鼠伤寒沙门菌sapC基因缺失株的构建及生物学特性分析

doi:10.11843/j.issn.0366-6964.2022.07.024

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (7): 2282-2289.doi: 10.11843/j.issn.0366-6964.2022.07.024

鼠伤寒沙门菌sapC基因缺失株的构建及生物学特性分析

曹莉^1,3, 程如楠¹, 武周慧¹, 王家伟², 王瑜¹, 张永红¹, 吴清民², 王真^1*

1. 北京农学院动物科学技术学院, 兽医学(中兽医)北京市重点实验室, 北京 102206;
2. 中国农业大学动物医学院, 北京 100193;
3. 北京大学第三医院实验动物中心, 北京 100191

收稿日期:2021-10-26 出版日期:2022-07-23 发布日期:2022-07-23
通讯作者: 王真,主要从事动物疾病诊断和防治技术研究,E-mail:wangzhen3355@163.com
作者简介:曹莉(1994-),女,北京人,硕士生,主要从事病原菌功能基因研究,E-mail:18310258839@163.com
基金资助:
北京市教委青年拔尖人才项目(CIT&TCD201904052)；北京市优秀人才培养资助项目(2017000026833ZK18)；北京农学院青年基金项目(QNKJ202107)

Construction and Biological Characteristics of sapC Gene Deletion Strain of Salmonella Typhimurium

CAO Li^1,3, CHENG Runan¹, WU Zhouhui¹, WANG Jiawei², WANG Yu¹, ZHANG Yonghong¹, WU Qingmin², WANG Zhen^1*

1. Beijing Key Laboratory of Traditional Chinese Veterinary Medicine, Animal Science and Technology College, Beijing University of Agriculture, Beijing 102206, China;
2. College of Veterinary Medicine, China Agricultural University, Beijing 100193, China;
3. Animal Experiment Center, Peking University Third Hospital, Beijing 100191, China

Received:2021-10-26 Online:2022-07-23 Published:2022-07-23

摘要/Abstract

摘要： 旨在研究鼠伤寒沙门菌ABC转运膜蛋白SapC在沙门菌致病机制中的功能。本研究利用λ-Red重组技术构建了鼠伤寒沙门菌sapC基因缺失突变株SMΔsapC，对其进行生长特性、酸性应激试验、多黏菌素B敏感性试验、生物被膜检测、胞内存活和小鼠体内毒力试验。结果显示，基因缺失株SMΔsapC与亲本菌株和互补菌株相比，其生长速度无明显差异；在酸应激条件下，SMΔsapC存活率显著低于亲本菌株；sapC基因缺失降低了鼠伤寒沙门菌生物被膜的形成能力；同时，SMΔsapC基因缺失株在鼠源巨噬细胞内的增殖能力和小鼠体内的毒力显著低于亲本菌株。研究表明，sapC基因影响鼠伤寒沙门菌的抗酸能力、生物被膜形成能力，从而影响沙门菌在体内外的毒力。本研究为进一步阐释鼠伤寒沙门菌的致病机制奠定了基础。

关键词: 鼠伤寒沙门菌, 同源重组, sapC, 生物学特性

Abstract: This study aimed to study the function of sapC in ABC transporters of SalmonellaTyphimurium in its pathogenic mechanism. The sapC gene deletion mutant SM△sapC of S. Typhimurium was constructed by λ-Red mediated mutagenesis procedures, and the growth characteristics, acid stress test, polymyxin B sensitivity test, biofilm detection, intracellular survival and toxicity test in mice were performed. The results showed that the growth rate of the deletion strain SMΔsapC was not significantly different from that of the parent strain and the supplemented strain. Under acid stress condition, the survival rate of SMΔsapCsapC was significantly lower than that of the parent strain; the deletion of sapC gene enhanced the drug resistance of polymyxin B and decreased the ability of biofilm formation of S. Typhimurium. The proliferation ability of SMΔsapC strain in mouse macrophages and the virulence in mice were significantly lower than that of the parent strain. This study showed sapC gene decreased the acid resistance and biofilm formation ability of S. Typhimurium, thus reducing the virulence of Salmonella in vivo and in vitro. This study laid a foundation for further explaining the pathogenic mechanism of S.Typhimurium.

Key words: Salmonella Typhimurium, homologous recombination, sapC, biological characteristics

中图分类号:

S852.61

曹莉, 程如楠, 武周慧, 王家伟, 王瑜, 张永红, 吴清民, 王真. 鼠伤寒沙门菌sapC基因缺失株的构建及生物学特性分析[J]. 畜牧兽医学报, 2022, 53(7): 2282-2289.

CAO Li, CHENG Runan, WU Zhouhui, WANG Jiawei, WANG Yu, ZHANG Yonghong, WU Qingmin, WANG Zhen. Construction and Biological Characteristics of sapC Gene Deletion Strain of Salmonella Typhimurium[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2282-2289.

参考文献 30

[1]	FUCHE F J, SOW O, SIMON R, et al. Salmonella Serogroup C:current status of vaccines and why they are needed[J]. Clin Vaccine Immunol, 2016, 23(9):737-745.
[2]	ANSARI N, YAZDIAN-ROBATI R, SHAHDORDIZADEH M, et al. Aptasensors for quantitative detection of Salmonella Typhimurium[J]. Anal Biochem, 2017, 533:18-25.
[3]	GARAI P, GNANADHAS D P, CHAKRAVORTTY D. Salmonella enterica serovars Typhimurium and Typhi as model organisms:revealing paradigm of host-pathogen interactions[J]. Virulence, 2012, 3(4):377-388.
[4]	SUN H H, WAN Y P, DU P C, et al. The epidemiology of monophasic Salmonella Typhimurium[J]. Foodborne Pathog Dis, 2020, 17(2):87-97.
[5]	QI X L, LI P, XU X G, et al. Epidemiological and molecular investigations on Salmonella responsible for gastrointestinal infections in the Southwest of Shanghai from 1998 to 2017[J]. Front Microbiol, 2019, 10:2025.
[6]	HANCOCK R E W, SCOTT M G. The role of antimicrobial peptides in animal defenses[J]. Proc Natl Acad Sci U S A, 2000, 97(16):8856-8861.
[7]	DE CESARE C B, CRISTY S A, GARSIN D A, et al. Antimicrobial peptides:a new frontier in antifungal therapy[J]. mBio, 2020, 11(6):e02123-20.
[8]	GALLO R L, MURAKAMI M, OHTAKE T, et al. Biology and clinical relevance of naturally occurring antimicrobial peptides[J]. J Allergy ClinImmunol, 2002, 110(6):823-831.
[9]	BADER M W, NAVARRE W W, SHIAU W, et al. Regulation of Salmonella typhimurium virulence gene expression by cationic antimicrobial peptides[J]. Mol Microbiol, 2003, 50(1):219-230.
[10]	BRODSKY I E, ERNST R K, MILLER S I, et al. mig-14 is a Salmonella gene that plays a role in bacterial resistance to antimicrobial peptides[J]. J Bacteriol, 2002, 184(12):3203-3213.
[11]	RODAS P I, CONTRERAS I, MORA G C. Salmonella enterica serovar Typhi has a 4. 1 kb genetic island inserted within the sapABCDF operon that causes loss of resistance to the antimicrobial peptide protamine[J]. J Antimicrob Chemother, 2010, 65(8):1624-1630.
[12]	PARRA-LOPEZ C, NAER M T, GROISMAN E A. Molecular genetic analysis of a locus required for resistance to antimicrobial peptides in Salmonella typhimurium[J]. EMBO J, 1993, 12(11):4053-4062.
[13]	ROEPE P D, WEI L Y, HOFFMAN M M, et al. Altered drug translocation mediated by the MDR protein:direct, indirect, or both?[J]. J Bioenerg Biomembr, 1996, 28(6):541-555.
[14]	KRAUSS S, ZIPPERER A, WIRTZ S, et al. Secretion of and self-resistance to the novel fibupeptide antimicrobial Lugdunin by distinct ABC transporters in Staphylococcus lugdunensis[J]. Antimicrob Agents Chemother, 2020, 65(1):e01734-20.
[15]	YAMAGISHI A, NAKANO S, YAMASAKI S, et al. An efflux inhibitor of the MacAB pump in Salmonella enterica serovar typhimurium[J]. Microbiol Immunol, 2020, 64(3):182-188.
[16]	曹莉,孙路,孔梓安,等.肠炎沙门氏菌yjeA基因缺失株的构建及其生物学功能测定[J].北京农学院学报, 2020, 35(2):16-20.CAO L, SUN L, KONG Z A, et al. Construction and characterization of yjeA gene deletion strain of Salmonella enteritidis[J]. Journal of Beijing University of Agriculture, 2020, 35(2):16-20.
[17]	ARANDA C M A, SWANSON J A, LOOMIS W P, et al. Salmonella Typhimurium activates virulence gene transcription within acidified macrophage phagosomes[J]. Proc Natl Acad Sci U S A, 1992, 89(21):10079-10083.
[18]	GUTIÉRREZ S, FISCHER J, GANESAN R, et al. Salmonella Typhimurium impairs glycolysis-mediated acidification of phagosomes to evade macrophage defense[J]. PLoS Pathog, 2021, 17(9):e1009943.
[19]	YUKI K E, EVA M M, RICHER E, et al. Suppression of hepcidin expression and iron overload mediate Salmonella susceptibility in Ankyrin 1 ENU-induced mutant[J]. PLoS One, 2013, 8(2):e55331.
[20]	PAUDYAL N, PAN H, WU B B, et al. Persistent asymptomatic human infections by Salmonella enterica serovar Newport in China[J]. mSphere, 2020, 5(3):e00163-20.
[21]	COBBOLD R N, RICE D H, DAVIS M A, et al. Long-term persistence of multi-drug-resistant Salmonella enterica serovar Newport in two dairy herds[J]. J Am Vet Med Assoc, 2006, 228(4):585-591.
[22]	ZHAO X X, DAI Q L, ZHU D K, et al. Recombinant attenuated Salmonella Typhimurium with heterologous expression of the Salmonella Choleraesuis O-polysaccharide:high immunogenicity and protection[J]. Sci Rep, 2017, 7(1):7127.
[23]	KUMAR A, ALAM A, RANI M, et al. Biofilms:survival and defense strategy for pathogens[J]. Int J Med Microbiol, 2017, 307(8):481-489.
[24]	DULA S, AJAYEOBA T A, IJABADENIYI O A. Bacterial biofilm formation on stainless steel in the food processing environment and its health implications[J]. Folia Microbiol (Praha), 2021, 66(3):293-302.
[25]	CHEBOTAR'I V, KONCHAKOVA E D, EVTEEVA N I. Neutrophil dependent breakdown of biofilms formed by Staphylococcus aureus[J]. Zh Mikrobiol Epidemiol Immunobiol, 2012(1):10-15.
[26]	MANGALAPPALLI-ILLATHU A K, KORBER D R. Adaptive resistance and differential protein expression of Salmonella enterica serovar Enteritidis biofilms exposed to benzalkonium chloride[J]. Antimicrob Agents Chemother, 2006, 50(11):3588-3596.
[27]	CHIN K C J, TAYLOR T D, HEBRARD M, et al. Transcriptomic study of Salmonella enterica subspecies enterica serovar Typhi biofilm[J]. BMC Genomics, 2017, 18:836.
[28]	HU S F, YU Y G, LV Z Q, et al. Proteomics study unveils ROS balance in acid-adapted Salmonella Enteritidis[J]. Food Microbiol, 2020, 92:103585.
[29]	JIAO S S, ZHANG H J, LIAO M J, et al. Investigation of the potential direct and cross protection effects of sublethal injured Salmonella Typhimurium induced by radio frequency heating stress[J]. Food Res Int, 2021, 150:110789.
[30]	ARVIZU-MEDRANO S M, ESCARTÍN E F. Effect of acid shock with hydrochloric, citric, and lactic acids on the survival and growth of Salmonella typhi and Salmonella typhimurium in acidified media[J]. J Food Prot, 2005, 68(10):2047-2053.

鼠伤寒沙门菌sapC基因缺失株的构建及生物学特性分析

Construction and Biological Characteristics of sapC Gene Deletion Strain of Salmonella Typhimurium

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 30

相关文章 15

编辑推荐

Metrics

本文评价

[1]	李昭燕, 高江, 郭时惠, 赵茹茜, 马文强. 猫过敏原检测方法与控制措施的研究进展[J]. 畜牧兽医学报, 2023, 54(6): 2272-2279.
[2]	刘青, 吴少鹏, 石彬, 邵红霞, 钱琨, 叶建强, 秦爱建. 鸡CTLA-4蛋白在昆虫细胞中的表达及其单克隆抗体的制备[J]. 畜牧兽医学报, 2023, 54(6): 2596-2604.
[3]	秦蕾, 吴慧敏, 徐琦琦, 陈万昭, 王东, 李宏博, 夏盼盼, 刘泽鹏, 夏利宁. 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.
[4]	李德豪, 李宜穗, 马静云, 孙媛, 张祥斌. 一株沙门菌温和噬菌体PEA2-3生物学特性及基因组分析[J]. 畜牧兽医学报, 2023, 54(4): 1632-1640.
[5]	蒋增海, 滕霖, 贺安文, 刘言言, 乐敏, 何启盖. 猪产业链中鼠伤寒沙门菌及沙门菌血清型4,[5],12:i:-基因组学分析[J]. 畜牧兽医学报, 2023, 54(3): 1199-1209.
[6]	李晓涵, 杨伏春, 刘芮, 高立, 崔红玉, 张艳萍, 刘长军, 祁小乐, 王笑梅, 高玉龙, 李凯. 表达鸡传染性法氏囊病病毒VP2基因的重组火鸡疱疹病毒的构建及生物学特性分析[J]. 畜牧兽医学报, 2023, 54(2): 656-662.
[7]	李莉莉, 陈凯风, 陈兵, 周洲平, 王南威, 瞿孝云, 徐成刚, 廖明, 张建民. STM1827在鼠伤寒沙门菌生物被膜形成及环境应激中的调控作用[J]. 畜牧兽医学报, 2023, 54(12): 5207-5217.
[8]	韩生义, 李玲霞, 李淑萍, 胡国元, 李生庆. 沙门菌噬菌体SP3的生物学特性及基因组分析[J]. 畜牧兽医学报, 2023, 54(12): 5228-5239.
[9]	杨梦林, 郑世奇, 彭凯, 王玮, 黄燕华, 彭杰. 鸽源鼠伤寒沙门菌的分离鉴定及致病性分析[J]. 畜牧兽医学报, 2023, 54(11): 4880-4888.
[10]	岑鑫, 阳亭亭, 赵尊福, 文永平, 张焕容. 沙门菌烈性噬菌体的分离鉴定、生物学特性及基因组分析[J]. 畜牧兽医学报, 2022, 53(8): 2677-2688.
[11]	张娜, 王菲, 葛锡民, 赵桂苹, 文杰, 李庆贺. 抑制USP7活性对鸡抗沙门菌感染的影响[J]. 畜牧兽医学报, 2022, 53(7): 2396-2402.
[12]	刘畅, 刘教, 陈进, 王勉之, 熊文广, 曾振灵. 1株可裂解bla_NDM-5及mcr-1阳性大肠杆菌噬菌体的分离与生物学特性[J]. 畜牧兽医学报, 2022, 53(3): 857-866.
[13]	张临, 卢芳, 付恒峰, 姜西迪, 魏祺灵, 漆彩丽, 高海侠, 李琳. 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.
[14]	孙培皓, 赵鑫哲, 吴涵潇, 吕策, 杨利国, 梁爱心. 奶牛乳腺上皮细胞的原代培养及其生物学特性分析[J]. 畜牧兽医学报, 2022, 53(2): 447-458.
[15]	吴居业, 卢宝春, 祝宇凡, 贾坤. 血小板凝血酶蛋白1对犬乳腺肿瘤细胞体外生物学特性的影响分析[J]. 畜牧兽医学报, 2022, 53(12): 4470-4481.