Molecular characterization and analysis of the ATPase ASNA1 homolog gene of Eimeria tenella in a drug sensitive strain and drug resistant strains.
Molecular characterization and analysis of the ATPase ASNA1 homolog gene of Eimeria tenella in a drug sensitive strain and drug resistant strains.
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DOI：
10.1016/j.ijpddr.2021.02.005
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发表时间：
2021-04
期刊：
影响因子：
--
通讯作者：
Han H
中科院分区：
文献类型：
--
作者：
Yu Y;Dong H;Zhao Q;Zhu S;Liang S;Wang Q;Wang H;Yu S;Huang B;Han H
药物的广泛使用加剧了柔嫩艾美耳球虫对抗球虫药物的耐药性。使用RNA-seq，我们先前发现了E. tenella（EtASNA 1）在耐药株和药物敏感株（DS）中差异表达。本研究采用Western blotting和实时荧光定量PCR（qRT-PCR）技术分析了EtASNA 1在地克珠利耐药株（DZR）、马杜拉霉素耐药株（MRR）、盐霉素耐药株（SMR）和DS耐药株中的翻译和转录水平，发现EtASNA 1在3种耐药株中均高表达。qRT-PCR和western blotting结果也表明EtASNA 1的表达水平随着药物浓度的增加而增加，并且DZR菌株的转录水平高于DS菌株。此外，我们使用体内和体外试验来分析DZR、MRR和DS株感染鸡后EtASNA 1表达的变化，以及在药物存在下体外接种DF-1细胞。药物的加入导致表达上调。qRT-PCR和western blotting结果也表明EtASNA 1在第二代裂殖子（SM）和未孢子化卵囊（UO）中的表达水平显著高于其他两个发育阶段。免疫荧光定位结果表明，EtASNA 1蛋白分布于子孢子（SZ）和SM中，但SZ的亲触体除外。体外抑制实验表明，抗EtASNA 1抗体孵育显著抑制DF-1细胞的SZ侵袭。上述结果表明，EtASNA 1可能与大肠杆菌的宿主细胞侵袭有关。tenella，并可能参与E. tenella对某些药物产生抗药性。体外和体内培养以研究药物对EtASNA 1转录水平的影响。DS+，药物敏感菌株+地克珠利或马杜霉素; DS-，药物敏感菌株，无药物; DZR+，地克珠利耐药菌株+地克珠利; DZR-，地克珠利耐药菌株，无药物; MRR+，马杜霉素耐药菌株+马杜霉素; MRR-，马杜霉素耐药菌株，无药物。 ATP酶ASNA 1同源物的研究首次在E. tenella。·EtASNA 1在不同耐药株（地克珠利耐药株、马杜霉素耐药株）中的表达高于敏感株。tenella，可能与E. tenella。·在第二代裂殖子中观察到的高表达可能与E. tenella。·EtASNA 1可能参与子孢子侵入。
The widespread use of drugs has exacerbated the resistance of Eimeria tenalla to anti-coccidial drugs. Using RNA-seq, we previously found the ATPase ASNA1 homolog of E. tenella (EtASNA1) was differentially expressed in resistant strains and drug sensitive (DS) strain. In our study, we used western blotting and quantitative real-time PCR (qRT-PCR) to analyze the translational and transcriptional levels of EtASNA1 in a diclazuril-resistant (DZR) strain, maduramicin-resistant (MRR) strain, salinomycin-resistant (SMR) strain, and DS strain and found EtASNA1 was highly expressed in three drug-resistant strains. The qRT-PCR and western blotting results also showed that the expression levels of EtASNA1 increased with increasing drug concentration, and the transcription levels of the DZR strains isolated from the field were higher than those of the DS strain. In addition, we used in vivo and in vitro tests to analyze the changes of EtASNA1 expression after DZR, MRR, and DS strain infections in chickens, and in vitro inoculation of DF-1 cells in the presence of drugs. The addition of drugs caused expression to be upregulated. The results of qRT-PCR and western blotting also showed that the expression levels of EtASNA1 in second-generation merozoites (SM) and unsporulated oocysts (UO) were significantly higher than those in the other two developmental stages. The immunofluorescence localization of EtASNA1 indicated that the protein was distributed throughout the sporozoites (SZ) and SM, except for the refractile bodies of SZ. In vitro inhibition experiments showed that anti-EtASNA1 antibody incubation significantly inhibited SZ invasion of DF-1 cells. The above results showed that EtASNA1 may be related to host cell invasion of E. tenella and may be involved in the development of E. tenella resistance to some drugs. In vitro and in vivo culture to study the effect of drugs on transcript levels of EtASNA1. DS+, drug-sensitive strains + diclazuril or maduramicin; DS-, drug-sensitive strains, drug-free; DZR+, diclazuril-resistant strains + diclazuril; DZR-, diclazuril-resistant strains, drug-free; MRR+, maduramicin-resistant strains + maduramicin; MRR-, maduramicin-resistant strains, drug-free. ATPase ASNA1 homolog was first studied in E. tenella. •EtASNA1 was highly expressed in different drug-resistant strains (diclazuril-resistant strains, maduramicin-resistant strains) compare to drug sensitive strain of E. tenella, and it might be related to drug resistance of E. tenella. •The high expression observed in second-generation merozoites may be associated with the propagation and development of E. tenella. •EtASNA1 may be involved in sporozoites invasion.
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