Taming the Trypansoma cruzi pathogen by genetic manipulation.

BARRIO A.B., ZAGO M. PAOLA, CIACCIO M., PEREZ BRANDÁN C.AND BASOMBRÍO M.A.

Ashburn, Virginia USA

Simposio; Meeting of HHMI International Research Scholars; 2006

With the complete sequencing of the Trypanosoma cruzi genome, we now know that the organism is diploid, with an average difference of 2.2% between allelic protein sequences, most divergence occurs at the intergenic regions, and the number of genes is close to 12,000, with an annotated dataset of 60.4 Mb per haploid genome. The list of singleand multiple-copy genes is growing. Recently, we attempted to attenuate the virulence of T. cruzi by genetic manipulation. We designed plasmids for targeted deletion by homologous recombination and retransfection of missing genes. We identified genetic rearrangements by PCR, Southern blot, or restriction analysis. By testing the modified parasites in mouse models, we were able to demonstrate the effect of gene rearrangements upon virulence and immunogenicity. A case in point is the calmodulin-ubiquitin (cub) gene. A single copy is placed between a long tandem array of calmodulin and ubiquitin genes. The cub gene encodes 208 amino acids of unknown function, with calcium-binding domains, but is essential for parasite survival. After elimination of one cub copy, a mutant, named TulCub8, was selected in which infectivity was profoundly altered as compared with the wild type, producing no infections or very low parasite loads in highly susceptible mice. This alteration was stable after serial mouse propagation. A cub retransfection vector restored the infectivity of the mutant. TulCub8 behaved as an experimentally attenuated vaccine. A single inoculum of 105 epimastigotes prevented further virulent T. cruzi infection. Thus, studies on cub show that one copy of a gene can be deleted, causing loss of virulence, and then restored, causing recovery of virulence. To make trypanosomatids attenuated by single or combined gene deletions, we are in the process of producing mutants of other diploid, single-copy T. cruzi genes studied in our lab, such as lyt, gp-72, and dhfr-ts.