Knockout of a putative Enoyl-CoA Hydratase gene in Trypanosoma cruzi generates attenuated parasites that induce CD8+ memory T cells in infected mice and confer protection against virulent parasite challenge

DAN XU, CECILIA PÉREZ BRANDÁN, RICK TARLETON

Athens, Georgia, USA

Simposio; 18th Annual Molecular Parasitology/Vector Biology Symposium; 2008

Trypanosoma cruzi ech1 and ech2 genes encode putative enoyl-CoA hydratase/isomerase family proteins. Transcriptome analysis and quantitative RT-PCR data of T. cruzi revealed that whereas the transcript for ech2 was upregulated in the amastigote stage, ech1 was detected to be most abundant in the epimastigote stage. Based on Multisite Gateway system, we generated knockout constructs for replacing ech genes through homologous recombination. We obtained parasites with 1 copy of ech1 gene and both copies of the ech2 gene deleted. Epimastigote forms of these parasites grew slightly slower than WT parasites and after converting to metacyclic trypomastigotes, they did not infect Vero cells as well as WT parasites. IFN-g-/- mice infected with the knockout parasites showed a significantly lower number of parasites circulating in peripheral blood. Acute infection of C57/BL6 mice with these knockout parasites induced a specific CD8+ T cell response to the same extent as WT parasites, as demonstrated by staining with MHC class I tetramers containing the T. cruzi specific peptide TSKB20. In the chronic phase, more than 50% of the T. cruzi specific CD8+ T cells examined expressed the central memory marker CD127hi, which is drastically higher than those in WT parasites infected mice. After immunosuppression, no circulating parasites in peripheral blood were observed in most of the mice infected with knockout parasites, whereas a high number of parasites was found in WT parasites infected mice, some of which even succumbed to death. Furthermore, vaccination of mice with these knockout parasites confered similar partial protection as vaccination with WT parasites against a challenge with virulent parasites. This study suggests that knocking out certain genes in T. cruzi may generate attenuated parasites that could be used as an effective experimental vaccine for Chagas disease.