ETT2结构基因epaPQR对禽致病性大肠杆菌生物学特性及致病性的影响

doi:10.11843/j.issn.0366-6964.2022.10.034

摘要/Abstract

摘要： 大肠杆菌Ⅲ型分泌系统2（Escherichia coli type III secretion system 2，ETT2）参与禽致病性大肠杆菌（avian pathogenic Escherichia coli，APEC）的致病作用。本研究旨在探究ETT2结构基因epaPQR对禽致病性大肠杆菌的生物学特性及致病作用的影响，为进一步阐明ETT2的致病机制提供依据。基于CRISPR-Cas9基因编辑技术，构建epaPQR基因缺失株和回复株，通过生长曲线测定、运动性和生物被膜形成能力等试验，分析epaPQR基因对APEC生物学特性的影响；通过血清杀菌与组织载菌量等试验分析epaPQR基因对APEC致病性的影响。结果表明，成功构建ETT2结构基因epaPQR基因缺失株和回复株，epaPQR基因缺失后，其生长能力和生物被膜形成能力并没有显著改变（P>0.05）；但epaPQR基因缺失后，其运动能力显著降低（P<0.05），在透射电镜下观察到缺失株鞭毛数量明显减少，通过荧光定量PCR发现，鞭毛T3SS结构基因及鞭毛输出蛋白基因的转录水平均显著下调（P<0.05）。epaPQR基因缺失后其抗血清杀菌能力显著增强（P<0.05），缺失株AE81ΔepaPQR在雏鸡体内不同器官的定殖能力显著降低。结果说明，ETT2结构基因epaPQR参与调控APEC的鞭毛形成，影响APEC抗血清杀菌能力以及在体内组织器官的定殖能力，表明epaPQR在APEC致病过程中发挥重要作用，本研究为深入探究ETT2功能和APEC致病机制提供参考。

关键词: 禽致病性大肠杆菌, ETT2, epaPQR基因, 生物学特性, 致病性

Abstract: Escherichia coli type III secretion system 2 (ETT2) is involved in the pathogenesis of avian pathogenic Escherichia coli (APEC). This study investigated the biological characteristics and pathogenic effects of ETT2 structural gene epaPQR on APEC in order to provide the basis for further understanding of the pathogenic effects of ETT2. The epaPQR mutant strain and complemented strain were constructed based on CRISPR-Cas9 gene editing technology. The effects of epaPQR gene on the biological characteristics of APEC were analyzed by detection of growth curve, motility and biofilm formation ability, etc. The effect of epaPQR gene on the pathogenicity of APEC was analyzed by serum bactericidal and tissue bacterial load tests. The results showed that the deletion and complemented strains of epaPQR gene of ETT2 were successfully constructed. Deletion of epaPQR gene did not significantly influence the growth and biofilm formation ability (P>0.05). After deletion of epaPQR gene, the motility was significantly reduced (P<0.05), and the number of flagella observed by transmission electron microscopy was significantly reduced. RT-qPCR showed that the transcription levels of flagellar T3SS structural genes and flagellar output protein genes were significantly down-regulated (P<0.05). After the deletion of epaPQR gene, its antiserum bactericidal ability was significantly increased (P<0.05), and the colonization ability of AE81ΔepaPQR strain in different organs of chicks was significantly decreased. The results indicated that epaPQR was involved in the regulation of APEC flagellar formation, and affected antiserum bactericidal ability of APEC, as well as the ability to colonize tissues and organs in vivo, suggested that epaPQR played an important role in pathogenesis of APEC. This study provides a reference for the further understanding of the function of ETT2 and the pathogenesis of APEC.

Key words: avian pathogenic Escherichia coli, ETT2, epaPQR gene, biological characteristics, pathogenicity

中图分类号:

S852.612

邵颖, 傅丹丹, 吴晓妍, 谷一, 宋祥军, 涂健, 祁克宗. ETT2结构基因epaPQR对禽致病性大肠杆菌生物学特性及致病性的影响[J]. 畜牧兽医学报, 2022, 53(10): 3631-3641.

SHAO Ying, FU Dandan, WU Xiaoyan, GU Yi, SONG Xiangjun, TU Jian, QI Kezong. Effect of the ETT2 Structural Gene epaPQR on the Biological Properties and Pathogenicity of Avian Pathogenic Escherichia coli[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3631-3641.

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