Molecular characterization and functional analysis of Eimeria tenella citrate synthase
Molecular characterization and functional analysis of Eimeria tenella citrate synthase
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DOI：
10.1007/s00436-020-07014-6
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发表时间：
2021-01
影响因子：
2
通讯作者：
Haixia Wang;Qiping Zhao;Shunhai Zhu;Hui Dong;Shuilan Yu;Qingjie Wang;Yu Yu-Yu;S. Liang;Huanzhi Zhao;Bing Huang;H. Han
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文献类型：
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作者：
Haixia Wang;Qiping Zhao;Shunhai Zhu;Hui Dong;Shuilan Yu;Qingjie Wang;Yu Yu-Yu;S. Liang;Huanzhi Zhao;Bing Huang;H. Han
鸡球虫病是由艾美耳属专性细胞内原生动物寄生虫引起的，是集约化饲养家禽业的主要寄生虫病。由于抗球虫药的广泛使用，耐药性已成为不可避免的问题。在我们之前的研究中，通过RNA序列发现，与药物敏感菌株相比，柔嫩艾美耳球虫柠檬酸合酶（EtCS）在两种耐药菌株（地克珠利耐药菌株和马杜霉素耐药菌株）中表达上调。本研究克隆表达了EtCS并获得了其多克隆抗体。采用实时定量聚合酶链（qPCR）反应和蛋白质印迹分析敏感菌株和三种耐药菌株中EtCS的转录和翻译水平。与敏感菌株相比，地克珠利和马杜霉素耐药菌株中EtCS的转录均显着上调，但盐霉素耐药菌株中EtCS的转录没有显着差异。三种耐药菌株的翻译水平没有显着差异。间接免疫荧光结果表明，EtCS主要位于除后折光体之外的子孢子细胞质中，以及裂殖子的细胞质和表面。抗rEtCS抗体对E．柔嫩子孢子对DF-1细胞的侵袭抑制率达83%以上。通过免疫荧光测定证实了该蛋白与鸡巨噬细胞 (HD11) 细胞的结合。当用rEtCS处理巨噬细胞时，巨噬细胞的一氧化氮分泌和细胞增殖显着减少。这些结果表明EtCS可能与大肠杆菌对宿主细胞的侵袭有关。球虫并参与球虫对某些药物耐药性的发展。
Chicken coccidiosis, caused by an obligate intracellular protozoan parasite of the genusEimeria, is a major parasitic disease in the intensively reared poultry industry. Due to the widespread use of anticoccidial drugs, resistance has become an inevitable problem. In our previous study,Eimeria tenellacitrate synthase (EtCS) was found to be up-expressed in two drug-resistant strains (diclazuril-resistant and maduramycin-resistant strains) compared to drug-sensitive strain by RNA sequence. In this study, we cloned and expressedEtCS and obtain its polyclonal antibodies. Quantitative real-time polymerase chain (qPCR) reactions and Western blots were used to analyze the transcription and translation levels ofEtCS in sensitive and three drug-resistant strains. Compared with the sensitive strain, the transcription ofEtCS was both significantly upregulated in diclazuril-resistant and maduramycin-resistant strains, but was not significantly different in salinomycin-resistant strain. No significant difference was seen in translation level in the three drug-resistant strains. Indirect immunofluorescence indicated thatEtCS was mainly located in the cytoplasm of sporozoites except for posterior refractile bodies and in the cytoplasm and surface of merozoites. Anti-rEtCS antibody has inhibitory effects onE. tenellasporozoite invasion of DF-1 cells and the inhibition rate is more than 83%. Binding of the protein to chicken macrophage (HD11) cells was confirmed by immunofluorescence assays. When macrophages were treated with rEtCS, secretion of nitric oxide and cell proliferation of the macrophages were substantially reduced. These results showed thatEtCS may be related to host cell invasion ofE. tenellaand involve in the development ofE.tenellaresistance to some drugs.