Huang Wenhao, Zhu Shunhai, Chen Ting, Zhao Qiping, Dong Hui, Huang Bing, Yao Yawen, Liu Zhan, Yu Yu, Han Hongyu
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Minhang, Shanghai, 200241, People's Republic of China.
College of Life Sciences, Shanghai Normal University, Shanghai, 200234, People's Republic of China.
Parasitol Res. 2022 Jun;121(6):1749-1760. doi: 10.1007/s00436-022-07508-5. Epub 2022 Apr 2.
Chicken coccidiosis is an extremely common and lethally epidemic disease caused by Eimeria spp. The control measures of coccidiosis depend mainly on drugs. However, the ensuing drug resistance problem has brought considerable economic loss to the poultry industry. In our previous study, comparative transcriptome analyses of a drug-sensitive (DS) strain and two drug-resistant strains (diclazuril-resistant (DZR) and maduramicin-resistant (MRR) strains) of Eimeria tenella were carried out by transcriptome sequencing. The expression of glyceraldehyde-3-phosphate dehydrogenase of E. tenella (EtGAPDH) was upregulated in the two resistant strains. In this study, we cloned and characterized EtGAPDH. Indirect immunofluorescence localization was used to observe the distribution of EtGAPDH in E. tenella. The results showed that the protein was distributed mainly on the surface of sporozoites and merozoites, and in the cytoplasm of merozoites. qPCR was performed to detect the transcription level of EtGAPDH in the different developmental stages of the E. tenella DS strain. The transcription level of EtGAPDH was significantly higher in second-generation merozoites than in the other three stages. The transcription level of EtGAPDH in the different drug-resistant strains and DS strain of E. tenella was also analyzed by qPCR. The results showed that the transcription level was significantly higher in the two drug-resistant strains (MRR and DZR) than in the DS strain. As the concentration of diclazuril and maduramicin increased, the transcription levels also increased. Western blot results showed that EtGAPDH protein was upregulated in the DZR and MRR strains. Enzyme activity showed that the enzyme activity of EtGAPDH was higher in the two resistant strains than in the DS strain. These results showed that EtGAPDH possess several roles that separate and distinct from its glycolytic function and maybe involved in the development of E. tenella resistance to anticoccidial drugs.
鸡球虫病是由艾美耳属原虫引起的一种极为常见且具有致死性的流行性疾病。球虫病的防控措施主要依赖药物。然而，随之而来的耐药性问题给家禽业带来了巨大的经济损失。在我们之前的研究中，通过转录组测序对柔嫩艾美耳球虫的一个药物敏感（DS）株和两个耐药株（地克珠利耐药（DZR）株和马杜霉素耐药（MRR）株）进行了比较转录组分析。柔嫩艾美耳球虫的甘油醛-3-磷酸脱氢酶（EtGAPDH）在这两个耐药株中的表达上调。在本研究中，我们克隆并鉴定了EtGAPDH。采用间接免疫荧光定位法观察EtGAPDH在柔嫩艾美耳球虫中的分布。结果表明，该蛋白主要分布在子孢子和裂殖子表面以及裂殖子的细胞质中。进行qPCR检测柔嫩艾美耳球虫DS株不同发育阶段EtGAPDH的转录水平。EtGAPDH在第二代裂殖子中的转录水平显著高于其他三个阶段。还通过qPCR分析了柔嫩艾美耳球虫不同耐药株和DS株中EtGAPDH的转录水平。结果表明，两个耐药株（MRR和DZR）中的转录水平显著高于DS株。随着地克珠利和马杜霉素浓度的增加，转录水平也升高。蛋白质印迹结果显示，EtGAPDH蛋白在DZR和MRR株中上调。酶活性显示，两个耐药株中EtGAPDH的酶活性高于DS株。这些结果表明，EtGAPDH具有与其糖酵解功能不同的多种作用，可能参与了柔嫩艾美耳球虫对抗球虫药物耐药性的形成。
