鼠伤寒沙门菌ompW基因缺失株与回补株的构建及其生物学特性

doi:10.11843/j.issn.0366-6964.2025.04.034

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (4): 1865-1875.doi: 10.11843/j.issn.0366-6964.2025.04.034

鼠伤寒沙门菌ompW基因缺失株与回补株的构建及其生物学特性

葛雷¹^,²^,³(), 仇汝龙¹^,²^,³, 范志宇¹^,²^,³, 胡波¹^,²^,³, 魏后军¹^,²^,³, 陈萌萌¹^,²^,³, 宋艳华¹^,²^,³, 李一鸣¹^,²^,³, 徐为中¹^,²^,³, 王芳¹^,²^,³^,*()

1. 江苏省农业科学院兽医研究所, 南京 210014
2. 农业农村部兽用生物制品工程重点实验室, 南京 210014
3. 兽用生物制品(泰州)国泰技术创新中心, 泰州 225300

收稿日期:2024-05-31 出版日期:2025-04-23 发布日期:2025-04-28
通讯作者: 王芳 E-mail:luckilt@163.com;rwangfang@126.com
作者简介:葛雷(1992-), 男, 江苏南京人, 助理研究员, 博士, 主要从事动物致病菌致病机制研究, E-mail: luckilt@163.com
基金资助:
现代农业产业技术体系建设专项资金(CARS-43-C-1)

Construction and Biological Characterization of ompW Gene Deletion and Complemented Strain of Salmonella Typhimurium

GE Lei¹^,²^,³(), QIU Rulong¹^,²^,³, FAN Zhiyu¹^,²^,³, HU Bo¹^,²^,³, WEI Houjun¹^,²^,³, CHEN Mengmeng¹^,²^,³, SONG Yanhua¹^,²^,³, LI Yiming¹^,²^,³, XU Weizhong¹^,²^,³, WANG Fang¹^,²^,³^,*()

1. Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
2. Key Laboratory for Veterinary Bio-Product Engineering, Ministry of Agriculture, Nanjing 210014, China
3. Guotai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China

Received:2024-05-31 Online:2025-04-23 Published:2025-04-28
Contact: WANG Fang E-mail:luckilt@163.com;rwangfang@126.com

摘要/Abstract

摘要：

鼠伤寒沙门菌(Salmonella Typhimurium)是最为常见的沙门菌血清型，严重危害畜牧业发展及公共卫生安全。外膜蛋白OmpW是由ompW基因编码的营养物质特异性运输通道，发挥特殊的铁离子外排功能，与细菌耐药性、致病性等密切相关。但其在沙门菌中的功能尚未见研究。为探究外膜蛋白OmpW对鼠伤寒沙门菌的影响，本试验以鼠伤寒沙门菌ATCC 14028为野生株(WT14028)，利用λ-Red同源重组技术构建ompW基因缺失株(14028ΔompW)，同时构建回补株(14028ΔompW: : ompW)，通过检测生长特性、生物被膜形成能力、环境耐受性、耐药性等方面探究鼠伤寒沙门菌ompW基因的生物学特性，并通过小鼠感染试验探究该基因对沙门菌致病性的影响。结果表明，缺失株与野生株菌落形态无显著差异；缺失株在对数生长期显著慢于野生株，但到达稳定期无显著差异；缺失株生物被膜形成能力下降31.73%±6.43%；在酸性(pH=3)、碱性(pH=10)及高盐(1 mol·L^-1 NaCl)环境下，缺失株存活率显著低于野生株；但在高铁(>0.625 g·L^-1 FeCl₃)环境下，缺失株存活率更高；缺失株对氨基糖苷类抗生素庆大霉素、卡那霉素的敏感性显著增强，对其他如氨苄西林、四环素等抗生素敏感性无显著变化；小鼠感染试验表明，与野生株相比，缺失株感染诱发的腹泻率更低、脾肿大及肠道损伤更轻微，且细菌移位能力也显著下降。综上，本研究成功构建了鼠伤寒沙门菌ompW基因缺失株及回补株，试验表明该基因与菌株生长、环境耐受性、耐药性、致病性等方面密切相关。本研究为进一步开展沙门菌的致病与耐药机制的研究奠定了基础。

关键词: 鼠伤寒沙门菌, ompW基因, 缺失株, 生物学特性, 致病性

Abstract:

Salmonella Typhimurium is the most common serotype of Salmonella, which seriously endangers the development of animal husbandry and public health safety. Outer membrance protein OmpW, encoded by ompW, is a special nutrient transport channel that has iron ion efflux function and is closely related to drug resistance, pathogenicity and other factors. However, the function of OmpW in Salmonella has not been studied yet. To investigate the effect of OmpW in Salmonella Typhimurium, this experiment used Salmonella Typhimurium ATCC 14028 (WT14028) as a wild strain, constructed ompW gene deletion strain (14028ΔompW) using λ-Red homologous recombination technology and constructed complemented strain (14028ΔompW: : ompW). Growth characteristics, biofilm formation ability, environmental tolerance and antimicrobial resistance were detected to investigate the biological characteristics of ompW. And the impact on the pathogenicity was explored through mouse infection experiments. The results showed that there was no significant difference in colony morphology between the deletion strain and the wild strain. The deletion strain showed significantly slower in logarithmic growth period than the wild strain, but there is no significant difference in stable period. The biofilm formation ability of the deletion strain decreased by 31.73%±6.43%. In acidic (pH=3), alkaline (pH=10) and high salt (1 mol·L^-1 NaCl) environments, the survival rate of the deletion strain was significantly lower than that of the wild strain. But in high iron (>0.625 g·L^-1 FeCl₃) environment, the survival rate of the deletion strain was higher. The sensitivity of the deletion strain to aminoglycoside antibiotics such as gentamicin and kanamycin was significantly enhanced, but there was no significant change in sensitivity to other antibiotics such as ampicillin and tetracycline. The results of the mouse infection experiments showed that the deletion strain induced lower diarrhea rate, milder splenomegaly and intestinal damage compared with the wild strain. And the bacterial translocation ability of the deletion strain also significantly decreased. In summary, this study successfully constructed ompW gene deletion strain and complement strain of Salmonella typhimurium, and experiments showed that ompW is closely related to bacterial growth, environmental tolerance, drug resistance, pathogenicity, and other aspects. This study lays the foundation for further research on the pathogenic mechanism and drug resistance mechanism of Salmonella.

Key words: Salmonella Typhimurium, ompW gene, deletion strain, biological characterization, pathogenicity

中图分类号:

S852.61

葛雷, 仇汝龙, 范志宇, 胡波, 魏后军, 陈萌萌, 宋艳华, 李一鸣, 徐为中, 王芳. 鼠伤寒沙门菌ompW基因缺失株与回补株的构建及其生物学特性[J]. 畜牧兽医学报, 2025, 56(4): 1865-1875.

GE Lei, QIU Rulong, FAN Zhiyu, HU Bo, WEI Houjun, CHEN Mengmeng, SONG Yanhua, LI Yiming, XU Weizhong, WANG Fang. Construction and Biological Characterization of ompW Gene Deletion and Complemented Strain of Salmonella Typhimurium[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1865-1875.

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参考文献 32

1	PETERSEN E , MILLER S I . The cellular microbiology of salmonellae interactions with macrophages[J]. Cell Microbiol, 2019, 21 (11): e13116.
2	陈治海. 蓬安县猪源沙门氏菌的分离鉴定及药物敏感性分析[D]. 重庆: 西南大学, 2020.
	CHEN Z H. Isolationg, identifiaction and antimicrobial susceptibility analysis of pig-associated Salmonella in Peng'an county[D]. Chongqing: Southwest University, 2020. (in Chinese)
3	秦蕾, 吴慧敏, 徐琦琦, 等. 外源MDR鼠伤寒沙门菌对健康小鼠肠道菌群的影响[J]. 畜牧兽医学报, 2023, 54 (5): 2158- 2169. doi: 10.11843/j.issn.0366-6964.2023.05.036
	QIN L , WU H M , XU Q Q , et al. Effect of exogenous drug-resistant Salmonella Typhimurium on intestinal flora in healthy mice[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54 (5): 2158- 2169. doi: 10.11843/j.issn.0366-6964.2023.05.036
4	郭帅, 范志宇, 王芳, 等. 家兔肠炎沙门氏菌病流行病学调查及防控[J]. 中国动物传染病学报, 2019, 27 (2): 42- 47.
	GUO S , FAN Z Y , WANG F , et al. Epidemiological investigation and prevention of Salmonella enteritidis in rabbits[J]. Chinese Journal of Animal Infectious Diseases, 2019, 27 (2): 42- 47.
5	姚素霞, 郝瑞娥, 王洋, 等. 2014—2017年山西省沙门氏菌分子分型及耐药性研究[J]. 中国人兽共患病学报, 2021, 37 (9): 815- 820.
	YAO S X , HAO R E , WANG Y , et al. Analysis of antimicrobial susceptibility and molecular typing of Salmonella in Shanxi Province during 2014-2017[J]. Chinese Journal of Zoonoses, 2021, 37 (9): 815- 820.
6	赵昕. 丁香酚对鼠伤寒沙门氏菌的抗菌作用及机制研究[D]. 扬州: 扬州大学, 2022.
	ZHAO X. The antibacterial activity and mechanism of eugenol against S. typhimurium[D]. Yangzhou: Yangzhou University, 2022. (in Chinese)
7	魏胜男. 基于纳米复合材料的鼠伤寒沙门氏菌可视化检测方法的建立与评价[D]. 长春: 吉林大学, 2023.
	WEI S N. Establishment and evaluation of visual detection methods based on nanocomposites for Salmonella typhimurium[D]. Changchun: Jilin University, 2023. (in Chinese)
8	KONOVALOVA A , KAHNE D E , SILHAVY T J . Outer membrane biogenesis[J]. Annu Rev Microbiol, 2017, 71, 539- 556. doi: 10.1146/annurev-micro-090816-093754
9	ROLLAUER S E , SOORESHJANI M A , NOINAJ N , et al. Outer membrane protein biogenesis in gram-negative bacteria[J]. Philos Trans Roy Soc B Biol Sci, 2015, 370 (1679): 20150023. doi: 10.1098/rstb.2015.0023
10	ZHANG P F , YE Z C , YE C , et al. OmpW is positively regulated by iron via Fur, and negatively regulated by SoxS contribution to oxidative stress resistance in Escherichia coli[J]. Microb Pathog, 2020, 138, 103808. doi: 10.1016/j.micpath.2019.103808
11	XIAO M F , LAI Y , SUN J , et al. Transcriptional regulation of the outer membrane porin gene ompW reveals its physiological role during the transition from the aerobic to the anaerobic lifestyle of Escherichia coli[J]. Front Microbiol, 2016, 7, 799.
12	吴贤斌, 邹海杰, 田丽花, 等. 大肠杆菌外膜蛋白ompW基因敲除菌的构建及其对两种抗生素的敏感性[J]. 微生物学报, 2012, 52 (8): 1021- 1026.
	WU X B , ZOU H J , TIAN L H , et al. Construction of ompW knock-out mutants of Escherichia coli to increase sensitivity to neomycinsulphate and ampicilin[J]. Acta Microbiologica Sinica, 2012, 52 (8): 1021- 1026.
13	李宝宝, 张振兴, 黄海峰, 等. 羊源多杀性巴氏杆菌ompW基因的克隆及生物信息学分析[J]. 中国畜牧兽医, 2019, 46 (1): 28- 36.
	LI B B , ZHANG Z X , HUANG H F , et al. Cloning and bioinformatics analysis of ompW gene from Pasteurella multocida in goat[J]. China Animal Husbandry & Veterinary Medicine, 2019, 46 (1): 28- 36.
14	张临, 卢芳, 付恒峰, 等. 鼠伤寒沙门菌BaeSR对氟喹诺酮类药物耐药性的调控机制[J]. 畜牧兽医学报, 2022, 53 (3): 894- 903. doi: 10.11843/j.issn.0366-6964.2022.03.022
	ZHANG L , LU F , FU H F , et al. Regulation mechanism of BaeSR on fluoroquinolones resistance in Salmonella typhimurium[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53 (3): 894- 903. doi: 10.11843/j.issn.0366-6964.2022.03.022
15	令狐远凤, 潘永, 杨阳, 等. 沙门氏菌sRNA RybB及伴侣蛋白Hfq对孔蛋白OmpW表达的调控作用[J]. 中国家禽, 2023, 45 (6): 30- 35.
	LINGHU Y F , PAN Y , YANG Y , et al. Regulation of sRNA RybB and chaperone Hfq of Salmonella on expression of porin OmpW[J]. China Poultry, 2023, 45 (6): 30- 35.
16	LIEBERMAN L A . Outer membrane vesicles: a bacterial-derived vaccination system[J]. Front Microbiol, 2022, 13, 1029146. doi: 10.3389/fmicb.2022.1029146
17	夏银河, 王坤芃, 刘盼盼, 等. 鸭源鸡杆菌ompW、gtxA双基因缺失株的构建及其相关特性分析[J]. 微生物学报, 2020, 60 (10): 2311- 2322.
	XIA Y H , WANG K P , LIU P P , et al. Construction and characterization of Gallibacterium anatis ΔompW ΔgtxA double deletion strain[J]. Acta Microbiologica Sinica, 2020, 60 (10): 2311- 2322.
18	张哲玮, 代小童, 韦祖丹, 等. 猪多杀性巴氏杆菌OmpW、TbpA蛋白的原核表达及免疫原性研究[J]. 中国畜牧兽医, 2023, 50 (4): 1489- 1498.
	ZHANG Z W , DAI X T , WEI Z D , et al. Study on prokaryotic expression and immunogenicity of OmpW and TbpA proteins in swine Pasteurella multocida[J]. China Animal Husbandry & Veterinary Medicine, 2023, 50 (4): 1489- 1498.
19	HONG H , PATEL D R , TAMM L K , et al. The outer membrane protein OmpW forms an eight-stranded β-barrel with a hydrophobic channel[J]. J Biol Chem, 2006, 281 (11): 7568- 7577.
20	张家莉, 段世宇, 令狐远凤, 等. 鼠伤寒沙门菌ompA基因缺失株的构建及生物学特性的分析[J]. 中国动物传染病学报, 2022, 30 (6): 27- 33.
	ZHANG J L , DUAN S Y , LINGHU Y F , et al. Construction of ompA gene deletion strain of Salmonella typhimurium and analysis of its biological characteristics[J]. Chinese Journal of Animal Infectious Diseases, 2022, 30 (6): 27- 33.
21	薄雅宁, 庄添瑞, 高玥, 等. 布鲁氏菌外膜蛋白OmpW家族蛋白生物信息预测[J]. 现代医学与健康研究, 2022, 6 (23): 13- 17.
	BO Y N , ZHUANG T R , GAO Y , et al. Prediction of biological information of outer membrane protein OmpW family of Brucella[J]. Modern Medicine and Health Research, 2022, 6 (23): 13- 17.
22	高吉娜. OmpW在阪崎克罗诺杆菌逆境胁迫中的作用探讨[D]. 合肥: 合肥工业大学, 2017.
	GAO J N. Exploration of the role on OmpW in Cronobacter sakazakii under environmental stresses[D]. Hefei: Hefei University of Technology, 2017. (in Chinese)
23	叶智鸧. 铁离子对大肠杆菌外膜蛋白OmpW的调控机制研究[D]. 杭州: 浙江理工大学, 2015.
	YE Z C. Regulatory mechanisms of iron ions on Escherichia coli outer membrane protein OmpW[D]. Hangzhou: Zhejiang Sci-Tech University, 2015. (in Chinese)
24	LIN X M , WU L N , LI H , et al. Downregulation of Tsx and ompW and upregulation of ompX are required for iron homeostasis in Escherichia coli[J]. J Proteome Res, 2008, 7 (3): 1235- 1243.
25	马月龙, 赖泽迎, 冯荆城, 等. 外膜蛋白ompC过表达提高大肠杆菌对庆大霉素敏感性研究[J]. 福建农业科技, 2022, 53 (12): 21- 25.
	MA Y L , LAI Z Y , FENG J C , et al. Study on the enhancement of the sensitivity of Escherichia coli to gentamicin by the overexpression of outer membrane protein ompC[J]. Fujian Agricultural Science and Technology, 2022, 53 (12): 21- 25.
26	GIORDANO N P , CIAN M B , DALEBROUX Z D . Outer membrane lipid secretion and the innate immune response to gram-negative bacteria[J]. Infect Immun, 2020, 88 (7): e00920- 19.
27	郭容, 齐瑜, 李洋洋, 等. 外膜蛋白OmpR对副溶血弧菌致病特性的影响[J]. 微生物学报, 2022, 62 (9): 3410- 3420.
	GUO R , QI Y , LI Y Y , et al. Influence of outer membrane protein OmpR on pathogenicity of Vibrio parahaemolyticus[J]. Acta Microbiologica Sinica, 2022, 62 (9): 3410- 3420.
28	白雪瑞, 王权, 陈永军, 等. 副溶血弧菌ompA基因缺失株的生物学特性及致病性分析[J]. 南京农业大学学报, 2018, 41 (5): 902- 910.
	BAI X R , WANG Q , CHEN Y J , et al. Biological characteristics and pathogenicity of an ompA mutant of Vibrio parahaemolyticus[J]. Journal of Nanjing Agricultural University, 2018, 41 (5): 902- 910.
29	许姝. 禽致脑膜炎型大肠杆菌外膜蛋白OmpA突破血脑屏障致病机制的研究[D]. 扬州: 扬州大学, 2020.
	XU S. Pathogenic mechanism of OmpA from avian pathogenic Escherichia coli for blood-brain barrier breakthrough[D]. Yangzhou: Yangzhou University, 2020. (in Chinese)
30	段世宇, 杨阳, 令狐远凤, 等. 沙门菌外膜蛋白相关基因缺失株的致病性[J]. 福建农林大学学报(自然科学版), 2022, 51 (2): 224- 229.
	DUAN S Y , YANG Y , LINGHU Y F , et al. Pathogenicity of Salmonella strains with gene deletion related to outer membrane protein[J]. Journal of Fujian Agriculture and Forestry University (Natural Science Edition), 2022, 51 (2): 224- 229.
31	FU D D , ZHENG Q Q , WU X Y , et al. The transcriptional regulator EtrA mediates ompW contributing to the pathogenicity of avian pathogenic Escherichia coli[J]. Vet Microbiol, 2023, 283, 109775.
32	SCHMITT B L , LEAL B F , LEYSER M , et al. Increased ompW and ompA expression and higher virulence of Acinetobacter baumannii persister cells[J]. BMC Microbiol, 2023, 23 (1): 157.

引物名称 Primers	序列(5′→3′) Sequence (5′→3′)	产物大小/bp Length of product
F1	$\underline{{\rm{TGACCCCGCCTGGCGGGGTCTTTTTTCGTCACAACATCACGCGA}}}$-ATATGAATATCCTCCTTAG	1 160
F2	$\underline{{\rm{TATTACTTCATTGTGGGTATATTCATCACGCTTTTATAACCATAACG}}}$-TGTAGGCTGGAGCTGCTTCG
F3	TTCGTCACAACATCACGCGA	890(重组失败)
F4	CCTTAGCCAGCCTTTATCGC	1 105(重组成功)
F5	ACCGAGCTCGAATTCGTAAT	2 987
F6	TCTAGAGTCGACCTGCAGGC
F7	$\underline{{\rm{GCATGCCTGCAGGTCGACTCTAGA}}}$TTAGAAACGATAGCCTGCCGAGA	690
F8	$\underline{{\rm{CATGATTACGAATTCGAGCTCGGT}}}$ATGGAGCGGGTATGAAAAAAT
F9	TTAGAAACGATAGCCTGCCGAGAAC	650
F10	ATGGAGCGGGTATGAAAAAATTTAC
F11	CTATCGGCGAAGTCACAGC	836
F12	CAGGGCAACAGGAGCAGAC

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鼠伤寒沙门菌ompW基因缺失株与回补株的构建及其生物学特性

Construction and Biological Characterization of ompW Gene Deletion and Complemented Strain of Salmonella Typhimurium

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