College of Veterinary Medicine, Jilin Agricultural University, Changchun 130118, China; Jilin Provincial Key Laboratory of Animal Microecology and Healthy Breeding, Jilin Agricultural University, Changchun 130118, China; Engineering Research Center of Microecological Vaccines (Drugs) for Major Animal Diseases, Ministry of Education, Jilin Agricultural University, Changchun 130118, China.
Vet Parasitol. 2024 Dec;332:110322. doi: 10.1016/j.vetpar.2024.110322. Epub 2024 Oct 2.
The Apicomplexa parasitic phylum rhoptry neck protein 2 (RON2) plays a key role in the process of invading host cells. Eimeria tenella, an intracellular protozoan shares a similar conserved invasion pattern. However, whether E. tenella RON2 participates in the process of invading the host intestinal epithelium is poorly understood. In this study, the sequence of EtRON2 was analyzed and expressed. The expression of the truncated extracellular N-terminal fragment of EtRON2 (403-700 aa, designated EtRON2) with a molecular mass of 38.3 kDa. EtRON2 in the sporozoite protein was detected at 151.4 kDa by rabbit anti-rEtRON2 antibody. Immunofluorescence results showed that EtRON2 was mainly localized to the nucleus and apex of the E. tenella sporozoite. qPCR results showed that the highest expression level of EtRON2 was detected in sporulated oocysts compared with other developmental stages of E. tenella. In vitro invasion inhibition assays showed that the capacity of sporozoites to invade DF-1 cells was significantly inhibited after pretreatment with the rabbit anti-rEtRON2 antibody. Silencing the EtRON2 gene by RNA interference (RNAi) significantly inhibited EtRON2 expression and significantly reduced the invasion of DF-1 cells by sporozoites. In vivo experiments revealed a significant decrease parasite burden and oocyst outputs in chicks after infection with EtRON2 gene-silenced sporozoites by cloacal inoculation. Recombinant EtRON2 (rEtRON2) immunizes chicks effectively against E. tenella infection by inducing humoral immunity and upregulating IFN-γ and CD8 T lymphocytes. Furthermore, chicks exhibited increased relative weight gain rates, lower cecum lesion scores, and reduced oocyst outputs during the E. tenella challenge. H&E staining showed that the cecum tissue of chicks immunized with rEtRON2 showed relatively mild histopathological changes. In conclusion, the results of this study demonstrated that EtRON2 plays a key role in E. tenella invasion of the host intestinal epithelium and provides a potential target for vaccines against E. tenella infection.
顶复门寄生虫罗氏小体颈蛋白 2（RON2）在入侵宿主细胞的过程中起着关键作用。艾美耳球虫是一种细胞内原生动物，具有相似的保守入侵模式。然而，艾美耳球虫 RON2 是否参与入侵宿主肠上皮细胞的过程尚不清楚。在本研究中，分析并表达了 EtRON2 的序列。通过兔抗 rEtRON2 抗体检测到大小为 38.3 kDa 的 EtRON2 截断的胞外 N 端片段（403-700 aa，命名为 EtRON2）的表达。在艾美耳球虫孢子虫蛋白中，EtRON2 检测到 151.4 kDa。免疫荧光结果表明，EtRON2 主要定位于艾美耳球虫孢子的核和顶端。qPCR 结果显示，与艾美耳球虫的其他发育阶段相比，在孢子化卵囊中转录水平最高。体外入侵抑制试验表明，用兔抗 rEtRON2 抗体预处理后，孢子的入侵能力显著受到抑制。通过 RNA 干扰（RNAi）沉默 EtRON2 基因，显著抑制了 EtRON2 的表达，并显著降低了孢子对 DF-1 细胞的入侵。体内实验显示，在经肛门接种 EtRON2 基因沉默的孢子后，鸡的寄生虫载量和卵囊产量明显降低。重组 EtRON2（rEtRON2）通过诱导体液免疫和上调 IFN-γ和 CD8 T 淋巴细胞，有效免疫小鸡免受艾美耳球虫感染。此外，在艾美耳球虫攻毒期间，免疫 rEtRON2 的小鸡表现出相对较高的体重增长率、较低的盲肠病变评分和卵囊产量降低。H&E 染色显示，用 rEtRON2 免疫的鸡的盲肠组织显示出相对较轻的组织病理学变化。总之，本研究结果表明，EtRON2 在上皮细胞的入侵过程中发挥着关键作用，为艾美耳球虫感染的疫苗提供了潜在的靶标。
