MicroRNA profiling and regulatory network analysis in the definitive host intestine upon Toxoplasma gondii infection

This study provides the first comprehensive analysis of microRNA (miRNA) expression dynamics in the feline small intestine during Toxoplasma gondii infection. Through small RNA sequencing of ileal epithelium from infected cats across primary (6, 10, 14 days post-infection) and secondary infection time points, 2,666 miRNAs were identified, including 91 novel candidates. A temporally dynamic pattern of differentially expressed miRNAs was observed, with peaks coinciding with active oocyst shedding. Key miRNAs such as hsa-miR-199b-5p and ssc-miR-199b-5p were persistently downregulated, and their interactions with target genes like CYTH1 and COQ7 were predicted. Functional enrichment analysis revealed significant involvement of miRNA targets in critical pathways including Rap1 and AMPK signaling, which are associated with cell adhesion, metabolism, and developmental processes. Experimental validation confirmed the novel_538-CNN2 interaction via dual-luciferase assay, and qPCR corroborated sequencing results. These findings suggest that T. gondii orchestrates a sophisticated post-transcriptional regulatory program in its definitive host to modify the intestinal environment, facilitating oocyst production and shedding, thereby providing new insights into host-parasite interactions and potential intervention strategies.本研究首次针对刚地弓形虫（Toxoplasma gondii）感染期间家猫小肠内微小RNA（miRNA）的表达动态展开全面分析。研究人员通过对初次感染（感染后6、10、14天）及二次感染各时间点的感染家猫回肠上皮开展小RNA测序，共鉴定出2666个miRNA，其中包含91个新候选miRNA。研究观察到差异表达miRNA呈现时间动态表达模式，其表达峰值与活跃卵囊排出阶段相吻合。关键miRNA如hsa-miR-199b-5p与ssc-miR-199b-5p持续下调，且预测其可与CYTH1、COQ7等靶基因产生相互作用。功能富集分析显示，miRNA靶基因显著富集于Rap1信号通路与AMPK信号通路等关键通路，这些通路与细胞黏附、代谢及发育过程密切相关。实验验证通过双荧光素酶报告实验确认了novel_538与CNN2的相互作用，定量聚合酶链反应（qPCR）也验证了测序结果的可靠性。本研究结果表明，刚地弓形虫可在其终宿主体内构建一套精密的转录后调控程序，以改造肠道微环境，促进卵囊生成与排出，该发现为宿主与寄生虫的互作机制及潜在干预策略提供了全新见解。