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嗜水气单胞菌Sec分泌系统亚基SecD的生理功能研究

傅钰瑛 张丽珊 SRINWASAN Ramanathan 李碗芯

傅钰瑛,张丽珊,SRINWASAN Ramanathan,等. 嗜水气单胞菌Sec分泌系统亚基SecD的生理功能研究 [J]. 福建农业学报,2023,38(5):598−606 doi: 10.19303/j.issn.1008-0384.2023.05.011
引用本文: 傅钰瑛,张丽珊,SRINWASAN Ramanathan,等. 嗜水气单胞菌Sec分泌系统亚基SecD的生理功能研究 [J]. 福建农业学报,2023,38(5):598−606 doi: 10.19303/j.issn.1008-0384.2023.05.011
FU Y Y, ZHANG L S, SRINWASAN R, et al. Physiological Functions of SecD in Aeromonas hydrophila [J]. Fujian Journal of Agricultural Sciences,2023,38(5):598−606 doi: 10.19303/j.issn.1008-0384.2023.05.011
Citation: FU Y Y, ZHANG L S, SRINWASAN R, et al. Physiological Functions of SecD in Aeromonas hydrophila [J]. Fujian Journal of Agricultural Sciences,2023,38(5):598−606 doi: 10.19303/j.issn.1008-0384.2023.05.011

嗜水气单胞菌Sec分泌系统亚基SecD的生理功能研究

doi: 10.19303/j.issn.1008-0384.2023.05.011
基金项目: 福建省教育厅中青年项目(JAT210724);中国博士后科学基金面上资助(2019M662214)
详细信息
    作者简介:

    傅钰瑛(1989−),女,博士,讲师,研究方向:病原微生物耐药与致病机制(E-mail:2021003@fjcpc.edu.cn

    通讯作者:

    李碗芯(1990−),女,博士,助理研究员,研究方向:细菌耐药性(E-mail: liwanxin@fjmu.edu.cn

  • 中图分类号: Q93-3

Physiological Functions of SecD in Aeromonas hydrophila

  • 摘要:   目的  系统研究嗜水气单胞菌Sec分泌系统亚基SecD(Protein-export membrane protein,SecD)的生理功能。  方法  利用pRE112自杀载体,通过同源重组的方法,构建secD基因缺失株(标记为∆secD)。以嗜水气单胞菌野生型菌株为对照组,分别采用绵羊血、牛奶固体培养基测定∆secD 的溶血性和胞外蛋白酶活性;结晶紫染色法结合多功能酶标仪测定生物被膜形成能力;利用全自动生长曲线仪进行细菌酸碱及高渗透压耐受性分析;采用二倍稀释法系统评估细菌抗生素耐药性。  结果  与嗜水气单胞菌野生型菌株相比,发现∆secD生物被膜形成能力降低,溶血性、胞外蛋白酶活性显著增强,碱性和高渗透压环境胁迫的耐受性增强,对盐酸土霉素、四环素、依诺沙星和美罗培南的MIC提高4倍,对环丙沙星和诺氟沙星的MIC提高2倍,而对红霉素和头孢噻肟钠的MIC分别降低了2倍和4倍。  结论  发现Sec分泌系统的亚基SecD参与嗜水气单胞菌毒力因子、抗生素耐药蛋白等的跨膜转运,对以Sec分泌系统的亚单位为靶标开发新一代抗菌药物提供理论依据,对预防和控制嗜水气单胞菌引起疾病的发生和传播可能具有重要的科学意义。
  • 图  1  目的基因同源臂及引物设计

    Figure  1.  Design of homologous arm and primer of target gene

    图  2  ΔsecD缺失株的验证

    M:5000 Marker;1:以嗜水气单胞菌为模板,P5、P6为引物的扩增产物;2:以敲除菌为模板,P5、P6为引物的扩增产物;3:以嗜水气单胞菌为模板,P7、P8为引物的扩增产物;4:以敲除菌为模板,P7、P8为引物的扩增产物。

    Figure  2.  Verification of ΔsecD strain

    M: 5000 Marker; 1: PCR products by P5 and P6 primers with A.hydrophila template; 2: PCR products by P5 and P6 primers with ΔsecD template; 3: PCR products by P7 and P8 primers with A.hydrophila template; 4: PCR products by P7 and P8 primers with ΔsecD template.

    图  3  ΔsecD生理表型

    A:生长曲线检测;B:生物被膜形成能力检测;C:溶血性检测;D:胞外蛋白酶活性检测;****表示P<0.0001。

    Figure  3.  Physiological phenotype of ΔsecD strain

    A: Growth curve examination; B: Biofilm forming ability ; C: Hemolytic activity;D: Extracellular protease activity; **** means P<0.0001.

    图  4  斑马鱼嗜水气单胞菌攻毒存活率

    Figure  4.  Survival rate of zebrafish after challenge by A.hydrophila

    图  5  ΔsecD耐受性检测

    A~E:pH为5、6、7、8、9处理;F:4% NaCl处理;

    Figure  5.  Tolerance of A.hydrophila and ΔsecD strain

    A–E: treatments of pH 5, 6, 7, 8, 9; F: treatment of 4% NaCl.

    表  1  secD基因敲除引物序列

    Table  1.   Primers for secD knockout

    编号
    No.
    寡核苷酸序列(5′-3′)
    Oligonucleotide sequence (5′-3′)
    描述
    Discription
    P1catgaattcccgggagagctcGGGACGCGATCGAGCAGG左臂上游引物
    P2aacatgtctcttccGCTGTGCTCCTCGGGAGATT左臂下游引物
    P3cacagcGGAAGAGACATGTTTCAGATTCTACATT右臂上游引物
    P4cgatcccaagcttcttctagaGGCCAGACGCCATTCGAA右臂下游引物
    P5GTGTTAAATCGCTATCCGCTGTGGA目的基因上游引物
    P6TTAGATCGGCAGCTTGTCGAGG目的基因下游引物
    P7CGATTGCTACACCTGCAAGAACTACA左臂外侧验证上游引物
    P8GATGTACTCGGTATCCCCCTTGC右臂外侧验证下游引物
    下载: 导出CSV

    表  2  ΔsecD抗生素耐药性检测结果

    Table  2.   Antibiotic resistance of A.hydrophila and ΔsecD strain

    分类
    Category
    名称
    Name
    最低抑菌质量浓度
    MIC/(μg·mL−1
    分类
    Category
    名称
    Name
    最低抑菌质量浓度
    MIC/(μg·mL−1
    野生株
    A.hydrophila
    缺失株
    ΔsecD
    野生株
    A.hydrophila
    缺失株
    ΔsecD
    四环素类
    Tetracyclines
    盐酸土霉素
    Oxytetracycline
    hydrochloride
    2.5 10 氨基糖苷类
    Aminoglycosides
    链霉素
    Streptomycin
    20 20
    四环素
    Tetracycline
    2.5 10 硫酸新霉素
    Neomycin sulfate
    6.25 6.25
    盐酸金霉素
    Chlortetracycline
    hydrochloride
    20 20 卡那霉素
    Kanamycin
    12.5 12.5
    多西霉素
    Doxycycline
    10 10 硫酸安普霉素
    Apramycin Sulfate
    25 25
    强力霉素
    Doxycycline
    10 10 庆大霉素
    Gentamicin
    1.25 1.25
    大环内酯类
    Macrolides
    红霉素
    Erythromycin
    32 16 硫酸巴龙霉素
    Paromomycin sulfate
    10 10
    罗红霉素
    Roxithromycin
    32 32 妥布霉素
    Tobramycin
    1.25 1.25
    阿奇霉
    Azithromycin
    0.5 0.5 壮观霉素
    Spectinomycin
    32 32
    那他霉素
    Natamycin
    10 10 阿米卡星
    Amikacin
    12.8 12.8
    乙酰螺旋霉素
    Acetylspiramycin
    512 512 利福霉素类
    Rifamycins
    利福平
    Rifampicin
    6.4 6.4
    奎诺酮类
    Quinolones
    恩诺沙星
    Enrofloxacin
    0.125 0.125 头孢菌素类
    Cephalosporins
    头孢美唑钠
    Cefmetazole
    8 8
    依诺沙星
    Enoxacin
    0.0125 0.0500 头孢米诺
    Cefminox
    64 64
    培氟沙星
    Pefloxacin
    0.125 0.125 头孢唑啉钠
    Cefazolin
    256 256
    环丙沙星
    Ciprofloxacin
    0.00625 0.01250 头孢噻肟钠
    Cefotaxime
    1.2500 0.3125
    巴洛沙星
    Balofloxacin
    0.625 0.625 头孢他啶
    Ceftazidime
    128 128
    萘啶酸
    Nalidixic acid
    0.5 0.5 头孢曲松钠
    Ceftriaxone
    0.25 0.25
    左氧氟沙星
    Levofloxacin
    0.015625 0.015625 多肽类
    Polypeptides
    硫酸粘菌素
    Colistin
    5 5
    氧氟沙星
    Ofloxacin
    0.025 0.025 硫酸多粘菌素
    B Polymyxin B
    6.25 6.25
    诺氟沙星
    Norfloxacin
    0.02 0.04 碳青霉烯类
    Carbapenems
    美罗培南
    Meropenem
    0.0625 0.2500
    莫西沙星
    Moxifloxacin
    0.0625 0.0625 亚胺培南
    Imipenem
    128 128
    下载: 导出CSV
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出版历程
  • 收稿日期:  2022-09-26
  • 修回日期:  2023-03-09
  • 网络出版日期:  2023-04-14
  • 刊出日期:  2023-05-28

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