Intracellular growth and metacyclogenesis defects in Trypanosoma cruzi carrying a targeted deletion of a Tc52 protein-encoding allele

We have identified previously a Trypanosoma cruzi gene encoding a protein n amed Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family involved in thiol-disulphide redox reactio ns. Furthermore, we have reported that Tc52 also played a role in T. cruzi- associated immunosuppression observed during Chagas' disease, In an effort to understand further the biological role of Tc52, we used a gene-targeted deletion strategy to create T. cruzi mutants, Although T, cruzi tolerates d eletion of one wild-type Tc52 allele, deletion of both genes is a lethal ev ent, indicating that at least one active Tc52 gene is required for parasite survival. Monoallelic disruption of Tc52(Tc52(+/-)) resulted in the produc tion of T, cruzi lines that express less Tc52 mRNA and produced lower amoun ts of Tc52 protein compared with wild-type cells, In axenic cultures, growt h rates of epimastigote forms bearing an interrupted allele were not differ ent from those of wild-type parasites. Furthermore, monoallelic disruption of the Tc52 gene did not modify the growth rate of epimastigotes or their s ensitivity to inhibition by benznidazole and nifurtimox, the two drugs used to treat Chagasic patients, Moreover, the antimonial drug SbIII, which is known, at least in Leishmania parasites, to be conjugated to a thiol and ex truded by an ATP-coupled pump, had a similar effect on wild-type and mutant parasites, being equally sensitive, Hence, parasite drug sensitivity was a lso observed in clones overexpressing the Tc52 protein as well as in those carrying an antisense plasmid construct, Surprisingly, a significant impair ment of the ability of epimastigotes carrying a Tc52 single gene replacemen t or antisense construct to differentiate into metacyclic trypomastigotes a nd to proliferate in vitro and in vivo was observed, whereas no significant enhancement of these biological properties was seen in the case of parasit es that overexpress Tc52 protein, Moreover, functional complementation of T c52(+/-) single mutant or selection of antisense revertant clones demonstra ted that the phenotype observed is a direct consequence of Tc52 gene manipu lation. Taken together, these results may suggest that Tc52 could participa te among other factors in the phenotypic expression of T. cruzi virulence.