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

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

1.
福建船政交通职业学院安全与环境学院，福建　福州　350007
2.
福建农林大学生命科学学院，福建　福州　350002
3.
福建农林大学福建省农业生态过程与安全监控重点实验室，福建　福州　350002
4.
福建医科大学公共卫生学院，福建　福州　350122

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
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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

Physiological Functions of SecD in Aeromonas hydrophila

FU Yuying^1
,,
ZHANG Lishan^{2, 3},
SRINWASAN Ramanathan^{2, 3},
LI Wanxin^{4
, ,}

1.
School of Safety and Environment, Fujian Chuanzheng Communications College, Fuzhou, Fujian　350007, China
2.
School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
3.
Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, Fuzhou, Fujian　350002, China
4.
School of Public Health, Fujian Medical University, Fuzhou, Fujian　350122, China

摘要

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。
- 嗜水气单胞菌 /
- SecD /
- Sec分泌系统 /
- 生理功能
Abstract: Objective Physiological functions of the Sec pathway subunit secD of Aeromonas hydrophila were studied. Method An secD-knockout A.hydrophila was constructed by homologous recombination with pRE112 suicide vector. Effects of the genetic deletion on the physiological phenotypes including hemolytic and extracellular protease activities were determined on sheep’s blood and skim milk agar plates; on the biofilm formation, by crystal violet staining combined with SpectraMax® i3; on the bacterial acid-base and hyperosmolarity tolerance, according to the growth curve; and on the minimal inhibitory concentration (MIC) of antibiotics, using the broth microdilution method. Result Compared to wild-type, the constructed ∆secD A.hydrophila showed no significant differences in growth and tolerance to pH 5, 6, 7, and 8. The activities of hemolytic and extracellular protease, as well as the resistance to high osmotic pressure or pH 9 were enhanced. The biofilm forming ability significantly declined. And the resistance to antibiotics varied, as the MIC increased 4 times on oxytetracycline, tetracycline, enoxacin, or meropenem and two times on ciprofloxacin and norfloxacin, but decreased 2 times on erythromycin and 4 times on cefotaxime. Conclusion The Sec pathway subunit secD was confirmed to be involved in the transport of virulence factors and resistance to antibiotics in A.hydrophila. It could be a key element in studies for developing antimicrobial applications and prevention of disease infected by the pathogen.
- Aeromonas hydrophila /
- secD /
- Sec pathway /
- physiological functions

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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

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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.

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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.

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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

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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.

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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
P1	catgaattcccgggagagctcGGGACGCGATCGAGCAGG	左臂上游引物
P2	aacatgtctcttccGCTGTGCTCCTCGGGAGATT	左臂下游引物
P3	cacagcGGAAGAGACATGTTTCAGATTCTACATT	右臂上游引物
P4	cgatcccaagcttcttctagaGGCCAGACGCCATTCGAA	右臂下游引物
P5	GTGTTAAATCGCTATCCGCTGTGGA	目的基因上游引物
P6	TTAGATCGGCAGCTTGTCGAGG	目的基因下游引物
P7	CGATTGCTACACCTGCAAGAACTACA	左臂外侧验证上游引物
P8	GATGTACTCGGTATCCCCCTTGC	右臂外侧验证下游引物

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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）
分类 Category	名称 Name	野生株 A.hydrophila	缺失株 ΔsecD	分类 Category	名称 Name	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	多肽类 Polypeptides	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	碳青霉烯类 Carbapenems	亚胺培南 Imipenem	128	128

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