CONSTRUCTION AND BIOLOGICAL CHARACTERISTICS ANALYSIS OF CPXRA MUTANT IN AEROMONAS HYDROPHILA
-
摘要: 为了探讨Cpx系统在嗜水气单胞菌生长及毒力等方面发挥的作用, 利用融合PCR和基因同源重组原理, 以自杀质粒pRE112为载体构建缺失57—1879 bp序列的cpxR-A基因簇突变株 Δcpx, 然后比较缺失株和野生株在生长、生物膜形成、应激耐受及毒力等生物学特性方面的差异。普通PCR及荧光定量PCR结果验证了突变株中cpxRA基因簇片段的部分缺失, 表明突变株构建成功; 生物学特性研究结果显示, 突变株在形态、生长、生物膜形成及毒力等方面与野生株没有显著差异, 两者主要在应对高渗透压、SDS (十二烷基磺酸钠)刺激及含有EDTA (乙二胺四乙酸二钠)或多黏菌素B环境表现不同。结果表明Cpx双组分系统在嗜水气单胞菌应对外界刺激方面扮演着重要角色, 但在毒力方面则可能处于次要地位。Abstract: To investigate the roles of two Cpx component systems in the growth and virulence of Aeromonas hydrophila, we constructed the cpxRA gene cluster mutant Δcpx with the deletion of 57—1879 bp using suicide plasmid pRE112 as the vector. This procedure was based on fusion PCR and gene homologous recombination principles. Through electrophoresis and fluorescence quantitative PCR, the partial deletion of the cpxRA gene cluster in the mutant was confirmed. Then we compared the differences between mutant and wild strains in biological characteristics including growth, biofilm formation, stress tolerance and virulence. The results showed that the mutant had no significant difference with the wild strain in morphology, growth, biofilm formation and virulence. The main differences existed in the response to high osmotic pressure, SDS (Sodium dodecyl sulfate), EDTA (Ethylene Diamine Tetraacetic Acid) and polymyxin B stimulation. This study reveals that the Cpx system of Aeromonas hydrophila is involved in the response to external stimulus factors, and plays a relatively minor role in virulence.
-
Keywords:
- Aeromonas hydrophila /
- cpxRA /
- pRE112 /
- Stress tolerance
-
-
![]()
图 2 基因缺失株的构建和鉴定
图A中, M1. DL2000 Marker; 1、2. 上游片段F1; 3、4. 下游片段F2; 5、6.融合片段F1F2; 图B中, 1—3是重组质粒pRE-Δcpx双酶切的3个重复, M1. DL2000 Marker; 图C中, M1. DL2000 Marker; M2. DL5000 Marker; 1—4. 以cpx-incheckF/R为引物PCR扩增; 5—8. 以cpx-5O/3O为引物PCR扩增; 9—12. 以cpx-outcheckF/R为引物PCR扩增; 1、2、5、6、9、10. 以野生株基因组为模板PCR扩增; 3、4、7、8、11、12.以突变株基因组为模板PCR扩增
Figure 2. Construction and identification of cpxRA mutant strain
Fig. A: M1. DL2000 Marker; 1, 2. Upstream fragment F1; 3, 4. downstream fragment F2; 5, 6. fusion fragment F1F2; Fig.B: 1—3. recombination plasmid pRE-Δcpx; M1. DL2000 Marker; Fig.C: M1. DL2000 Marker; M2. DL5000 Marker; 1—4. PCR amplification using cpx-incheckF/R as primers; 5—8. PCR amplification using cpx-5O/3O as primers; 9—12. PCR amplification using cpx-outcheckF/R as primers; 1, 2, 5, 6, 9, 10. PCR amplification using wild strain genome as template; 3, 4, 7, 8, 11, 12. PCR amplification using mutant genome as template
![]()
图 3 互补株的鉴定
1—3. 以待鉴定菌株基因组为模板PCR扩增; G. 以野生株基因组为模板PCR扩增; Δ.以突变株基因组为模板PCR扩增; p. 以互补质粒pACYC-CΔcpx为模板PCR扩增; M. DL5000/2000 Marker
Figure 3. Identification of cpxRA complementary strain
1—3. PCR amplification using candidate strains genome as template; G. PCR amplification using wild strain genome as template; Δ. PCR amplification using mutant genome as template; p. PCR amplification using complement plasmid pACYC-CΔcpx as template; M. DL5000/2000 Marker
![]()
图 9 斑马鱼浸泡攻毒累积存活曲线
Figure 9. The survival curve of zebrafish attacked by Aeromonas hydrophila strains soak
表 1 本研究中使用的引物
Table 1 Primers used in this study
引物Primers 序列Sequences (5′—3′) 用途Usage cpx-5O CGCTCTAGACAGGTCGGAGCGGTAGT Amplification of up-stream of gene cpxR:F1 cpx-5I CGAAAGAAGGGCAGGAACTCGGTGAGCAACTGGGT cpx-3O CCCGGTACCTTGGTGGTACAGGCGAAT Amplification of down-stream of gene cpxA:F2 cpx-3I ACCCAGTTGCTCACCGAGTTCCTGCCCTTCTTTCG cpx-incheck-F TTGAGCAGGGAGGAGATG Identification of cpxRA mutant cpx-incheck-R TGTCATCCCACTCAAACCC cpx-outcheck-F AGACCTCCTCCTGACCT Identification of cpxRA mutant cpx-outcheck-R TGCACCGATTCATAGC PRE-check-F TTCGTCTCAGCCAATC Sequencing the recombinant plasmid pRE-Δcpx pRE-check-R TGGTGCGTACCGGGTTG com-F CCCAAGCTTGGTAATCAGCAGGGTGGC Construction of cpxRA complementary strain com-R GTGAGCATGCGAGTCTGCTCAGCCGATG 注: 引物序列下划线部分表示酶切位点Note: The underline primer sequence represents restriction site 
下载: 导出CSV
表 2 荧光定量PCR引物
Table 2 Primers used in qRT-PCR
引物编号
Primers引物序列
Sequences (5′—3′)编码基因
CDScpxR-F GCTGCTGGACGTGATGATGC CpxR cpxR-R CGTTGACCCGATCCTGGC cpxA_F GCTGTTGCTGGTGGTGGC CpxA cpxA-R AATCCTGATTGGGGTCTG 16s-F CAACCCCTGTCCTTTGTT 16S rRNA 16s-R TTTGGGATTCGCTCACTA alt-F TGCTGGAGCTGAGCTTTG Alt alt-R CTGTCCTTGAGGGAGTCG hly-F GATGGCATCGGTGGCATA Hly hly-R CGCTGGACGAAGAGTCGG aer-F TAACCCGGCCCCATTATT Aer aer-R CGGCAGAGCCCGTCTATC env-F TCTCCTATGCCACCTTCT EnvZ env-R TCGGCGTCTTCACTCAAC omp-F TCAGGCTGAAGTTCTCAC OmpR omp-R AATACAAGGTTCTGGTCG QseB-F GTCACGGGCGGTGAGGAT QseB QseB-R TCAAGAGCGAGGAGTTTG QseC-F GAGATGAGCCACCACAGC QseC QseC-R CAACAACGTCACCAAGGA dsbA-F TTCCTTGCTGCCATGCTG DsbA dsbA-R GTTGGGCGCTACCGGTCT RpoE-F TTCGCCACCGCCATAGTA RpoE RpoE-R CGCAGGAGGCATTCATCA ExbD1-F CTGCTTGTCTGGCTGGTT ExbD1 ExbD1-R TGGTGCTGCTCATCGTCT b561-F GAGCATCAGGGGGGAGAG B561 b561-R ATGGAGTTCAGGGACATC FimA F AAGTGGGGAAGAGATCGTG FimA FimA R ATAAAGTCGGCGGAGGCAT PilA F GAATTGATGATCGTGGTCG PilA PilA R CGCTGTTGCTATGTTTGCC ycfS F GTCGATCCCTATTTGCCAA YcfS ycfS R CACCATCACCTCGTTTTTA HK-F CGCAGCAGAGCCATCCACAA Histidine HK-R AGTCCGCCGATCCACCACAT kinases
下载: 导出CSV
表 3 斑马鱼腹腔注射累积死亡情况
Table 3 The cumulative death of zebrafish injected A. hydrophila strains by intraperitoneal
菌株
Strain菌液浓度(cfu/尾)
Concentration累积死亡数
Cumulative death number累积死亡率
Cumulative mortality rate (%)1 2 3 4 5 6 7 Wild 6450 1 14 15 15 15 15 15 100 3225 0 12 13 13 13 13 13 86.67 1613 1 6 12 12 12 12 12 80 807 0 7 7 7 7 8 8 53.33 Δcpx 6600 1 13 15 15 15 15 15 100 3300 3 12 13 14 14 14 14 93.33 1650 2 7 11 11 11 11 11 73.33 825 0 9 9 9 9 9 9 60.00
下载: 导出CSV
-
[1] Thune R L, Stanley L A, Cooper R K. Pathogenesis of gram-negative bacterial infections in warm water fish [J]. Annual Reviews of Fish Diseases, 1993, 3: 145—185
[2] Popoff M. Aeromonas. Krieg N R, editor. Bergy’s Manual of Systematic Bacteriology, vol. 1. Baltimore [M]. Williams &Wilkins, 1984, 545—548
[3] Altwegg M, Geiss H K. Aeromonas as a human pathogen [J]. Critical Reviews in Microbiology, 1989, 16: 253—286
[4] Austin B, Austin D A. Bacterial fish pathogens. Disease of Farmed and Wild Fish [M]. 5th ed Chichester: Springer Praxis, 2012, 119—146
[5] Cipriano R C. Aeromonas hydrophila and Motile Aeromonad Septicemias of Fish [M]. Fish Disease Leaflet, 2001, 68
[6] Conrad M. Cross-scale information processing in evolution, development and intelligence [J]. Biosystems, 1996, 38(2—3): 97—109
[7] Raivio T L, Silhavy T J. Transduction of envelope stress in Escherichia coli by the Cpx two-component system [J]. Journal of Bacteriology, 1997, 179(24): 7724—7733
[8] 张旭杰, 杨五名, 李彤彤, 等. 湖北地区暴发病池塘中嗜水气单胞菌的遗传多样性和毒力特征研究. 水生生物学报, 2013, 37(3): 458—466 Zhang X J, Yang W M, Li T T, et al. The genetic diversity and virulence characteristics of Aeromonas hydrophila isolated from fishponds with disease outbreaks in Hubei province [J]. Acta Hydrobiologica Sinica, 2013, 37(3): 458—466
[9] Ho S N, Hunt H D, Horton R M, et al. Site-directed mutagenesis by overlap extension using the polymerase chain-reaction [J]. Gene, 1989, 77(1): 51—59
[10] Hu Y H, Liu C S, Hou J H, et al. Identification, characterization, and molecular application of a virulence-associated autotransporter from a pathogenic Pseudomonas fluorescens strain [J]. Applied and Environmental Microbiology, 2009, 75(13): 4333—4340
[11] Kristin S, Emina C, Elke H, et al. Molecular and proteome analyses highlight the importance of the Cpx envelope stress system for acid stress and cell wall stability in Escherichia coli [J]. Microbiology Open, 2016, 5(4): 582—596
[12] 李爱华. 我国鱼类病原菌耐药性、耐药质粒及几种药物抗菌作用的研究. 中国科学院水生生物研究所博士学位论文. 武汉. 1998 Li A H. The study of the drug resistance, resistance plasmid of fish pathogenic bacteria, and the antibacterial action of several drug in China [D]. Ph D thesis, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan. 1998
[13] Acosta N, Pukatzki S, Raivio T L. The Vibrio cholerae Cpx envelope stress response senses and mediates adaptation to low iron [J]. Journal of Bacteriology, 2015, 197: 262—276
[14] Raivio T L, Leblanc S K, Price N L, et al. The Escherichia coli Cpx envelope stress response regulates genes of diverse function that impact antibiotic resistance and membrane integrity [J]. Journal of Bacteriology, 2013, 195: 2755—2767
[15] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–ΔΔCt) method [J]. Methods, 2001, 25(4): 402—408
[16] McEwen J, Silverman P. Chromosomal mutations of Escherichia coli that alter expression of conjugative plasmid functions [J]. Proceedings of the National Academy of Sciences of the United States of America, 1980, 77: 513—517
[17] Cosma C L, Danese P N, Carlson J H, et al. Mutational activation of the Cpx signal transduction pathway of Escherichia coli suppresses the toxicity conferred by certain envelope-associated stresses [J]. Molecular Microbiology, 1995, 18: 491—505
[18] De W P, Lin E C. Cpx Two-Component Signal Transduction in Escherichia coli: Excessive CpxR-P levels underlie CpxA* phenotypes [J]. Journal of Bacteriology, 2000, 182(5): 1423—1426
[19] De W P, Kwon O, Lin E C. The CpxRA signal transduction system of Escherichia coli: Growth-related autoactivation and control of unanticipated target operons [J]. Journal of Bacteriology, 1999, 181(21): 6772—6778
[20] Gal-Mor O, Segal G. Identification of cpxR as a positive regulator of icm and dot virulence genes of Legionella pneumophila [J]. Journal of Bacteriology, 2003, 185(16): 4908—4919
[21] Jubelin G, Vianney A, Beloin C, et al. Cpx R/Omp R interplay regulates curli gene expression in response to osmolarity in Escherichia coli [J]. Journal of Bacteriology, 2005, 187(6): 2038—2049
[22] Gerken H, Misra R. MzrA-EnvZ interactions in the periplasm influence the EnvZ/OmpR two-component regulon [J]. Journal of Bacteriology, 2010, 192(23): 6271—6278
[23] Blanvillain S, Meyer D, Boulanger A, et al. Plant carbohydrate scavenging through tonB-dependent receptors: a feature shared by phytopathogenic and aquatic bacteria [J]. PLoS One, 2007, 2: e224
[24] Schauer K, Gouget B, Carrière M, et al. Novel nickel transport mechanism across the bacterial outer membrane energized by the TonB/ExbB/ExbD machinery [J]. Molecular Microbiology, 2007, 63: 1054—1068
[25] Humphreys S, Rowley G, Stevenson A, et al. Role of the two-component regulator Cpx AR in the virulence of Salmonella entetica serotype typhimurium [J]. Infection and Immunity, 2004, 72(8): 4654—4661
[26] 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]. Infection and Immunity, 2013, 81(5): 1450—1459
计量
- 文章访问数: 1836
- HTML全文浏览量: 563
- PDF下载量: 38