Abstract
Background Trypanosoma cruzi is a protozoan parasite that causes severe disease in millions of habitants of developing countries. Currently there is no vaccine to prevent this disease and the available drugs have the consequences of side effects. Live vaccines are likely to be more effective in inducing protection than recombinant proteins or DNA vaccines; however, safety problems associated to their use have been pointed out. In recent years, increasing knowledge on the molecular genetics of Trypanosomes has allowed the identification and elimination of genes that may be necessary for parasite infectivity and survival. In this sense, targeted deletion or disruption of specific genes in the parasite genome may protect against such reversion to virulent genotypes.Methods and FindingsBy targeted gene disruption we generated monoallelic mutant parasites for the dhfr-ts gene in a T. cruzi strain that has been shown to be naturally attenuated. In comparison to T. cruzi wild type epimastigotes, impairment in growth of dhfr-ts+/− mutant parasites was observed and mutant clones displayed decreased virulence in mice. Also, a lower number of T. cruzi-specific CD8+ T cells, in comparison to those induced by wild type parasites, was detected in mice infected with mutant parasites. However, no remarkable differences in the protective effect of TCC wild type versus TCC mutant parasites were observed. Mice challenged with virulent parasites a year after the original infection with the mutant parasites still displayed a significant control over the secondary infection.ConclusionThis study indicates that it is possible to generate genetically attenuated T. cruzi parasites able to confer protection against further T. cruzi infections.
Highlights
Chagas disease is one of the major health problems in Latin and Central America, where an estimated of 7.7 million people are infected [1]
Tulahuen strains of T. cruzi were used, as well as two mutant clones derived from the Tulahuen strain carrying a targeted mutation of one dhfr-ts allele [24]
Generation of TCC dhfr-ts mutant parasites Using constructs targeted for the interruption of the dhfr-ts gene, single-allele knockout parasites for the TCC strain of T. cruzi could be achieved by electroporation with the plasmid pBSdh1f8Neo and selection in 300 mg/ml of G418, as it was previously shown for the Tulahuen strain of this parasite [24]
Summary
Chagas disease is one of the major health problems in Latin and Central America, where an estimated of 7.7 million people are infected [1] This disease is the consequence of the infection by the protozoan parasite Trypanosoma cruzi. In recent years, increasing knowledge on the molecular genetics of Trypanosomes has allowed the identification and elimination of genes that may be necessary for parasite infectivity and survival. In this sense, targeted deletion or disruption of specific genes in the parasite genome may protect against such reversion to virulent genotypes
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