空肠弯曲菌Cia效应蛋白参与黏附侵袭细胞和激活炎性细胞因子的研究

Campylobacter jejuni Cia effectors are involved in adhesion and invasion of host cells and activation of inflammatory cytokines

摘要:

【背景】空肠弯曲菌(Campylobacter jejuni)是世界范围内最重要的食源性病原菌之一，在家禽盲肠内通常共生定殖，成为感染人的主要来源，目前该菌在家禽肠道内的定殖机制尚未阐明。【目的】探究空肠弯曲菌不同效应蛋白在参与黏附侵袭肠上皮细胞、免疫细胞及激活炎性细胞因子中的作用，解析该菌在鸡肠道内定殖的机制，为其防控提供潜在的作用靶标。【方法】首先利用同源重组技术构建空肠弯曲菌NCTC 11168菌株不同效应蛋白CiaB、CiaC、CiaD和CiaI的基因缺失突变株，测定突变株对细菌生物膜形成和自身凝集能力的影响，进而测定突变株对肠上皮细胞Caco-2的黏附和侵袭能力，以及对鸡巨噬细胞系HD11的侵袭能力，最后利用RT-qPCR鉴定突变株感染HD11细胞对炎性细胞因子表达的影响。【结果】构建空肠弯曲菌不同效应蛋白CiaB、CiaC、CiaD和CiaI的基因缺失突变株；∆ciaD突变株生物膜形成能力降低，∆ciaC、∆ciaD和∆ciaI突变株自身凝集能力增强；∆ciaB突变株黏附侵袭Caco-2细胞能力增强，而∆ciaC、∆ciaD和∆ciaI突变株黏附侵袭Caco-2以及侵袭HD11细胞能力均显著降低；空肠弯曲菌感染HD11显著提高不同炎性细胞因子表达水平，∆ciaB、∆ciaC和∆ciaD突变株显著降低不同炎性因子表达水平，而∆ciaI突变株显著增强不同炎性因子表达水平。【结论】空肠弯曲菌不同Cia效应蛋白在黏附侵袭肠上皮细胞、免疫细胞及激活炎性细胞因子表达中发挥不同的作用，本研究为解析空肠弯曲菌与宿主肠道上皮相互作用机制提供了理论依据，并为该菌的防控提供了潜在靶标。

关键词:空肠弯曲菌;Cia效应蛋白;黏附;侵袭;炎性细胞因子

Abstract:

[Background] Campylobacter jejuni, one of the major foodborne pathogens worldwide, can colonize the cecum of poultry, becoming the main source of infection in humans. Currently, the mechanism of C. jejuni colonization in avian intestine has not been elucidated. [Objective] To investigate the roles of different Cia effectors of C. jejuni in the adhesion and invasion of intestinal epithelial cells and immune cells, as well as the activation of inflammatory cytokines, thus elucidating the mechanism of C. jejuni colonization in chicken intestine and providing potential targets for the prevention and control of this pathogen. [Methods] Firstly, homologous recombination was employed to construct gene knockout mutants of different effectors (CiaB, CiaC, CiaD, and CiaI) of the wild-type strain (WT, C. jejuni NCTC 11168). The effects of cia knockout on the biofilm formation and autoagglutination were determined. Subsequently, the adhesion and invasion of cia mutants on intestinal epithelial cell line Caco-2, as well as their invasion in the chicken macrophage line HD11, were determined. Finally, RT-qPCR was employed to determine the expression levels of inflammatory cytokines in HD11 cells infected by the mutants. [Results] The cia gene knockout mutants of C. jejuni were successfully constructed. Compared with WT, ∆ciaD showed reduced biofilm formation, while ∆ciaC, ∆ciaD, and ∆ciaI demonstrated enhanced autoagglutination. The mutant ∆ciaB showcased enhanced adhesion and invasion of Caco-2 cells, while ∆ciaC, ∆ciaD, and ∆ciaI exhibited reduced abilities to adhere and invade Caco-2 cells and invade HD11 cells. The infection of HD11 cells by C. jejuni WT significantly up-regulated the expression levels of different inflammatory cytokines, while ∆ciaB, ∆ciaC, and ∆ciaD showed down-regulated expression levels of different inflammatory cytokines. In contrast, ∆ciaI presented up-regulated expression levels of different inflammatory cytokines. [Conclusion] Different Cia effectors of C. jejuni play different roles in the adhesion and invasion of intestinal epithelial cells and immune cells, as well as the activation of inflammatory cytokine expression. The findings provided a theoretical basis for understanding the interaction mechanism between C. jejuni and host intestinal epithelium, and mining potential targets for the prevention and control of this bacterium.

Key words:Campylobacter jejuni;Cia effector;adhesion;invasion;inflammatory cytokine