Trypanosoma cruzi: use of herpes simplex virus-thymidine kinase as a negative selectable marker

Trypanosoma cruzi, the protozoan that causes Chagas' disease, was transfected with a fusion gene of hygromycin phosphotransferase and herpes simplex virus-thymidine kinase, HyTK. Transfectants selected in hygromycin had thymidine kinase activity, whereas controls did not. In vitro growth of the mammalian life-stage forms, amastigotes and trypomastigotes, was inhibited 98% by the nucleoside analogue ganciclovir (5 micrograms/ml). Growth of the insect-stage form, epimastigotes, was not inhibited by ganciclovir (up to 250 micrograms/ml) or other nucleoside analogues. Intracellular uptake of ganciclovir by epimastigotes was found to be 10-fold less than that by amastigotes. Mice infected with the HyTK-expressing parasites and treated with ganciclovir had a statistically significant reduction of parasitemia by 57%; however, complete eradication of parasites was not achieved. The parasites recovered from the treated mice continued to be susceptible to ganciclovir in vitro. Parasite clones with higher expression of thymidine kinase were more sensitive to ganciclovir, suggesting that greater expression of the thymidine kinase gene may lead to parasites that can be fully eradicated from infected experimental animals.