pSLT质粒的转移及其对肠道细胞凋亡的影响

doi:10.19586/j.2095-2341.2025.0090

生物技术进展 ›› 2026, Vol. 16 ›› Issue (2): 388-394.DOI: 10.19586/j.2095-2341.2025.0090

• 研究论文 • 上一篇

pSLT质粒的转移及其对肠道细胞凋亡的影响

洪甜¹(), 赵璐璐¹, 杜一凡², 李静雯¹, 陈尔凝¹, 杜美红¹()

^1.北京市科学技术研究院分析测试研究所（北京市理化分析测试中心），北京　100094
^2.北京城市学院生物医药学部，北京　100094

收稿日期:2025-07-29 接受日期:2025-12-25 出版日期:2026-03-25 发布日期:2026-04-27
通讯作者: 杜美红
作者简介:洪甜 E-mail: htian1025@163.com；
基金资助:
北京市科学技术研究院创新工程项目(25CA011)

Transfer of the pSLT Plasmid and its Impact on Intestinal Cell Apoptosis

Tian HONG¹(), Lulu ZHAO¹, Yifan DU², Jingwen LI¹, Erning CHEN¹, Meihong DU¹()

^1.Institute of Analysis and Testing，Beijing Academy of Science and Technology （Beijing Center for Physical & Chemical Analysis），Bejing 100094，China
^2.School of Biomedicine，Beijing City University，Beijing 100094，China

Received:2025-07-29 Accepted:2025-12-25 Online:2026-03-25 Published:2026-04-27
Contact: Meihong DU

摘要/Abstract

摘要：

pSLT质粒是主要存在于鼠伤寒沙门氏菌株中的一种特异性毒力质粒，探讨了沙门氏菌pSLT毒力质粒水平基因转移（horizontal gene transfer，HGT）的可行性，及其对细胞坏死和凋亡的影响。采用λRed同源重组技术，将pKD46质粒来源的Kana抗性基因整合插入沙门氏菌的pSLT质粒中；以沙门氏菌（ATCC 14028）为供体菌，大肠杆菌（ATCC 25922）为受体菌，通过M9培养基平板接合法进行接合转移，获得的接合菌在含有卡那霉素的麦康凯液体培养基中进行产酸产气培养验证，划线培养取单菌落进行菌液PCR鉴定和测序鉴定比对；以人结肠癌细胞HCT116为肠道代表菌，将接合菌以感染复数（multiplicity of infection，MOI）100进行作用，通过流式细胞术检测细胞坏死和凋亡情况。结果发现，含有卡那霉素的营养琼脂平板上有单菌落生长，Kana抗性基因片段成功重组插入pSLT；接合菌在含有卡那霉素的麦康凯液体培养基中有产酸产气现象，pSLT^Kana质粒接合转移至大肠杆菌；与对照组相比，ATCC 25922处理组未造成Annexin V/PI双阳性细胞比例的显著变化（P>0.05），ATCC 25922 pSLT和ATCC 14028处理组均表现出显著降于Annexin V/PI双阳性细胞比例（P<0.01）。通过构建ATCC 14028 pSLT^Kana重组菌株，证实了沙门氏菌携带的pSLT毒力质粒向大肠杆菌进行水平基因转移的可行性，ATCC 25922 pSLT^Kana对肠道细胞的坏死和凋亡存在潜在的抑制作用，为临床治疗和公共卫生安全监管提供了重要的科学数据和思路。

关键词: 沙门氏菌, pSLT, 大肠杆菌, 接合转移

Abstract:

The pSLT plasmid is a specific virulence plasmid primarily found in Salmonella enterica serovar Typhimurium strains. This study aimed to explore the feasibility of horizontal gene transfer （HGT） of the Salmonella pSLT virulence plasmid and investigate its effects on cell necrosis and apoptosis. The Kanamycin resistance gene derived from plasmid pKD46 was integrated into the Salmonella pSLT plasmid using λRed homologous recombination technology. Salmonella enterica serovar Typhimurium （ATCC 14028） served as the donor strain， and Escherichia coli （ATCC 25922） as the recipient strain. Conjugation was performed using the M9 minimal medium plate mating method. The obtained transconjugants were cultured in Kanamycin-supplemented MacConkey broth to verify acid and gas production. Single colonies were isolated， followed by confirmatory PCR and sequencing for the inserted Kana gene. Human colon carcinoma cells （HCT116）， representing intestinal cells， were infected with transconjugants at a multiplicity of infection （MOI） of 100. Cell necrosis and apoptosis were assessed using flow cytometry （Annexin V/PI staining）. The results showed that single colonies grew on nutrient agar plates containing Kanamycin， confirming successful recombination and insertion of the Kana gene fragment into pSLT （creating pSLT^Kana）. Transconjugants exhibited acid and gas production in Kanamycin-supplemented MacConkey broth， demonstrating the successful conjugative transfer of the pSLT^Kana plasmid to Escherichia coli. Compared to the control group， the ATCC 25922 （wild-type E. coli） treatment group did not cause a significant change （P>0.05） in the proportion of Annexin V/PI double-positive cells （indicating late apoptosis/necrosis）. In contrast， both the ATCC 25922 pSLT^Kana （E. coli harboring pSLT） and ATCC 14028 （Salmonella with native pSLT） treatment groups showed a significant reduction （P<0.01） in the proportion of Annexin V/PI double-positive cells. By constructing the ATCC 14028 pSLT^Kana recombinant strain， this study confirmed the feasibility of HGT of the pSLT virulence plasmid from Salmonella to E. coli. The ATCC 25922 pSLT^Kana strain exhibited a potential inhibitory effect on necrosis and apoptosis in intestinal cells. These findings provide crucial scientific data and insights for clinical treatment strategies and public health safety oversight.

Key words: Salmonella, pSLT, Escherichia coli, conjugation transfer

中图分类号:

Q935

洪甜, 赵璐璐, 杜一凡, 李静雯, 陈尔凝, 杜美红. pSLT质粒的转移及其对肠道细胞凋亡的影响[J]. 生物技术进展, 2026, 16(2): 388-394.

Tian HONG, Lulu ZHAO, Yifan DU, Jingwen LI, Erning CHEN, Meihong DU. Transfer of the pSLT Plasmid and its Impact on Intestinal Cell Apoptosis[J]. Current Biotechnology, 2026, 16(2): 388-394.

图/表 6

图1 pKD46质粒上的EcoRⅠ酶切位点

Fig. 1 EcoRⅠ restriction site in the pKD46 plasmid

图2 EcoRⅠ单酶切pKD46质粒注：M—DL2000 marker；1—pKD46质粒；2—EcoRⅠ单酶切后的片段。

Fig. 2 EcoRⅠ digestion of pKD46 plasmid

图3 PCR产物琼脂糖凝胶电泳注：M—DL2000 marker；1~2—同源片段PCR产物。

Fig. 3 Agarose gel electrophoresis of PCR products

图4 Sanger测序鉴定pSLTkana质粒A：使用Kana-jd-F进行的正向测序；B：使用Kana-jd-R进行的反向测序；红框表示Kana基因序列，无框线为pSLT基因序列。

Fig. 4 Identification of the pSLTkana plasmid by Sanger sequencing

图5 麦康凯培养基培养接合菌A：大肠杆菌、沙门氏菌和接合体系在麦康凯液体培养基中的生长情况。1—含有卡那霉素的麦康凯培养基中接种ATCC 25922；2—不含有卡那霉素的麦康凯培养基中接种ATCC 25922；3—含有卡那霉素的麦康凯培养基中接种ATCC 14028 pSLTkana；4—含有卡那霉素的麦康凯培养基中接种40 μL的接合体系；5—含有卡那霉素的麦康凯培养基中接种1 mL的接合体系。B：沙门氏菌（左）和大肠杆菌（右）在麦康凯琼脂培养基上的生长情况。C：40 μL接合体系在麦康凯琼脂上的生长情况。D：1 mL接合体系在麦康凯琼脂上的生长情况

Fig. 5 Cultivation of Zygomycetes on MacConkey agar

图6 人结肠癌细胞的坏死和凋亡检测注：***表示在P<0.001水平上有统计学意义。

Fig. 6 Apoptosis detection in human colorectal cancer cells

参考文献 27

[1]	HILEY L, GRAHAM R M A, JENNISON A V. Genetic characterisation of variants of the virulence plasmid, pSLT, in Salmonella enterica serovar Typhimurium provides evidence of a variety of evolutionary directions consistent with vertical rather than horizontal transmission[J/OL]. PLoS One, 2019, 14(4): e0215207[2026-02-11]. .
[2]	AHMER B M, TRAN M, HEFFRON F. The virulence plasmid of Salmonella typhimurium is self-transmissible[J]. J. Bacteriol., 1999, 181(4): 1364-1368.
[3]	GARCÍA-QUINTANILLA M, RAMOS-MORALES F, CASADESÚS J. Conjugal transfer of the Salmonella enterica virulence plasmid in the mouse intestine[J]. J. Bacteriol., 2008, 190(6): 1922-1927.
[4]	TENAILLON O, SKURNIK D, PICARD B, et al.. The population genetics of commensal Escherichia coli [J]. Nat. Rev. Microbiol., 2010, 8(3): 207-217.
[5]	WU C, YANG F, ZHONG H, et al.. Obesity-enriched gut microbe degrades myo-inositol and promotes lipid absorption[J]. Cell Host Microbe, 2024, 32(8): 1301-1314.
[6]	LYNCH J P, GOERS L, LESSER C F. Emerging strategies for engineering Escherichia coli Nissle 1917-based therapeutics[J]. Trends Pharmacol. Sci., 2022, 43(9): 772-786.
[7]	DE OLIVEIRA ALVES N, DALMASSO G, NIKITINA D, et al.. The colibactin-producing Escherichia coli alters the tumor microenvironment to immunosuppressive lipid overload facilitating colorectal cancer progression and chemoresistance[J/OL]. Gut Microbes, 2024, 16(1): 2320291[2025-10-11]. .
[8]	GURBATRI C R, ARPAIA N, DANINO T. Engineering bacteria as interactive cancer therapies[J]. Science, 2022, 378(6622): 858-864.
[9]	SPRAGGE F, BAKKEREN E, JAHN M T, et al.. Microbiome diversity protects against pathogens by nutrient blocking[J/OL]. Science, 2023, 382(6676): eadj3502[2025-10-11]. .
[10]	DENAMUR E, CLERMONT O, BONACORSI S, et al.. The population genetics of pathogenic Escherichia coli [J]. Nat. Rev. Microbiol., 2021, 19(1): 37-54.
[11]	LIU B, FUREVI A, PEREPELOV A V, et al.. Structure and genetics of Escherichia coli antigens[J]. FEMS Microbiol. Rev., 2020, 44(6): 655-683.
[12]	LI W, BAI X, SHENG H, et al.. Conjugative transfer of mcr-1-bearing plasmid from Salmonella to Escherichia coli in vitro on chicken meat and in mouse gut[J/OL]. Food Res. Int., 2022, 157: 111263[2025-12-01]. .
[13]	TIAGO V L L. Transferência horizontal de genes de resistência à Colistina em Salmonella enterica e Escherichia coli de Animais Produtores de Alimento[D]. Coimbra: Universidade de Coimbra, 2022.
[14]	刘艺云,邓利敏,岳慧颖,等.质粒接合转移及其抑制剂的研究进展[J].生物技术通报,2022,38(9):35-46.
	LIU Y Y, DENG L M, YUE H Y, et al.. Research progress in plasmid conjugation and its inhibitors[J]. Biotechnol. Bull., 2022, 38(9): 35-46.
[15]	ARES-ARROYO M, COLUZZI C, ROCHA E P C. Origins of transfer establish networks of functional dependencies for plasmid transfer by conjugation[J]. Nucleic Acids Res., 2023, 51(7): 3001-3016.
[16]	BENZ F, HALL A R. Host-specific plasmid evolution explains the variable spread of clinical antibiotic-resistance plasmids[J/OL]. Proc. Natl. Acad. Sci. USA, 2023, 120(15): e2212147120[2025-10-11]. .
[17]	FENG Z, WANG L, GUAN Q, et al.. Acinetobacter baumannii coordinates central metabolism, plasmid dissemination, and virulence by sensing nutrient availability[J/OL]. mBio, 2023, 14(6): e02276-e02223[2025-10-11]. .
[18]	CAMACHO E M, SERNA A, MADRID C, et al.. Regulation of finP transcription by DNA adenine methylation in the virulence plasmid of Salmonella enterica [J]. J. Bacteriol., 2005, 187(16): 5691-5699.
[19]	SÁNCHEZ-ROMERO M A, MÉRIDA-FLORIANO Á, CASADESÚS J. Corrigendum: copy number heterogeneity in the virulence plasmid of Salmonella enterica [J/OL]. Front. Microbiol., 2021, 11: 645054[2025-10-11]. .
[20]	WANG H, JOFFRÉ E. Plasmid copy number as a modulator in bacterial pathogenesis and antibiotic resistance[J/OL]. NPJ Antimicrob. Resist., 2025, 3(1): 72[2026-02-11]. .
[21]	王品舒,岳瑾,乔岩,等.Bc、Bt、Ba 质粒研究进展[J].生物技术进展,2013,3(3):196-200.
	WANG P S, YUE J, QIAO Y, et al.. Plasmids research progress of bc, bt and Ba[J]. Curr. Biotechnol., 2013, 3(3): 196-200.
[22]	李伟. mcr-1基因在食源性沙门氏菌及大肠杆菌中的转移机制[D].杨凌:西北农林科技大学,2021.
[23]	赵月.恩诺沙星对氟喹诺酮类耐药基因qnrS向沙门菌转移的影响[D].武汉:华中农业大学,2021.
[24]	曹晨阳,苏丽,李梅,等.小檗碱和绿原酸对质粒介导的耐药基因接合转移的影响[J].食品安全质量检测学报,2022,13(23):7725-7733.
	CAO C Y, SU L, LI M, et al.. Effects of berberine and chlorogenic acid on plasmid mediated conjugative transfer of drugresistance genes[J]. J. Food Saf. Qual., 2022, 13(23): 7725-7733.
[25]	盛焕精,李怡谰,王泽维,等.IncI1和IncN质粒阳性沙门氏菌耐药及质粒接合转移特征[J].食品科学,2020,41(18):77-84.
	SHENG H J, LI Y L, WANG Z W, et al.. Characteristics of antibiotic resistance and plasmid conjugative transfer of IncI1 and IncN plasmid positive Salmonella [J]. Food Sci., 2020, 41(18): 77-84.
[26]	ZHAO H, ZHANG X, ZHANG N, et al.. The interplay between Salmonella and host: mechanisms and strategies for bacterial survival[J/OL]. Cell Insight, 2025, 4(2): 100237[2025-12-21]. .
[27]	DONG K, ZHU Y, DENG Q, et al.. Salmonella pSLT-encoded effector SpvB promotes RIPK3-dependent necroptosis in intestinal epithelial cells[J/OL]. Cell Death Discov., 2022, 8(1): 44[2025-10-11]. .

pSLT质粒的转移及其对肠道细胞凋亡的影响

Transfer of the pSLT Plasmid and its Impact on Intestinal Cell Apoptosis

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