A novel serine/threonine protein phosphatase type 5 from second-generation merozoite of Eimeria tenella is associated with diclazuril-induced apoptosis
A novel serine/threonine protein phosphatase type 5 from second-generation merozoite of Eimeria tenella is associated with diclazuril-induced apoptosis
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来自柔嫩艾美耳球虫第二代裂殖子的新型丝氨酸/苏氨酸蛋白磷酸酶 5 型与地克珠利诱导的细胞凋亡相关
DOI：
10.1007/s00436-013-3336-0
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发表时间：
2013-04-01
影响因子：
2
通讯作者：
Xue, Fei-qun
中科院分区：
文献类型：
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作者：
Zhou, Bian-hua;Wang, Hong-wei;Xue, Fei-qun
筛选抗球虫药物作用靶点对于开发新的抗球虫药物和揭示球虫耐药的分子基础具有重要意义。地克珠利具有高效、安全等优点，在家禽养殖业中得到了广泛的应用。为了研究柔嫩艾美耳球虫（Eimeria tenella）第二代裂殖子5型丝氨酸/苏氨酸蛋白磷酸酶（EtPP 5）在地克珠利抗鸡球虫活性中的作用，采用逆转录聚合酶链反应（RT-PCR）和快速扩增cDNA末端的方法克隆了EtPP 5。透射电镜观察第二代裂殖子超微结构变化，实时荧光定量PCR检测EtPP 5 mRNA表达水平。结果表明，克隆的EtPP 5 cDNA全长（2，495 bp）包含1，647 bp的开放阅读框，编码548个氨基酸残基的多肽，分子量为60.82 kDa，理论等电点为5.89。EtPP 5的分子分析揭示了C-末端磷酸酶结构域和扩展的N-末端tetratricopeptide重复基序的存在，这是蛋白磷酸酶的典型特征。该cDNA序列已提交至GenBank数据库，登录号为JX 987508。EtPP 5与已发表的弓形虫PP 5同源基因（GenBank XP_002364442.1）的同源性为89%。透射电镜观察到地克珠利引起第二代裂殖子超微结构的变化。实时荧光定量PCR分析显示，与对照组相比，地克珠利处理组EtPP 5 mRNA表达水平显著下调51.4%。EtPP 5与先前描述的其他生物体的PP 5的高度相似性，以及其在地克珠利诱导的第二代裂殖子中的下调表达和与凋亡的联系，表明它可能在理解地克珠利诱导的裂殖子凋亡的信号转导机制中起重要作用。
Screening the anticoccidial drug targets is very important for developing novel drugs and revealing the molecular basis of drug resistance in coccidia. Due to high effectivity and safety, diclazuril was used widely in the poultry industry. To assess the roles of the serine/threonine protein phosphatase type 5 of second-generation merozoites in Eimeria tenella (EtPP5) in the anticoccidial activity of diclazuril against chicken coccidiosis, EtPP5 was cloned using reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Ultrastructural changes in second-generation merozoites and mRNA expression level of EtPP5 were monitored by transmission electron microscopy (TEM) and quantitative real-time PCR, respectively. The results showed that the full length of the cloned EtPP5 cDNA (2,495 bp) encompassed a 1,647-bp open reading frame encoding a polypeptide of 548 residues with an estimated molecular mass of 60.82 kDa and a theoretical isoelectric point of 5.89. Molecular analysis of EtPP5 reveals the presence of a C-terminal phosphatase domain and an extended N-terminal tetratricopeptide repeat motif, a typical feature of protein phosphatases. The cDNA sequence has been submitted to the GenBank database with accession number JX987508. EtPP5 shared 89% homology with the published sequence of a PP5 ortholog of Toxoplasma gondii at the amino acid level (GenBank XP_002364442.1). TEM observed that diclazuril induced ultrastructural changes in second-generation merozoites. Quantitative real-time PCR analysis showed that compared with the control group, the level of EtPP5 mRNA expression was significantly downregulated by 51.4% by diclazuril treatment. The high similarity of EtPP5 to previously described PP5 of other organisms, as well as its downregulated expression and connection with apoptosis in the second-generation merozoites induced by diclazuril, suggests that it could act an important role in understanding the signaling mechanism underlining the diclazuril-induced merozoites apoptosis.