Instituto de Investigaciones Biomédicas, Departamento de Inmunología, Universidad Nacional Autónoma de México, 04510, Mexico City, Mexico.
Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, 07360, Mexico City, Mexico.
Parasitol Res. 2020 Nov;119(11):3803-3815. doi: 10.1007/s00436-020-06891-1. Epub 2020 Oct 2.
Trypanosoma cruzi is the etiological agent of Chagas disease, whose clinical outcome ranges from asymptomatic individuals to chronic fatal megasyndromes. Despite being central to pathogenesis, the regulation of parasite virulence factors' expression remains largely unknown. In this work, the relative expression of several parasite virulence factors between two TcI strains (Ninoa, low virulence and Qro, high virulence) was assessed by qRT-PCR of total and of polysome-associated mRNA, as well as by western blots. Trypomastigotes were also incubated with specific anti-sense morpholino oligonucleotides to block the translation of a selected virulence factor, calreticulin, in both strains. Ninoa trypomastigotes showed significantly lower levels of trypomastigote-decay acceleration factor, complement regulatory protein, complement C2 receptor inhibitor trispanning, and glycoproteins 82 and 90 mRNAs compared with Qro. There was a significantly lower recruitment of complement regulatory protein and complement C2 receptor inhibitor trispanning mRNAs to polysomes and higher recruitment of MASP mRNA to monosomes in Ninoa strain. Calreticulin mRNA displayed both a higher total mRNA level and recruitment to translationally active polysomes in the Ninoa strain (low virulence) than in the Qro strain (high virulence). When calreticulin was downregulated by ≈ 50% by anti-sense morpholino oligonucleotides, a significant decrease of parasite invasion in mammalian cells was found in both strains. Calreticulin downregulation, however, only increased significantly the activation of the complement system by Ninoa trypomastigotes. These results suggest a role for the regulation of virulence factors' gene expression in the differential virulence among T. cruzi strains. Furthermore, a possible function of calreticulin in parasite invasion not related to its binding to complement factors is shown.
克氏锥虫是恰加斯病的病原体，其临床结果从无症状个体到慢性致命巨症候群不等。尽管寄生虫毒力因子的表达调控在发病机制中起着核心作用，但这种调控在很大程度上仍然未知。在这项工作中，通过 qRT-PCR 对总 mRNA 和多核糖体相关 mRNA 以及 Western blot 检测，评估了两种 TcI 株（低毒力的 Ninoa 和高毒力的 Qro）之间几种寄生虫毒力因子的相对表达。还使用针对特定反义形态发生素寡核苷酸来阻断两种菌株中钙网蛋白这一选定毒力因子的翻译，以检测 Ninoa 鞭毛体的翻译。与 Qro 相比，Ninoa 鞭毛体的鞭毛体衰减加速因子、补体调节蛋白、补体 C2 受体抑制剂跨膜和糖蛋白 82 和 90 mRNAs 的水平明显较低。在 Ninoa 菌株中，补体调节蛋白和补体 C2 受体抑制剂跨膜 mRNAs 向多核糖体的募集显著降低，而 MASP mRNA 向单核糖体的募集显著增加。Ninoa 株（低毒力）的钙网蛋白 mRNA 总水平和向翻译活性多核糖体的募集均高于 Qro 株（高毒力）。当钙网蛋白被反义形态发生素寡核苷酸下调约 50%时，两种菌株中的寄生虫对哺乳动物细胞的侵袭均显著降低。然而，只有在 Ninoa 鞭毛体中，钙网蛋白的下调才会显著增加补体系统的激活。这些结果表明，寄生虫毒力因子基因表达的调控在克氏锥虫株之间的差异毒力中起着作用。此外，还表明钙网蛋白在寄生虫入侵中的作用可能与其与补体因子的结合无关。
