Kawase Osamu, Nishikawa Yoshifumi, Bannai Hiroshi, Igarashi Makoto, Matsuo Tomohide, Xuan Xuenan
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.
Parasitol Int. 2010 Jun;59(2):211-6. doi: 10.1016/j.parint.2010.02.001. Epub 2010 Feb 6.
Toxoplasma gondii is an obligate intracellular protozoan parasite that invades a wide range of host cells. The parasite releases a large variety of proteins from a secretory organelle, microneme, and the secretion is essential for the parasite invasion. We cloned a secreted protein with an altered thrombospondin repeat of Toxoplasma gondii (TgSPATR), which was the homologue of Plasmodium SPATRs. Immunofluorescence double staining experiment revealed that TgSPATR was co-localized with a microneme protein, MIC2, and immuno-electron microscopic (IEM) analysis detected TgSPATR in the microneme-like structure. TgSPATR secretion was induced by ethanol, while an intracellular Ca(2+) chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), suppressed the ethanol-induced secretion, suggesting the secretion was Ca(2+)-dependent, similarly to known microneme proteins. Furthermore, TgSPATR, existed on outer surface of the parasites, was detected by incomplete membrane permeabilization by saponin and immunofluorescent antibody test (IFAT). Both TgSPATR and MIC2 were detected on outer surface of extracellular parasites, but not of intracellular single parasites, suggesting they were similarly secreted during early stages of parasite invasion. Therefore, TgSPATR is probably new member of microneme protein and maybe involved in parasite invasion.
刚地弓形虫是一种专性细胞内原生动物寄生虫，可侵入多种宿主细胞。该寄生虫从分泌细胞器微线体释放出大量蛋白质，且这种分泌对于寄生虫的入侵至关重要。我们克隆了一种具有改变的血小板反应蛋白重复序列的刚地弓形虫分泌蛋白（TgSPATR），它是疟原虫SPATR的同源物。免疫荧光双染色实验表明，TgSPATR与一种微线体蛋白MIC2共定位，免疫电子显微镜（IEM）分析在微线体样结构中检测到了TgSPATR。乙醇可诱导TgSPATR分泌，而细胞内Ca(2+)螯合剂1,2-双（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸四乙酰甲酯（BAPTA-AM）可抑制乙醇诱导的分泌，这表明该分泌与已知的微线体蛋白一样是Ca(2+)依赖性的。此外，通过皂苷不完全透膜和免疫荧光抗体试验（IFAT），在寄生虫外表面检测到了TgSPATR。在细胞外寄生虫的外表面检测到了TgSPATR和MIC2，但在细胞内单个寄生虫的外表面未检测到，这表明它们在寄生虫入侵早期以类似方式分泌。因此，TgSPATR可能是微线体蛋白的新成员，可能参与寄生虫入侵。
