禽致病性大肠杆菌Hcp2b对雏鸡气管黏膜细胞因子-细胞因子受体相互作用通路的影响

doi:10.11843/j.issn.0366-6964.2021.03.018

摘要/Abstract

摘要： T6SS （type VI secretion system）是革兰阴性菌中常见的一种分泌系统，其效应蛋白Hcp2b作用机制迄今仍未明晰。本研究以禽致病性大肠杆菌（avian pathogenic Escherichia coli，APEC） Hcp2b蛋白为研究主体，旨在探究Hcp2b蛋白在APEC感染鸡气管黏膜过程中发挥的作用及机制。采用Red重组方法以质粒pKD3为模板构建hcp2b缺失株，pSTV-28质粒连接hcp2b目的片段构建重组载体，导入感受态Δhcp2b菌株构建回复株，并对Δhcp2b菌株的生长曲线进行测定。7日龄雏鸡气管感染hcp2b缺失株及野生株，感染后12、24 h收集鸡气管黏膜细胞进行转录组学测序及生物信息学分析。结果表明，hcp2b缺失株及回复株构建成功，hcp2b基因缺失对菌株的生长性能无影响。hcp2b基因缺失株感染气管黏膜后，mRNA的表达谱发生变化，感染后12 h，有144个基因表达量发生变化（上调差异基因87个，下调57个）；感染后24 h，有135个基因表达量发生变化（上调差异基因79个，下调56个）。生物信息学分析发现差异基因富集在Cytokine-cytokine receptor interaction、Protein processing in endoplasmic reticulum等信号通路。hcp2b基因缺失未影响APEC的生长特性，hcp2b基因缺失后影响雏鸡气管黏膜Cytokine-cytokine receptor interaction通路基因mRNA转录水平的变化，IL-1β、IL-12b的表达均上调。该结果为APEC的致病机制研究提供了理论依据。

关键词: 禽致病性大肠杆菌, APEC, hcp2b, 气管黏膜细胞, 转录组

Abstract: T6SS (type VI secretion system) is a common secretion system in Gram-negative bacteria. And its mechanism of effector protein Hcp2b is still unclear. The Hcp2b protein of avian pathogenic Escherichia coli (APEC) is the main subject of this study, which aims to explore the role and mechanism of Hcp2b protein in the process of APEC infection of chicken tracheal mucosa. The Red recombination method was used to construct the hcp2b-deleted strain by using the plasmid pKD3 as the template. And the pSTV-28 plasmid was connected to the hcp2b target fragment to construct the recombinant vector, which was introduced to the competent Δhcp2b strain to construct the hcp2b-reverted strain. Then, we measured the growth curve of Δhcp2b strain. Seven-day-old chicks were infected with the hcp2b-deleted strain and the wild strain by tracheal injection. The infected tracheal mucosal cells were collected at 12 and 24 h after infection and subjected to transcriptomics sequencing and bioinformatics analysis. The results showed that the Δhcp2b strain and Chcp2b strain were successfully constructed. And the deletion of hcp2b gene did not affect the growth performance of the strain. The expression profile of the mRNA of the tracheal mucosa changed after the hcp2b gene-deleted strain infected. Twelve hours after infection, the expression of 144 genes changed (87 differential genes were up-regulated and 57 were down-regulated); Twenty-four hours after infection, 135 gene expressions changed (79 differential genes up-regulated and 56 down-regulated). Bioinformatics analysis found that differential genes were enriched in signaling pathways such as Cytokine-cytokine receptor interaction, Protein processing in endoplasmic reticulum, etc. The deletion of hcp2b gene does not affect the growth characteristics of APEC. After the deletion of hcp2b gene, it affects the change of mRNA transcription level of Cytokine-cytokine receptor interaction pathway gene in chick tracheal mucosa. And the expression of IL-1β and IL-12b are all up-regulated. This study provides a theoretical basis for the study of the pathogenic mechanism of APEC.

Key words: avian pathogenic Escherichia coli, APEC, hcp2b, tracheal mucosal cells, transcriptome

中图分类号:

S852.612

宋祥军, 沈啸, 蒋胡艳, 陈哲, 刘华, 邵颖, 涂健, 祁克宗. 禽致病性大肠杆菌Hcp2b对雏鸡气管黏膜细胞因子-细胞因子受体相互作用通路的影响[J]. 畜牧兽医学报, 2021, 52(3): 742-751.

SONG Xiangjun, SHEN Xiao, JIANG Huyan, CHEN Zhe, LIU Hua, SHAO Ying, TU Jian, QI Kezong. Effect of Avian Pathogenic Escherichia coli Hcp2b on the Cytokine-Cytokine Receptor Interaction Pathway in Chick Tracheal Mucosa[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(3): 742-751.

参考文献 42

[1]	DOU X H, GONG J S, HAN X G, et al. Characterization of avian pathogenic Escherichia coli isolated in eastern China[J]. Gene, 2016, 576(2):244-248.
[2]	SONG X J, QIU M Y, JIANG H Y, et al. ybjX mutation regulated avian pathogenic Escherichia coli pathogenicity though stress-resistance pathway[J]. Avian Pathol, 2020, 49(2):144-152.
[3]	MU X H, GAO R X, XIAO W H, et al. EntE, EntS and TolC synergistically contributed to the pathogenesis of APEC strain E058[J/OL]. Microb Pathog, 2020, 141:103990.[2020-06-01]. https://www.researchgate.net/publication/338722382_EntE_EntS_and_TolC_synergistically_contri-buted_to_the_pathogenesis_of_APEC_strain_E058.
[4]	NAKAZATO G, DE CAMPOS T A, STEHLING E G, et al. Virulence factors of avian pathogenic Escherichia coli (APEC)[J]. Pesq Vet Bras, 2009, 29(7):479-486.
[5]	HEJAIR H M A, MA J L, ZHU Y C, et al. Role of outer membrane protein T in pathogenicity of avian pathogenic Escherichia coli [J]. Res Vet Sci, 2017, 115:109-116.
[6]	GUERRA P R, HERRERO-FRESNO A, PORS S E, et al. The membrane transporter PotE is required for virulence in avian pathogenic Escherichia coli (APEC) (APEC)[J]. Vet Microbiol, 2018, 216:38-44.
[7]	VERMA R, ROJAS T C G, MALUTA R P, et al. Role of hypothetical protein YicS in the pathogenicity of avian pathogenic Escherichia coli in vivo and in vitro[J]. Microbiol Res, 2018, 214:28-36.
[8]	YI Z F, WANG D, XIN S H, et al. The CpxR regulates type VI secretion system 2 expression and facilitates the interbacterial competition activity and virulence of avian pathogenic Escherichia coli [J]. Vet Res, 2019, 50(1):40.
[9]	JOURENT L, CASCALES E. The Type VI secretion system in Escherichia coli and related species[J/OL]. EcoSal Plus, 2016, 7(1):doi:10.1128/ecosalplus.ESP-0009-2015.[2020-06-01]. https://hal-amu.archives-ouvertes.fr/hal-01778564/file/EcoSal-2016.pdf.
[10]	GAYTÁN M O, MARTÍNEZ-SANTOS V I, SOTO E, et al. Type three secretion system in attaching and effacing pathogens[J/OL]. Front Cell Infect Microbiol, 2016, 6(129):doi:10.3389/fcimb.2016.00129.[2020-06-01]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5073101/.
[11]	HACHANI A, WOOD T E, FILLOUX A. Type VI secretion and anti-host effectors[J]. Curr Opin Microbiol, 2016, 29:81-93.
[12]	WEN H Y, GENG Z, GAO Z Q, et al. Characterization of the Pseudomonas aeruginosa T6SS PldB immunity proteins PA5086, PA5087 and PA5088 explains a novel stockpiling mechanism[J]. Acta Crystallogr F Struct Biol Commun, 2020, 76(5):222-227.
[13]	ZONG B B, ZHANG Y Y, WANG X R, et al. Characterization of multiple type-VI secretion system (T6SS) VgrG proteins in the pathogenicity and antibacterial activity of porcine extra-intestinal pathogenic Escherichia coli [J]. Virulence, 2019, 10(1):118-132.
[14]	BASLER M, PILHOFER M, HENDERSON G P, et al. Type VI secretion requires a dynamic contractile phage tail-like structure[J]. Nature, 2012, 483(7388):182-186.
[15]	HU T J, CHEN R, Zhang L Z, et al. Balanced role of T3SS and T6SS in contribution to the full virulence of Edwardsiella piscicida[J]. Fish Shellfish Immunol, 2019, 93:871-878.
[16]	VETTIGER A, BASLER M. Type VI secretion system substrates are transferred and reused among sister cells[J]. Cell, 2016, 167(1):p. 99-110.
[17]	JANA B, SALOMON D. Type VI secretion system:a modular toolkit for bacterial dominance[J]. Future Microbiol, 2019, 14:1451-1463.
[18]	ANDERSSON J A, SHA J, EROVA T E, et al. Identification of new virulence factors and vaccine candidates for Yersinia pestis[J/OL]. Front Cell Infect Microbiol, 2017, 7:448.[2020-06-01]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5650977/pdf/fcimb-07-00448.pdf.
[19]	GALLIQUE M, DECOIN V, BARBEY C, et al. Contribution of the Pseudomonas fluorescens MFE01 type VI secretion system to biofilm formation[J/OL]. PLoS One, 2017, 12(1):e0170770.[2020-06-01]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5256989/pdf/pone.0170770.pdf.
[20]	PENG Y, WANG X R, SHOU J, et al. Roles of Hcp family proteins in the pathogenesis of the porcine extraintestinal pathogenic Escherichia coli type VI secretion system[J/OL]. Sci Rep, 2016. 6:26816.[2020-06-01]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882540/pdf/srep26816.pdf.
[21]	彭颖. 猪源肠外致病性大肠杆菌六型分泌系统hcp基因缺失株的构建及功能研究[D]. 武汉:华中农业大学, 2015.PENG Y. Construction and function study of pig-derived extraintestinal pathogenic Escherichia coli type VI secretion system hcp gene deletion strain[D]. Wuhan:Huazhong Agricultural University, 2015. (in Chinese)
[22]	DE PACE F, NAKAZATO G, PACHECO A, et al. The type VI secretion system plays a role in type 1 fimbria expression and pathogenesis of an avian pathogenic Escherichia coli strain[J]. Infect Immun, 2010, 78(12):4990-4998.
[23]	RAMÍREZ M R, ALMANZA Y, GARCÍA S, et al. Adherence and invasion of avian pathogenic Escherichia coli to avian tracheal epithelial cells[J]. World J Microbiol Biotechnol, 2009, 25(6):1019-1023.
[24]	POURBAKHSH S A, DHO-MOULIN M, BRÉE A, et al. Localization of the in vivo expression of P and F1 fimbriae in chickens experimentally inoculated with pathogenic Escherichia coli[J]. Microb Pathog, 1997, 22(6):331-341.
[25]	WAICKMAN A T, KELLER H R, KIM T H, et al. The cytokine receptor IL-7Rα impairs IL-2 receptor signaling and constrains the in vitro differentiation of Foxp3+ Treg cells[J/OL]. iScience, 2020, 23(8):101421.[2020-06-01]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424196/.
[26]	SONG X J, JIANG H Y, QI Z, et al. APEC infection affects cytokine-cytokine receptor interaction and cell cycle pathways in chicken trachea[J]. Res Vet Sci, 2020, 130:144-152.
[27]	SONG X J, HOU M M, JIANG H Y, et al. Hcp2a of type VI secretion system contributes to IL8 and IL1β expression of chicken tracheal epithelium by affecting APEC colonization[J]. Res Vet Sci, 2020, 132:279-284.
[28]	XUE M, RAHEEM M A, GU Y, et al. The KdpD/KdpE two-component system contributes to the motility and virulence of avian pathogenic Escherichia coli[J]. Res Vet Sci, 2020, 131:24-30.
[29]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J]. Methods, 2001, 25(4):402-408.
[30]	NAVARRO-GARCIA F, RUIZ-PEREZ F, CATALDI Á, et al. Type VI secretion system in pathogenic Escherichia coli:structure, role in virulence, and acquisition[J]. Front Microbiol, 2019, 10:1965.
[31]	SI M R, WANG Y, ZHANG B, et al. The type VI secretion system engages a redox-regulated dual-functional heme transporter for zinc acquisition[J]. Cell Rep, 2017, 20(4):949-959.
[32]	马家乐. Ⅵ型分泌系统参与大肠杆菌致病进程及细菌间竞争机制[D].南京:南京农业大学,2017.MA J L. Type Ⅵ secretion system participating in the pathogenic process of Escherichia coli and the competition mechanism between bacteria[D]. Nanjing:Nanjing Agricultural College, 2017. (in Chinese)
[33]	DE PACE F, BOLDRIN DE PAIVA J, NAKAZATO G, et al. Characterization of IcmF of the type VI secretion system in an avian pathogenic Escherichia coli (APEC) strain[J]. Microbiology (Reading), 2011, 157(10):2954-2962.
[34]	ZMRHAL V, SLAMA P. Current knowledge about interactions between avian dendritic cells and poultry pathogens[J/OL]. Dev Comp Immunol, 2020, 104:103565.[2020-06-01]. https://www.sciencedirect.com/science/article/pii/S0145305X19304914?via%3Dihub.
[35]	邓志文, 王泽平, 李倩文, 等. 禽致病性大肠杆菌双组分系统phoP/Q对鞭毛Ⅲ型分泌系统基因的调控预测[J]. 畜牧兽医学报, 2019, 50(4):821-829.DENG Z W, WANG Z P, LI Q W, et al. Prediction of the regulation of flagell in type Ⅲ secretion system genes by two-component system phoP/Q in avian pathogenic Escherichia coli[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(4):821-829.
[36]	DING X Y, ZHANG Q, WANG H, et al. The different roles of hcp1 and hcp2 of the type VI secretion system in Escherichia coli strain CE129[J]. J Basic Microbiol, 2018, 58(11):938-946.
[37]	HARADA T, YOSHIOKA H, YOSHIDA S, et al. Increased interleukin-6 levels in peritoneal fluid of infertile patients with active endometriosis[J]. Am J Obstet Gynecol, 1997, 176(3):593-597.
[38]	LI L, WANG Y N, JIA H B, et al. The type VI secretion system protein AsaA in Acinetobacter baumannii is a periplasmic protein physically interacting with TssM and required for T6SS assembly[J/OL]. Sci Rep, 2019, 9(1):9438.[2020-06-01]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602968/.
[39]	MOL N, PENG L, ESNAULT E, et al. Avian pathogenic Escherichia coli infection of a chicken lung epithelial cell line[J]. Vet Immunol Immunopathol, 2019, 210:55-59.
[40]	范海涛,李恒,郭磊,等.白细胞介素1受体1和白细胞介素6双基因敲除小鼠的构建及基因型鉴定[J].国际免疫学杂志, 2018, 41(3):251-255.FAN H T, LI H, GUO L, et al. Construction and genotype identification of double gene knockout mice of interleukin 1 receptor 1 and interleukin 6[J]. International Immunology, 2018, 41(3):p.251-255.
[41]	AKDIS M, BURGLER S, CRAMERI R, et al. Interleukins, from 1 to 37, and interferon-γ:receptors, functions, and roles in diseases[J]. J Allergy Clin Immunol, 2011, 127(3):701-721.
[42]	UMEHARA H, BOLLM E, OKAZAKI T, et al. Fractalkine and vascular injury[J]. Trends Immunol, 2001, 22(11):602-607.