RESUMO
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects millions of individuals around the world. Although it has been known for more than a century, the study of T. cruzi has been a challenge, particularly due to the scarcity of tools for genome inquiries. Recently, strategies have been described allowing gene disruption in T. cruzi by the CRISPR/Cas9 nuclease system. Although these strategies demonstrated success in deleting some genes, several aspects could be improved to increase the efficiency of the CRISPR/Cas9 system in T. cruzi. Here, we report a strategy, based on adaptations and improvements of the two previously described systems, that results in efficient gene disruption that can be applied to any target, including the study of essential genes.
Assuntos
Sistemas CRISPR-Cas , Engenharia Genética/métodos , Trypanosoma cruzi/genéticaRESUMO
Chagas disease, caused by Trypanosoma cruzi, still affects millions of people around the world. No vaccines nor treatment for chronic Chagas disease are available, and chemotherapy for the acute phase is hindered by limited efficacy and severe side effects. The processes by which the parasite acquires infectivity and survives in different hosts involve tight regulation of gene expression, mainly post-transcriptionally. Nevertheless, chromatin structure/organization of trypanosomatids is similar to other eukaryotes, including histone variants and post-translational modifications. Emerging evidence suggests that epigenetic mechanisms also play an important role in the biology/pathogenesis of these parasites, making epigenetic targets suitable candidates to drug discovery. Here, we present the first comprehensive map of post-translational modifications of T. cruzi canonical and variant histones and show that its histone code can be as sophisticated as that of other eukaryotes. A total of 13 distinct modification types were identified, including rather novel and unusual ones such as alternative lysine acylations, serine/threonine acetylation, and N-terminal methylation. Some histone marks correlate to those described for other organisms, suggesting that similar regulatory mechanisms may be in place. Others, however, are unique to T. cruzi or to trypanosomatids as a group and might represent good candidates for the development of antiparasitic drugs.
Assuntos
Epigênese Genética , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Trypanosoma cruzi/genética , Acetilação , Acilação , Código das Histonas , Lisina/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Serina/metabolismo , Treonina/metabolismoRESUMO
The nuclear lamina is a structure that lines the inner nuclear membrane. In metazoans, lamins are the primary structural components of the nuclear lamina and are involved in several processes. Eukaryotes that lack lamins have distinct proteins with homologous functions. Some years ago, a coiled-coil protein in Trypanosoma brucei, NUP-1, was identified as the major filamentous component of its nuclear lamina. However, its precise role has not been determined. We characterized a homologous protein in Trypanosoma cruzi, TcNUP-1, and identified its in vivo DNA binding sites using a chromatin immunoprecipitation assay. We demonstrate for the first time that TcNUP-1 associates with chromosomal regions containing large non-tandem arrays of genes encoding surface proteins. We therefore suggest that TcNUP-1 is a structural protein that plays an essential role in nuclear organization by anchoring T. cruzi chromosomes to the nuclear envelope.
Assuntos
DNA de Protozoário/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas Nucleares/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/química , Imunoprecipitação da Cromatina , Clonagem Molecular , DNA de Protozoário/química , Proteínas de Ligação a DNA/metabolismo , Membrana Nuclear/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismoRESUMO
We have identified two zinc finger proteins of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease in humans. These proteins, named tcZFP1 and tcZFP2, share the unusual zinc finger motif (CCCH) found in a diverse range of RNA-binding proteins involved in various aspects of the control of cell homeostasis and differentiation. We report here the functional expression of a recombinant tcZFP1, and the relative affinity and stability of the specific complexes formed between the protein and synthetic oligoribonucleotides containing C-rich sequences.