猪源大肠杆菌高致病性毒力岛结构基因进化分析及其对TNF/NF-κB通路的影响

doi:10.11843/j.issn.0366-6964.2025.01.036

摘要/Abstract

摘要：

旨在研究猪源大肠杆菌(Escherichia coli，E. coli)高致病性毒力岛(high pathogenicity island，HPI)结构基因的进化趋势及其对TNF/NF-κB通路的影响。本研究运用MEGA等软件分析HPI结构基因的遗传进化，使用R语言功能包挖掘E.coli感染细胞的基因表达综合数据库(gene expression omnibus，GEO)，并进行差异基因富集分析；使用分子对接预测HPI-Ybt与TNF/NF-κB通路相关蛋白的结合；将E. coli及前期构建的HPI缺陷株E.coliΔHPI感染IPEC-J2细胞，运用qPCR、ELISA和Western blot对TNF/NF-κB通路中的关键调控点进行检测。系统进化树分析显示，分离株YUNNAN001的intb基因与GQ891729.1亲缘关系最近，而与CQ903045.1亲缘关系较远。KEGG富集发现TNF和NF-κB通路均参与了E. coli感染，且共同基因有10个；火山图及热图富集显示，E. coli感染显著促进了TNF/NF-κB通路中PTGS2、NF-κB、TRAF6等的表达(P < 0.05)；分子对接显示，HPI-Ybt能与同属于TNF/NF-κB通路的10个蛋白相结合，其中与MAP3K14、TNFAIP3的结合最强；感染E. coli组的NF-κB、PTGS2、TNF-α、TRAF6、TRIF、ATF4、cxcl2和IL-6 mRNA水平显著高于E. coliΔHPI组(P < 0.01)，且相对于E. coliΔHPI组，E. coli组显著激活了TNF/NF-κB通路蛋白的表达；此外，与E. coliΔHPI组相比，E. coli感染显著升高了NF-κB和IL-6的水平(P < 0.01)。结果提示，E. coli-HPI通过合成Ybt结合TNF/NF-κB通路中的关键蛋白，进而激活TNF/NF-κB通路。

关键词: 大肠杆菌, 高致病性毒力岛, 耶尔森杆菌素, 进化分析, TNF/NF-κB信号通路

Abstract:

This study was designed to study the evolutionary trend of structural genes of high pathogenicity island (HPI) of Escherichia coli (E. coli) and its effect on TNF/NF-κB pathway. In this study, MEGA and other software were used to analyze the genetic evolution of HPI structural genes, and R language function package was used to mine the gene expression omnibus (GEO) of E. coli infected cells, and differential gene enrichment analysis was performed. Molecular docking was used to predict the binding of HPI-Ybt to TNF/NF-κB pathway related proteins. E. coli and the previously constructed HPI deficient strain E. coliΔHPI were infected with IPEC-J2 cells, and qPCR, ELISA and Western blot were used to detect the key regulatory points in TNF/NF-κB pathway. Phylogenetic analysis showed that the intb gene of isolate YUNNAN001 was most closely related to GQ891729.1, but far from CQ903045.1. KEGG enrichment found that TNF and NF-κB pathways were both involved in E. coli infection, and there were 10 common genes. Volcano plot and heat map enrichment showed that E. coli infection significantly promoted the expression of PTGS2, NF-κB, TRAF6 and other proteins in TNF/NF-κB pathway (P < 0.05). Molecular docking showed that HPI-Ybt could bind to 10 proteins belonging to TNF/NF-κB pathway, among which MAP3K14 and TNFAIP3 were the strongest. The mRNA levels of NF-κB, PTGS2, TNF-α, TRAF6, TRIF, ATF4, cxcl2 and IL-6 in E. coli group were significantly higher than those in E. coliΔHPI group (P < 0.01), and compared with E. coliΔHPI group, E. coli group significantly activated the expression of TNF/NF-κB pathway proteins. In addition, E. coli infection significantly increased the levels of NF-κB and IL-6 compared with the E. coliΔHPI group (P < 0.01). The results suggest that E.coli-HPI activates TNF/NF-κB pathway by synthesizing Ybt and binding to key proteins in TNF/NF-κB pathway.

Key words: Escherichia coli, high pathogenicity island, Yersiniobactin, analysis of evolution, TNF/NF-κB pathway

中图分类号:

S852.612

王浩, 王世玉, 肖金龙, 沈珏, 潘天灵, 张靖松, 肖鹏, 高洪. 猪源大肠杆菌高致病性毒力岛结构基因进化分析及其对TNF/NF-κB通路的影响[J]. 畜牧兽医学报, 2025, 56(1): 392-403.

WANG Hao, WANG Shiyu, XIAO Jinlong, SHEN Jue, PAN Tianling, ZHANG Jingsong, XIAO Peng, GAO Hong. Evolutionary Analysis of High Pathogenicity Virulence Island Structure Gene of Escherichia coli from Pigs and Its Effect on TNF/NF-κB Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 392-403.

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基因 Genes	引物序列(5′→3′) Primer sequences	基因 Genes	引物序列(5′→3′) Primer sequences
PTGS2	TTCCAATCCATGTCAAAACCGT	TNF-α	CAGGCGGTGCCTATGTCTC
NM_011198	AGTCCGGGTACAGTCACACTT	NM_013693	CGATCACCCCGAAGTTCAGTAG
NF-κB	ATGGCAGACGATGATCCCTAC	ATF4	CCTGAACAGCGAAGTGTTGG
NM_008689	CGGAATCGAAATCCCCTCTGTT	NM_009716	TGGAGAACCCATGAGGTTTCAA
TRAF6	TACGATGTGGAGTTTGACCCA	cxcl2	CCAACCACCAGGCTACAGG
NM_009424	CACTGCTTCCCGTAAAGCCAT	NP_033166	GCGTCACACTCAAGCTCTG
TRIF	CGATCAAGACGGCCATGAGTC	IL-6	CTGCAAGAGACTTCCATCCAG
NM_173394	CTCGTCGGTGTCATCTTCTGC	NM_031168	AGTGGTATAGACAGGTCTGTTGG

蛋白 Protein	对接分数 Docking fraction	蛋白 Protein	对接分数 Docking fraction	蛋白 Protein	对接分数 Docking fraction
BIRC3	-7.3	NF-κB	-6.0	TNF	-7.3
CXCL2	-7.2	NFKBIA	-6.3	TNFAIP3	-7.8
MAP3K14	-8.2	PTGS2	-7.6	TRAF6	-6.7

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