Trypanosoma cruzi: Attenuation of virulence and protective immunogenicity after monoallelic disruption of the cub gene

BARRIO, A. B., VAN VOORHIS, W. C. AND BASOMBRIO, M.A.

EXPERIMENTAL PARASITOLOGY

Año: 2007 vol. 117 p. 382 - 389

0014-4894

Calmodulin–ubiquitin (cub) is a single-copy gene of Trypanosoma cruzi, which encodes a 208 aminoacid polypeptide of unknowncub) is a single-copy gene of Trypanosoma cruzi, which encodes a 208 aminoacid polypeptide of unknown 11 function, containing putative calcium-binding domains. After targeted deletion, a clone (TulCub8) was derived where one of the twofunction, containing putative calcium-binding domains. After targeted deletion, a clone (TulCub8) was derived where one of the two 12 alleles was disrupted. This clone displayed a sharp and stable loss of virulence for mice. Parasitemias after inoculation of 106 trypom-alleles was disrupted. This clone displayed a sharp and stable loss of virulence for mice. Parasitemias after inoculation of 106 trypom- 13 astigotes of the mutant, as compared to wild-type parasites were 68-fold lower (p = 0.018) in adult Swiss mice and 27-fold lowerastigotes of the mutant, as compared to wild-type parasites were 68-fold lower (p = 0.018) in adult Swiss mice and 27-fold lower 14 (p = 0.002) in newborn Balb/c mice. Epimastigote inocula of the mutant were strongly protective against infection by wild-type parasites.(p = 0.002) in newborn Balb/c mice. Epimastigote inocula of the mutant were strongly protective against infection by wild-type parasites. 15 Virulence was not restored by serial passage in mice, showing that the attenuated phenotype is stable and gene-conversion from the intactVirulence was not restored by serial passage in mice, showing that the attenuated phenotype is stable and gene-conversion from the intact 16 cub allele does not occur at an appreciable rate. Retransfection of the missing cub allele restored virulence. Complementation experi-cub allele does not occur at an appreciable rate. Retransfection of the missing cub allele restored virulence. Complementation experi- 17 ments showed that the intact cub gene is necessary for full expression of virulence.ments showed that the intact cub gene is necessary for full expression of virulence.