Table 1_MicroRNA warfare: how chickens combat Klebsiella variicola with gga-miR-2954.docx

Klebsiella variicola is a member of Klebsiella pneumoniae complex and an emerging zoonotic pathogen. As part of the lymphatic system, the spleen plays a pivotal role in destroying invading pathogens. Various microRNAs (miRNAs) are involved in host resistance to pathogens. However, specific miRNAs that act against K. variicola remain unknown. Therefore, RNA sequencing (RNA-Seq) of the miRNA profile of the chicken spleen was conducted to further clarify the host immune response to K. variicola infection. Challenge of 7-day-old chicks with K. variicola strain AHKV-S01 caused severe damage and enlargement of the spleen. In total, 22 differentially expressed (DE) miRNAs (fold change>2, q< 0.05) were identified. Functional annotation analysis of the target genes of DE miRNAs found that signaling pathways related to innate immunity, inflammation, and metabolism were significantly enriched. Notably, expression of gga-miR-2954 was significantly upregulated in the infection group as compared to the control group. In vitro, gga-miR-2954 directly repressed luciferase reporter gene activity by binding to the 3′ untranslated regions of STAB1. Overexpression of gga-miR-2954 in HD11 macrophages significantly inhibited expression of STAB1, which is involved in activation of several proinflammatory cytokines. K. variicola induced damage to the spleen by over activation of inflammatory and innate immune responses. The observed changes to the miRNA expression profile of the chicken spleen elucidate host immune responses to K. variicola infection, providing critical insights for developing novel therapeutic strategies to enhance chicken resistance against this pathogen.

变异性克雷伯菌（Klebsiella variicola）是肺炎克雷伯菌复合体（Klebsiella pneumoniae complex）的成员之一，同时也是一种新兴的人畜共患病原菌。脾脏作为淋巴系统的组成部分，在清除入侵病原菌的过程中发挥着关键作用。多种微小RNA（microRNAs, miRNAs）参与宿主对病原菌的免疫防御，但目前针对变异性克雷伯菌发挥拮抗作用的特异性miRNAs仍未明确。因此，本研究对鸡脾脏的miRNA表达谱开展RNA测序（RNA sequencing, RNA-Seq），以进一步阐明宿主对变异性克雷伯菌感染的免疫应答机制。以变异性克雷伯菌菌株AHKV-S01攻毒7日龄雏鸡后，可导致脾脏出现严重损伤与肿大。本研究共鉴定出22个差异表达（differentially expressed, DE）miRNAs（倍数变化>2，q<0.05）。对DE miRNAs的靶基因进行功能注释分析后发现，天然免疫、炎症及代谢相关的信号通路显著富集。值得注意的是，与对照组相比，感染组中gga-miR-2954的表达水平显著上调。体外实验显示，gga-miR-2954可通过结合STAB1的3'非翻译区（3' untranslated regions）直接抑制荧光素酶报告基因（luciferase reporter gene）的活性。在HD11巨噬细胞（macrophages）中过表达gga-miR-2954可显著抑制STAB1的表达，而STAB1参与多种促炎细胞因子（proinflammatory cytokines）的激活过程。变异性克雷伯菌可通过过度激活炎症与天然免疫应答，对脾脏造成损伤。本研究观察到的鸡脾脏miRNA表达谱变化，阐明了宿主对变异性克雷伯菌感染的免疫应答机制，为开发新型治疗策略以增强鸡群对该病原菌的抵抗力提供了重要理论依据。