Pastor-Fernández Iván, Regidor-Cerrillo Javier, Álvarez-García Gema, Marugán-Hernández Virginia, García-Lunar Paula, Hemphill Andrew, Ortega-Mora Luis M
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain.
Institute of Parasitology, Vetsuisse Faculty, University of Berne, Länggass-Strasse 122, CH-3012, Berne, Switzerland.
Parasit Vectors. 2016 Jun 21;9(1):352. doi: 10.1186/s13071-016-1620-4.
NTPases (also NTPDases) are enzymes with apyrase activity. They are widely distributed among eukaryotes, and also among members of the family Sarcocystidae. In Toxoplasma gondii, the TgNTPase accumulates in the dense granules, and has been commonly associated with the strain virulence. In the closely related Neospora caninum, the NcNTPase lacks nucleoside diphosphate hydrolase activity and appears to be more abundant in virulent isolates, indicating that it may contribute to the pathogenicity of neosporosis. However, so far no additional information on NcNTPase has been provided.
Herein, the NcNTPase coding sequences were analysed by different in silico and de novo sequencing approaches. A comparative analysis of NcNTPase and NcGRA7 in terms of protein dynamics, secretion, phosphorylation, and mRNA expression profiles during the tachyzoite lytic cycle was also carried out. Moreover, NcNTPase immunolocalization was analysed by confocal microscopy techniques over a set number of time-points.
We describe the presence of three different loci containing three copies of the NcNTPase within the Nc-Liv genome, and report the existence of up to four different NcNTPase alleles in Nc-Liv. We also provide evidence for the occurrence of diverse protein species of the NcNTPase by two-dimensional gel electrophoresis. Both NcNTPase and NcGRA7 were similarly up-regulated and secreted during the egress and/or early invasion phases, and were phosphorylated. However, its secretion was not affected by the addition of calcium modulators such as A23187 and ethanol. NcNTPase and NcGRA7 localized in dense granules and parasitophorous vacuole membrane throughout the lytic cycle, although differed in their inmunolocalization during early invasion and egress.
The present study reveals the complexity of the NcNTPase loci in N. caninum. We hypothesize that the expression of different isoforms of the NcNTPase protein could contribute to parasite virulence. Our findings showed regulation of expression, secretion and phosphorylation of NcNTPase suggesting a potential role for progression through the tachyzoites lytic cycle.
NTP酶（也称为NTP二磷酸酶）是具有腺苷三磷酸双磷酸酶活性的酶。它们广泛分布于真核生物以及肉孢子虫科成员中。在刚地弓形虫中，TgNTP酶积聚在致密颗粒中，并且通常与菌株毒力相关。在密切相关的犬新孢子虫中，NcNTP酶缺乏核苷二磷酸水解酶活性，并且在强毒株中似乎更为丰富，这表明它可能与新孢子虫病的致病性有关。然而，到目前为止，尚未提供关于NcNTP酶的更多信息。
在此，通过不同的电子分析和从头测序方法对NcNTP酶编码序列进行了分析。还对速殖子裂解周期中NcNTP酶和NcGRA7在蛋白质动力学、分泌、磷酸化和mRNA表达谱方面进行了比较分析。此外，通过共聚焦显微镜技术在一定数量的时间点上分析了NcNTP酶的免疫定位。
我们描述了在Nc-Liv基因组中存在三个不同的位点，其中包含三个NcNTP酶拷贝，并报告了Nc-Liv中存在多达四个不同的NcNTP酶等位基因。我们还通过二维凝胶电泳为NcNTP酶不同蛋白质种类的存在提供了证据。NcNTP酶和NcGRA7在逸出和/或早期侵入阶段同样上调并分泌，并且被磷酸化。然而，其分泌不受添加钙调节剂如A23187和乙醇的影响。在整个裂解周期中，NcNTP酶和NcGRA7定位于致密颗粒和寄生泡膜，尽管它们在早期侵入和逸出期间的免疫定位有所不同。
本研究揭示了犬新孢子虫中NcNTP酶基因座的复杂性。我们假设NcNTP酶蛋白不同异构体的表达可能有助于寄生虫的毒力。我们的研究结果显示了NcNTP酶表达、分泌和磷酸化调控，提示其在速殖子裂解周期进程中可能发挥潜在作用。
