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Abstract：
BACKGROUND: Babesia gibsoni, the causative agent of canine babesiosis, belongs to the phylum Apicomplexa. The development of in vitro culture technology has driven research progress in various kinds of omics studies, including transcriptomic analysis of Plasmodium spp. between in vitro and in vivo environments, which has prompted the observation of diagnostic antigens and vaccine development. Nevertheless, no information on Babesia spp. could be obtained in this respect, which greatly hinders the further understanding of parasite growth and development in the blood stage.
METHODS: In this study, considerable changes in the morphology and infectivity of continuous in vitro cultured B. gibsoni (Wuhan isolate) were observed compared to in vivo parasites. Based on these changes, B. gibsoni (Wuhan isolate) was collected from both in vivo and in vitro cultures, followed by total RNA extraction and Illumina transcriptome sequencing. The acquired differentially expressed genes (DEGs) were validated using qRT-PCR, and then functionally annotated through several databases. The gene with the greatest upregulation after in vitro culture was cloned from the genome of B. gibsoni (Wuhan isolate) and characterized by western blotting and indirect immunofluorescence assay for detecting the native form and cellular localization.
RESULTS: Through laboratory cultivation, multiple forms of parasites were observed, and the infectivity of in vitro cultured parasites in dogs was found to be lower. Based on these changes, Illumina transcriptome sequencing was conducted, showing that 377 unigenes were upregulated and 334 unigenes were downregulated. Notably, an AP2 transcription factor family, essential for all developmental stages of parasites, was screened, and the transcriptional changes in these family members were tested. Thus, the novel AP2 transcription factor gene (BgAP2-M) with the highest upregulated expression after in vitro adaptation was selected. This gene comprises an open reading frame (ORF) of 1989 base pairs encoding a full-length protein of 662 amino acids. BgAP2-M contains one AP2 domain and one ACDC conserved domain, which may be involved in the nuclear biology of parasites. The prepared polyclonal antibodies against the BgAP2-M peptides further detected a native size of ~ 73 kDa and were localized to the nuclei of B. gibsoni.
CONCLUSIONS: This study presents a thorough transcriptome analysis of B. gibsoni in vivo and in vitro for the first time, contributing to a detailed understanding of the effects of environmental changes on the growth and development of parasites in the blood stage. Moreover, it also provides a deeper investigation for the different members of the ApiAP2 transcription factor family as various life stage regulators in Babesia spp.
METHODS: In this study, considerable changes in the morphology and infectivity of continuous in vitro cultured B. gibsoni (Wuhan isolate) were observed compared to in vivo parasites. Based on these changes, B. gibsoni (Wuhan isolate) was collected from both in vivo and in vitro cultures, followed by total RNA extraction and Illumina transcriptome sequencing. The acquired differentially expressed genes (DEGs) were validated using qRT-PCR, and then functionally annotated through several databases. The gene with the greatest upregulation after in vitro culture was cloned from the genome of B. gibsoni (Wuhan isolate) and characterized by western blotting and indirect immunofluorescence assay for detecting the native form and cellular localization.
RESULTS: Through laboratory cultivation, multiple forms of parasites were observed, and the infectivity of in vitro cultured parasites in dogs was found to be lower. Based on these changes, Illumina transcriptome sequencing was conducted, showing that 377 unigenes were upregulated and 334 unigenes were downregulated. Notably, an AP2 transcription factor family, essential for all developmental stages of parasites, was screened, and the transcriptional changes in these family members were tested. Thus, the novel AP2 transcription factor gene (BgAP2-M) with the highest upregulated expression after in vitro adaptation was selected. This gene comprises an open reading frame (ORF) of 1989 base pairs encoding a full-length protein of 662 amino acids. BgAP2-M contains one AP2 domain and one ACDC conserved domain, which may be involved in the nuclear biology of parasites. The prepared polyclonal antibodies against the BgAP2-M peptides further detected a native size of ~ 73 kDa and were localized to the nuclei of B. gibsoni.
CONCLUSIONS: This study presents a thorough transcriptome analysis of B. gibsoni in vivo and in vitro for the first time, contributing to a detailed understanding of the effects of environmental changes on the growth and development of parasites in the blood stage. Moreover, it also provides a deeper investigation for the different members of the ApiAP2 transcription factor family as various life stage regulators in Babesia spp.
摘要：
背景：吉卜索尼巴贝斯，犬巴贝斯虫病的病原体，属于顶孔门。体外培养技术的发展推动了各种组学研究的进展,包括疟原虫的转录组学分析。在体外和体内环境之间，这促进了诊断抗原的观察和疫苗的开发。然而，没有关于巴贝西亚的信息。可以在这方面获得，这极大地阻碍了对寄生虫在血液阶段的生长发育的进一步了解。
方法：在本研究中，与体内寄生虫相比，观察到连续体外培养的B.gibsoni（武汉分离株）的形态和感染性发生了很大变化。基于这些变化,从体内和体外培养物中收集B.gibsoni（武汉分离株），然后进行总RNA提取和Illumina转录组测序。获得的差异表达基因（DEGs）使用qRT-PCR进行验证，然后通过几个数据库进行功能注释。从B.gibsoni（武汉分离株）的基因组中克隆了体外培养后表达最高的基因，并通过Western印迹和间接免疫荧光测定法进行了表征，以检测天然形式和细胞定位。
结果：通过实验室培养，观察到多种形式的寄生虫，发现体外培养的寄生虫在狗中的感染性较低。基于这些变化,进行了Illumina转录组测序，显示377个单基因上调，334个单基因下调。值得注意的是,AP2转录因子家族，对寄生虫的所有发育阶段都至关重要，被筛选，并测试了这些家族成员的转录变化。因此，选择体外适应后上调表达最高的新型AP2转录因子基因（BgAP2-M）。该基因包含1989个碱基对的开放阅读框(ORF),其编码662个氨基酸的全长蛋白质。BgAP2-M包含一个AP2域和一个ACDC保守域，这可能与寄生虫的核生物学有关。制备的针对BgAP2-M肽的多克隆抗体进一步检测到〜73kDa的天然大小,并定位到吉布氏芽孢杆菌的细胞核。
结论：本研究首次在体内和体外对双歧杆菌进行了全面的转录组分析，有助于详细了解环境变化对血液阶段寄生虫生长和发育的影响。此外，它还为ApiAP2转录因子家族的不同成员作为Babesiaspp的各种生命阶段调节因子提供了更深入的研究。
方法：在本研究中，与体内寄生虫相比，观察到连续体外培养的B.gibsoni（武汉分离株）的形态和感染性发生了很大变化。基于这些变化,从体内和体外培养物中收集B.gibsoni（武汉分离株），然后进行总RNA提取和Illumina转录组测序。获得的差异表达基因（DEGs）使用qRT-PCR进行验证，然后通过几个数据库进行功能注释。从B.gibsoni（武汉分离株）的基因组中克隆了体外培养后表达最高的基因，并通过Western印迹和间接免疫荧光测定法进行了表征，以检测天然形式和细胞定位。
结果：通过实验室培养，观察到多种形式的寄生虫，发现体外培养的寄生虫在狗中的感染性较低。基于这些变化,进行了Illumina转录组测序，显示377个单基因上调，334个单基因下调。值得注意的是,AP2转录因子家族，对寄生虫的所有发育阶段都至关重要，被筛选，并测试了这些家族成员的转录变化。因此，选择体外适应后上调表达最高的新型AP2转录因子基因（BgAP2-M）。该基因包含1989个碱基对的开放阅读框(ORF),其编码662个氨基酸的全长蛋白质。BgAP2-M包含一个AP2域和一个ACDC保守域，这可能与寄生虫的核生物学有关。制备的针对BgAP2-M肽的多克隆抗体进一步检测到〜73kDa的天然大小,并定位到吉布氏芽孢杆菌的细胞核。
结论：本研究首次在体内和体外对双歧杆菌进行了全面的转录组分析，有助于详细了解环境变化对血液阶段寄生虫生长和发育的影响。此外，它还为ApiAP2转录因子家族的不同成员作为Babesiaspp的各种生命阶段调节因子提供了更深入的研究。
