain that does not cause disease in immune-deficient animals."
Building on the vaccine possibility, the researchers immunized mice with a single dose of their mutant strain. Weeks later they challenged the immunized mice with a lethal dose of a highly virulent T. gondii parasite. "The mice were completely protected from a lethal challenge infection. "These mutants may offer great potential as a strategy for vaccine development," Fox said.
Touting the benefits of applying genetics to human pathogens, Bzik said, "the parasite carbamoyl phosphate synthetase II enzyme is an attractive target for the design of drugs to treat infections from this family of parasites. The pyrimidine pathway may ultimately prove to be a weakness of protozoan parasites."
Materials provided by Dartmouth Medical School. Note: Content may be edited for style and length.
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