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Background <p><i>Toxoplasma gondii</i>, 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 <i>T. gondii</i> infection remains unexplored.</p> Methods <p>Fifteen cats were divided into control, primary infection (6, 10, 14&#xa0;days post-infection, DPI), and secondary infection (SI) groups. Infection was confirmed via <i>B1</i> 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).</p> Results <p>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),&#xa0;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 <i>CYTH1</i> and <i>COQ7</i>. 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–<i>CNN2</i> interaction was experimentally validated.</p> Conclusions <p>This study provides the first comprehensive profile of miRNA expression in the feline small intestine during <i>T. gondii</i> infection. The temporal dynamics and specific dysregulation of miRNAs, coupled with enrichment in key pathways controlling cell adhesion and metabolism, suggest that <i>T. gondii</i> 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 <i>T. gondii</i> and its definitive hosts.</p> Graphical Abstract <p></p>

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Dynamic landscape of microRNA expression in the feline small intestine during Toxoplasma gondii infection

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月11日

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  • Bintao Zhai,
  • Bibo Bao,
  • Shi-chen Xie,
  • Hui Yang,
  • Yang Liu,
  • Weiwei Wang,
  • Yaxin Zhou,
  • Bing Li,
  • Junjun He,
  • Jiyu Zhang

摘要

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.

Graphical Abstract