Laboratory of Immunoparasitology "Dr. Mário Endsfeldz Camargo", Institute of Biomedical Sciences, Universidade Federal de Uberlândia, Uberlândia, Minas Gerais, Brazil.
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA.
Sci Rep. 2017 Jun 19;7(1):3768. doi: 10.1038/s41598-017-03978-1.
The development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. We have established a system specifically designed for Neospora caninum, and used this system as a heterologous platform for the expression of foreign genes. Plasmid constructs containing fluorescent proteins or targeted genes of Toxoplasma gondii, driven by N. caninum promoters, have yielded robust expression and correct trafficking of target gene products as assessed by immunofluorescence assays and Western blot analyses. Using this approach, we here demonstrated that N. caninum expressing T. gondii's GRA15 and ROP16 kinase are biologically active and induced immunological phenotypes consistent with T. gondii strains. N. caninum expressing TgGRA15 differentially disturbed the NF-κB pathway, inducing an increased IL-12 production. On the other hand, N. caninum expressing TgROP16 induced host STAT3 phosphorylation and consequent reduction of IL-12 synthesis. These results indicate that heterologous gene expression in N. caninum is a useful tool for the study of specific gene functions and may allow the identification of antigenic targets responsible for the phenotypic differences observed between these two closely related apicomplexan parasites. Additionally, these observations may prove to be useful for the development of vaccine protocols to control toxoplasmosis and/or neosporosis.
分子遗传学的发展极大地促进了对顶复门寄生虫相关生物学和病理学的研究。我们建立了一个专门针对新生隐球菌的系统，并将该系统用作表达外源基因的异源平台。含有荧光蛋白或刚地弓形虫靶基因的质粒构建体，由新生隐球菌启动子驱动，通过免疫荧光分析和 Western blot 分析评估，可实现靶基因产物的强表达和正确运输。通过这种方法，我们在此证明表达刚地弓形虫 GRA15 和 ROP16 激酶的新生隐球菌具有生物活性，并诱导与刚地弓形虫株一致的免疫表型。表达 TgGRA15 的新生隐球菌可差异干扰 NF-κB 通路，诱导 IL-12 产生增加。另一方面，表达 TgROP16 的新生隐球菌诱导宿主 STAT3 磷酸化，继而导致 IL-12 合成减少。这些结果表明，新生隐球菌中的异源基因表达是研究特定基因功能的有用工具，并且可能能够鉴定导致这两种密切相关的顶复门寄生虫之间观察到的表型差异的抗原靶标。此外，这些观察结果可能对制定控制弓形虫病和/或新生隐球菌病的疫苗方案证明有用。
