Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi

Abstract

BackgroundTrypanosoma cruzi, a kinetoplastid protozoan parasite that causes Chagas disease, infects approximately 15 million people in Central and South America. In contrast to the substantial in silico studies of the T. cruzi genome, transcriptome, and proteome, only a few genes have been experimentally characterized and validated, mainly due to the lack of facile methods for gene manipulation needed for reverse genetic studies. Current strategies for gene disruption in T. cruzi are tedious and time consuming. In this study we have compared the conventional multi-step cloning technique with two knockout strategies that have been proven to work in other organisms, one-step-PCR- and Multisite Gateway-based systems.ResultsWhile the one-step-PCR strategy was found to be the fastest method for production of knockout constructs, it does not efficiently target genes of interest using gene-specific sequences of less than 80 nucleotides. Alternatively, the Multisite Gateway based approach is less time-consuming than conventional methods and is able to efficiently and reproducibly delete target genes.ConclusionUsing the Multisite Gateway strategy, we have rapidly produced constructs that successfully produce specific gene deletions in epimastigotes of T. cruzi. This methodology should greatly facilitate reverse genetic studies in T. cruzi.

Knockout of the dhfr-ts Gene in Trypanosoma cruzi Generates Attenuated Parasites Able to Confer Protection against a Virulent Challenge

“…Apparently, duplications of the target gene or the whole chromosome may be taking place. Similar events showing target locus amplification were observed when trying to obtained null mutant T. cruzi parasites for the enoyl-CoA hydratase ( ech ) and UDP-Glcp 4′-epimerase (TcGALE) genes [24], [38]. Identifying the frequency at which duplication events take place could be important for targeted deletion protocols and for probing the plasticity of the genome of this intriguing parasite.…”

Oral Exposure to Trypanosoma cruzi Elicits a Systemic CD8+T Cell Response and Protection against Heterotopic Challenge

“…To test this hypothesis, we exposed mice to an attenuated strain of T. cruzi by the oral route and subsequently challenged them with virulent parasites. Mice received three doses of T. cruzi strain CL attenuated by the deletion of one allele of the enoyl-CoAhydratase-1 (ECH1) gene and both alleles of the ECH2 gene (66). Both ECH genes encode an enzyme involved in fatty acid oxidation, a process thought to be important in amastigote energy metabolism (2).…”

“…injection with 1,000 tissue culture trypomastigotes (TCT). Attenuated parasites (ECH1 ϩ/Ϫ ECH2 Ϫ/Ϫ ) were generated by targeting the tandem enoyl-coenzyme A (CoA) hydratase 1 and 2 genes in a high-throughput knockout system recently developed by our lab (66). These ECH1 ϩ/Ϫ ECH2 Ϫ/Ϫ parasites fail to establish persistent infection in C57B6/J mice (as determined by immunosuppression) (7) or in immunodeficient mice.…”

Gene-deleted live-attenuatedTrypanosoma cruziparasites as vaccines to protect against Chagas disease

“…The MultiSite Gateway cloning system has emerged as an efficient way to accelerate the time-consuming multiple gene cloning and to generate efficient constructions able to delete genes in the T. cruzi genome [133]. Mutants carrying only one copy of the ech1 gene (ECH1+/À) and none of the ech2 genes (ECH2 À/À ) were generated using Gateway constructions.…”