双组分系统CpxR影响禽致病性大肠杆菌的耐药性、抗血清杀菌能力及毒力

doi:10.11843/j.issn.0366-6964.2021.04.020

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (4): 1053-1060.doi: 10.11843/j.issn.0366-6964.2021.04.020

双组分系统CpxR影响禽致病性大肠杆菌的耐药性、抗血清杀菌能力及毒力

易正飞¹, 张耀东¹, 王瑶^1,2, 朱红¹, 陶程琳^1,3, AFAYIBO Dossêh Jean Apôtre¹, 李涛¹, 祁晶晶¹, 田明星¹, 丁铲¹, 于圣青^1*, 王少辉^1*

1. 中国农业科学院上海兽医研究所, 上海 200241;
2. 西南民族大学生命科学与技术学院, 成都 610041;
3. 扬州大学兽医学院, 扬州 225009

收稿日期:2020-09-02 出版日期:2021-04-23 发布日期:2021-04-25
通讯作者: 王少辉,主要从事畜禽细菌性传染病研究,Tel:021-34293412;E-mail:shwang0827@126.com;于圣青,主要从事畜禽细菌性传染病研究,Tel:021-34293461,E-mail:yus@shvri.ac.cn
作者简介:易正飞(1992-),男,安徽马鞍山人,硕士,主要从事禽致病性大肠杆菌致病机制研究,E-mail:zhengfeiyi666@163.com
基金资助:
国家自然科学基金项目（31972654；31872483）；上海市浦江人才计划项目（2019PJD057）

Two Component System CpxR Contributes to the Antibiotic Resistance, Serum Bactericidal Resistance and Virulence of Avian Pathogenic Escherichia coli

YI Zhengfei¹, ZHANG Yaodong¹, WANG Yao^1,2, ZHU Hong¹, TAO Chenglin^1,3, AFAYIBO Dossêh Jean Apôtre¹, LI Tao¹, QI Jingjing¹, TIAN Mingxing¹, DING Chan¹, YU Shengqing^1*, WANG Shaohui^1*

1. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China;
2. College of Life Science and Technology, Southwest Minzu University, Chengdu 610041, China;
3. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China

Received:2020-09-02 Online:2021-04-23 Published:2021-04-25

摘要/Abstract

摘要： CpxR是细菌中Cpx双组分系统（two component system，TCS）的反应调控蛋白，通过调控靶基因的转录表达，在细菌细胞膜稳定及毒力方面发挥作用。本研究旨在探究TCS CpxR对禽致病性大肠杆菌（avian pathogenic Escherichia coli，APEC）基本生物学特性、抗血清杀菌能力及致病性的影响。利用Red同源重组系统及互补质粒构建cpxR基因缺失株、互补株，然后比较分析野生株、基因缺失株与互补株的生长曲线、运动性、生物被膜形成能力、药物敏感性、抗血清杀菌能力、动物致病性的差异。结果显示：cpxR基因缺失株与野生株、互补株的生长速度和运动性能无明显差异，且缺失cpxR基因不影响APEC的生物被膜形成能力。然而，缺失CpxR导致APEC对阿米卡星和卡那霉素耐药性降低。血清杀菌试验结果显示，CpxR有助于APEC的抗血清杀菌能力。动物感染试验结果显示，野生株、cpxR基因缺失株和互补株对雏鸭的半数致死量（LD₅₀）分别为7.50×10⁵、7.50×10⁶、1.33×10⁶ CFU，表明CpxR缺失显著降低APEC的毒力。综上表明，TCS CpxR在APEC耐药性、抗血清杀菌能力及毒力方面发挥作用，为阐明APEC的环境适应性、生存能力及致病机制提供参考。

关键词: 禽致病性大肠杆菌, CpxR, 耐药性, 抗血清杀菌, 毒力

Abstract: CpxR is the response regulator of the Cpx two component system (TCS), which is involving in maintaining stability of membrane and virulence through regulating the transcriptional expression of target genes in bacteria. This study investigated the effects of TCS CpxR on the biological characteristics, serum bactericidal resistance and pathogenicity of avian pathogenic Escherichia coli (APEC). The cpxR gene mutant and complemented strains of APEC was constructed by Red homologous recombination system and complementation plasmid. Then, the growth curve, motility, biofilm formation, antibiotic sensitivity, serum bactericidal resistance, virulence among wild-type, cpxR mutant and complemented strains were compared and analyzed. There is no significant difference in growth rate and motility among wild-type, cpxR mutant and complemented strains. Moreover, CpxR did not affect the biofilm formation of APEC. However, the antibiotic sensitivity assays showed that cpxR mutant was less resistant to amikacin and kanamycin. The results indicated that of CpxR could enhance the survival ability of APEC in serum. Animal infection experiment showed that the median lethal dose (LD₅₀) of wild-type, cpxR mutant and complemented strains were 7.50×10⁵, 7.50×10⁶ and 1.33×10⁶ CFU respectively, indicating that cpxR mutant strain exhibited attenuated virulence in ducks. These data indicated that TCS CpxR contributes to the antibiotic resistance, serum bactericidal resistance and virulence of APEC, providing help for elucidating the environment adaptability, survival ability and pathogenic mechanism of APEC.

Key words: avian pathogenic Escherichia coli, CpxR, antibiotic resistance, serum bactericidal resistance, virulence

中图分类号:

S852.612

易正飞, 张耀东, 王瑶, 朱红, 陶程琳, AFAYIBO Dossêh Jean Apôtre, 李涛, 祁晶晶, 田明星, 丁铲, 于圣青, 王少辉. 双组分系统CpxR影响禽致病性大肠杆菌的耐药性、抗血清杀菌能力及毒力[J]. 畜牧兽医学报, 2021, 52(4): 1053-1060.

YI Zhengfei, ZHANG Yaodong, WANG Yao, ZHU Hong, TAO Chenglin, AFAYIBO Dossêh Jean Apôtre, LI Tao, QI Jingjing, TIAN Mingxing, DING Chan, YU Shengqing, WANG Shaohui. Two Component System CpxR Contributes to the Antibiotic Resistance, Serum Bactericidal Resistance and Virulence of Avian Pathogenic Escherichia coli[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(4): 1053-1060.

参考文献 32

[1]	RUSSO T A, JOHNSON J R. Proposal for a new inclusive designation for extraintestinal pathogenic isolates of Escherichia coli:ExPEC[J]. J Infect Dis, 2000, 181(5):1753-1754.
[2]	RODRIGUEZ-SIEK K E, GIDDINGS C W, DOETKOTT C, et al. Comparison of Escherichia coli isolates implicated in human urinary tract infection and avian colibacillosis[J]. Microbiology, 2005, 151(6):2097-2110.
[3]	MANGES A R. Escherichia coli and urinary tract infections:the role of poultry-meat[J]. Clin Microbiol Infect, 2016, 22(2):122-129.
[4]	BAUCHART P, GERMON P, BRÉE A, et al. Pathogenomic comparison of human extraintestinal and avian pathogenic Escherichia coli——search for factors involved in host specificity or zoonotic potential[J]. Microb Pathog, 2010, 49(3):105-115.
[5]	STOCK A M, ROBINSON V L, GOUDREAU P N. Two-component signal transduction[J]. Annu Rev Biochem, 2000, 69:183-215.
[6]	XU Y, ZHAO Z, TONG W H, et al. An acid-tolerance response system protecting exponentially growing Escherichia coli[J]. Nat Commun, 2020, 11:1496.
[7]	VOGT S L, SCHOLZ R, PENG Y, et al. Characterization of the Citrobacter rodentium Cpx regulon and its role in host infection[J]. Mol Microbiol, 2019, 111(3):700-716.
[8]	YAN K, LIU T, DUAN B Z, et al. The CpxAR two-component system contributes to growth, stress resistance, and virulence of Actinobacillus pleuropneu-moniae by upregulating wecA transcription[J]. Front Microbiol, 2020, 11:1026.
[9]	LIU M, HAO G J, LI Z, et al. CitAB two-component system-regulated citrate utilization contributes to Vibrio cholerae competitiveness with the gut microbiota[J]. Infect Immun, 2019, 87(3):e00746-18.
[10]	DE WULF P, MCGUIRE A M, LIU X Q, et al. Genome-wide profiling of promoter recognition by the two-component response regulator CpxR-P in Escherichia coli[J]. J Biol Chem, 2002, 277(29):26652-26661.
[11]	RAIVIO T L, SILHAVY T J. Transduction of envelope stress in Escherichia coli by the Cpx two-component system[J]. J Bacteriol, 1997, 179(24):7724-7733.
[12]	HIRAKAWA H, NISHINO K, HIRATA T, et al. Comprehensive studies of drug resistance mediated by overexpression of response regulators of two-component signal transduction systems in Escherichia coli[J]. J Bacteriol, 2003, 185(6):1851-1856.
[13]	CAO Q, FENG F F, WANG H, et al. Haemophilus parasuis CpxRA two-component system confers bacterial tolerance to environmental stresses and macrolide resistance[J]. Microbiol Res, 2018, 206:177-185.
[14]	DE LA CRUZ M A, PÉREZ-MORALES D, PALACIOS I J, et al. The two-component system CpxR/A represses the expression of Salmonella virulence genes by affecting the stability of the transcriptional regulator HilD[J]. Front Microbiol, 2015, 6:807.
[15]	DEBNATH I, NORTON J P, BARBER A E, et al. The Cpx stress response system potentiates the fitness and virulence of uropathogenic Escherichia coli[J]. Infect Immun, 2013, 81(5):1450-1459.
[16]	WANG S H, DAI J J, MENG Q M, et al. DotU expression is highly induced during in vivo infection and responsible for virulence and Hcp1 secretion in avian pathogenic Escherichia coli[J]. Front Microbiol, 2014, 5:588.
[17]	DATSENKO K A, WANNER B L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J]. Proc Natl Acad Sci USA, 2000, 97(12):6640-6645.
[18]	黄慧, 刘保光, 孙亚伟, 等. 鼠伤寒沙门菌cpxR和acrB双基因缺失菌株的构建及其对抗菌药物敏感性分析[J]. 畜牧兽医学报, 2016, 47(3):595-602.HUANG H, LIU B G, SUN Y W, et al. Construction of cpxR and acrB double gene deletion strain of salmonella enterica serovar typhimurium and analysis of its susceptibility to antibacterial agents[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(3):595-602. (in Chinese)
[19]	吴同垒, 苏硕青, 李巧玲, 等. 狐源沙门菌cpxR基因缺失菌株的构建及部分表型研究[J]. 中国兽医学报, 2019, 39(3):456-460.WU T L, SU S Q, LI Q L, et al. Construction of cpxR gene-knockout strain of Salmonella and its partial phenotype study[J]. Chinese Journal of Veterinary Science, 2019, 39(3):456-460. (in Chinese)
[20]	CLSI. Performance standards for antimicrobial susceptibility testing[M]. 28th ed. Wayne, PA:Clinical and Laboratory Standards Institute, 2018.
[21]	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, 50:40.
[22]	ZHUANG Q Y, WANG S C, LI J P, et al. A clinical survey of common avian infectious diseases in China[J]. Avian Dis, 2014, 58(2):297-302.
[23]	MITCHELL N M, JOHNSON J R, JOHNSTON B, et al. Zoonotic potential of Escherichia coli isolates from retail chicken meat products and eggs[J]. Appl Environ Microbiol, 2015, 81(3):1177-1187.
[24]	SUBRAMANIAM S, MVLLER V S, HERING N A, et al. Contribution of the Cpx envelope stress system to metabolism and virulence regulation in Salmonella enterica serovar Typhimurium[J]. PLoS One, 2019, 14(2):e0211584.
[25]	SHETTY D, ABRAHANTE J E, CHEKABAB S M, et al. Role of CpxR in biofilm development:Expression of key fimbrial, O-antigen and virulence operons of Salmonella enteritidis[J]. Int J Mol Sci, 2019, 20(20):5146.
[26]	JUBELIN G, VIANNEY A, BELOIN C, et al. CpxR/OmpR interplay regulates curli gene expression in response to osmolarity in Escherichia coli[J]. J Bacteriol, 2005, 187(6):2038-2049.
[27]	MATTER L B, ARES M A, ABUNDES-GALLEGOS J, et al. The CpxRA stress response system regulates virulence features of avian pathogenic Escherichia coli[J]. Environ Microbiol, 2018, 20(9):3363-3377.
[28]	KURABAYASHI K, HIRAKAWA Y, TANIMOTO K, et al. Role of the CpxAR two-component signal transduction system in control of fosfomycin resistance and carbon substrate uptake[J]. J Bacteriol, 2014, 196(2):248-256.
[29]	MAHONEY T F, SILHAVY T J. The Cpx stress response confers resistance to some, but not all, bactericidal antibiotics[J]. J Bacteriol, 2013, 195(9):1869-1874.
[30]	BATCHELOR E, WALTHERS D, KENNEY L J, et al. The Escherichia coli CpxA-CpxR envelope stress response system regulates expression of the porins ompF and ompC[J]. J Bacteriol, 2005, 187(16):5723-5731.
[31]	SERRUTO D, RAPPUOLI R, SCARSELLI M, et al. Molecular mechanisms of complement evasion:learning from Staphylococci and Meningococci[J]. Nat Rev Microbiol, 2010, 8(6):393-399.
[32]	MATSUDA K, CHAUDHARI A A, KIM S W, et al. Physiology, pathogenicity and immunogenicity of lon and/or cpxR deleted mutants of Salmonella Gallinarum as vaccine candidates for fowl typhoid[J]. Vet Res, 2010, 41(5):59.

双组分系统CpxR影响禽致病性大肠杆菌的耐药性、抗血清杀菌能力及毒力

Two Component System CpxR Contributes to the Antibiotic Resistance, Serum Bactericidal Resistance and Virulence of Avian Pathogenic Escherichia coli

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