Alcantara Monica Visnieski, Kessler Rafael Luis, Gonçalves Rosana Elisa Gonçalves, Marliére Newmar Pinto, Guarneri Alessandra Aparecida, Picchi Gisele Fernanda Assine, Fragoso Stenio Perdigão
Instituto Carlos Chagas/Fiocruz-PR, Rua Prof. Algacyr Munhoz Mader, 3775-CIC, Curitiba, PR, 81350-010, Brazil.
Vector Behavior and Pathogen Interaction Group, Centro de Pesquisas René Rachou, Fiocruz, Av. Augusto de Lima, 1715-Barro Preto, Belo Horizonte, MG, 30190-002, Brazil.
Mol Biochem Parasitol. 2018 Apr;221:1-9. doi: 10.1016/j.molbiopara.2018.01.006. Epub 2018 Feb 2.
In the protozoan parasite Trypanosoma cruzi - the causative agent of Chagas disease - gene expression control is mainly post-transcriptional, where RNA-binding proteins (RBPs) play a central role, by controlling mRNA stability, distribution and translation. A large variety of RBPs are encoded in the T. cruzi genome, including the CCCH-type zinc finger (CCCH ZnF) protein family, which is characterized by the presence of the C-X-C-X-C-X-H (CCCH) motif. In the related parasite T. brucei, CCCH ZnF proteins have been shown to control key differentiation steps in the parasite's life cycle. However, little is known about the CCCH ZnF proteins in T. cruzi. We have worked on the generation of T. cruzi mutants for CCCH ZnF proteins in an effort to shed light on the functions of these proteins in this parasite. Here, we characterize the expression and function of the CCCH ZnF protein TcZC3H31 of T. cruzi. TcZC3H31 is almost exclusively expressed in epimastigotes and metacyclic trypomastigotes, the parasite forms found in the invertebrate host. Importantly, we show that the epimastigote form of the T. cruzi knockout for the TcZC3H31 gene (TcZC3H31 KO) is incapable, both in vitro and in vivo (in infected triatomine insects), to differentiate into the metacyclic trypomastigote form, which is responsible for infection transmission from vectors to humans. The epimastigote forms recovered from the excreta of insects infected with TcZC3H31 KO parasites do not have the typical epimastigote morphology, suggesting that parasites are arrested in a mid-differentiation step. Also, epimastigotes overexpressing TcZC3H31 differentiate into metacyclics more efficiently than wild-type epimastigotes, in vitro. These data suggest that TcZC3H31 is an essential positive regulator of T. cruzi differentiation into the human-infective metacyclic form.
在原生动物寄生虫克氏锥虫（恰加斯病的病原体）中，基因表达控制主要发生在转录后阶段，其中RNA结合蛋白（RBP）通过控制mRNA的稳定性、分布和翻译发挥核心作用。克氏锥虫基因组中编码了多种RBP，包括CCCH型锌指（CCCH ZnF）蛋白家族，其特征是存在C-X-C-X-C-X-H（CCCH）基序。在相关寄生虫布氏锥虫中，CCCH ZnF蛋白已被证明可控制寄生虫生命周期中的关键分化步骤。然而，对于克氏锥虫中的CCCH ZnF蛋白知之甚少。我们致力于生成克氏锥虫CCCH ZnF蛋白的突变体，以阐明这些蛋白在该寄生虫中的功能。在此，我们对克氏锥虫的CCCH ZnF蛋白TcZC3H31的表达和功能进行了表征。TcZC3H31几乎只在无脊椎动物宿主中发现的寄生虫形式即上鞭毛体和循环后期锥鞭毛体中表达。重要的是，我们发现，在体外和体内（在感染的锥蝽昆虫中），敲除TcZC3H31基因的克氏锥虫（TcZC3H31 KO）的上鞭毛体形式无法分化为循环后期锥鞭毛体形式，而循环后期锥鞭毛体形式负责将感染从媒介传播给人类。从感染TcZC3H31 KO寄生虫的昆虫排泄物中回收的上鞭毛体形式没有典型的上鞭毛体形态，这表明寄生虫在分化中期阶段停滞。此外，在体外，过表达TcZC3H31的上鞭毛体比野生型上鞭毛体更有效地分化为循环后期锥鞭毛体。这些数据表明，TcZC3H31是克氏锥虫分化为人感染性循环后期形式的必需正向调节因子。
