Dynamic landscape of microRNA expression in the feline small intestine during Toxoplasma gondii infection
Bintao Zhai1, Bibo Bao1, Shi-Chen Xie2
1Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou, 730050, Gansu Province, PR China.
Background:
Toxoplasma gondii, an obligate intracellular parasite with felids as its definitive hosts, undergoes sexual reproduction and oocyst shedding in the feline small intestine, a critical stage for its transmission. Small non-coding RNAs, particularly microRNAs (miRNAs), are crucial post-transcriptional regulators in host-pathogen interactions, but their role in the definitive host's intestine during T. gondii infection remains unexplored.
Methods:
Fifteen cats were divided into control, primary infection (6, 10, 14 days post-infection, DPI), and secondary infection (SI) groups. Infection was confirmed via B1 gene polymerase chain reaction (PCR). Small RNA sequencing was performed on the ileal epithelium. Bioinformatics analyses identified known and novel miRNAs, differential expression, target genes, and enriched pathways. Key miRNA-messenger RNA (mRNA) interactions were validated by dual-luciferase assay, and sequencing results were confirmed by quantitative PCR (qPCR).
Results:
Successful infection was molecularly confirmed. Sequencing identified 2666 miRNAs (2575 known, 91 novel). A dynamic pattern of differentially expressed (DE) miRNAs was observed, with peaks at 6 DPI (126), 10 DPI (122), 14DPI (36) and SI DPI (237), coinciding with active oocyst shedding. Key miRNAs like hsa-miR-199b-5p and ssc-miR-199b-5p were persistently downregulated. Target prediction and network analysis revealed complex interactions, including miR-199b-5p targeting CYTH1 and COQ7. Functional enrichment highlighted significant involvement of target genes in the Rap1 and AMPK signaling pathways, as well as processes related to development and cellular organization. The novel_538-CNN2 interaction was experimentally validated.
Conclusions:
This study provides the first comprehensive profile of miRNA expression in the feline small intestine during T. gondii infection. The temporal dynamics and specific dysregulation of miRNAs, coupled with enrichment in key pathways controlling cell adhesion and metabolism, suggest that T. gondii could orchestrate a sophisticated post-transcriptional program in its definitive host to potentially modify the intestinal environment for successful oocyst production and shedding. These findings lay the groundwork for future functional studies regarding the interplay between T. gondii and its definitive hosts.
