CONICET | Buscador de Institutos y Recursos Humanos

Trypanosoma cruzi is a protozoan parasite and the causativeagent of Chagas´ disease. This parasite has a complex life cyclewith two intermediate hosts, a triatomine insect and a mammalianvertebrate. Nuclear gene organization in T. cruzi is atypical fromother eukaryotic cells. Genes arrange in long tandems that are transcribedpolycistronically from a small number of initiation sites. Thetranscription is driven by a limited number of basal nuclear factorsand its regulation depends on chromatin structure. Among the usualproteins that participate in epigenetic regulation, a small numberof genes coding for acetylases, deacetylases and bromodomainsare present in the T. cruzi genome. Bromodomains are conservedprotein modules capable of binding acetylated lysines and found inmany proteins associated with chromatin. T. cruzi has at least sixgenes that encode for bromodomain-containing factors (TcBDF1-6).The overall objective of the project is to explore bromodomains asdrug target in T. cruzi, by assaying their essentiality using a CRISPR/Cas9 gene deletion approach. Briefly, target sequences (SgRNA)were cloned into the expression plasmid pTREX-Cas9-neo.Parasites were transfected by electroporation and drug selected.Deletion of the target genes were determined by PCR. We obtainedmutated parasites for all the genes tested but only for Bdfx we gotthe homozygous population, which mean that this gene is not essentialfor epimastigotes growth. For the rest of the genes we detectedless amount of protein for mutated cultures but we couldn´t get homozygousnull mutants, and only one of them showed a detrimentalgrowth rate. We are evaluating the essentiality of these genes inthe other life cycle stages as well as testing the expression of dominant-negative mutants approach for some of the genes. Altogetherthese results suggest that except Bdfx, bromodomains genes couldbe crucial for T. cruzi normal growth.