铜绿假单胞菌潜在VI型分泌系统效应蛋白PA0423的鉴定及功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-1068

摘要/Abstract

摘要：

【目的】探讨VI型分泌系统（type VI secretion system, T6SS）新的效应蛋白及其功能。【方法】以铜绿假单胞菌PAO1为研究对象，通过基因敲除、免疫印迹、免疫共沉淀、种内和种间竞争、大肠杆菌毒性实验以及结晶紫生物膜等实验探索PA0423的分泌方式和功能。【结果】免疫印迹和免疫共沉淀实验表明PA0423可以与PA0262相互作用，且PA0423的分泌依赖于H2-T6SS；竞争实验表明PA0423是H2-T6SS依赖的抗菌效应蛋白，且具有种间竞争优势；PA0423具有大肠杆菌毒性且PA0422为其免疫蛋白；PA0423能够影响细菌生物膜的形成。【结论】PA0423为H2-T6SS依赖的抗菌效应蛋白，其具有杀菌功能且影响细菌生物膜的形成。

关键词: 铜绿假单胞菌, VI型分泌系统, PA0423, 效应蛋白

Abstract:

【Objective】The objective of this study is to investigate the novel effector proteins and their functions in the type VI secretion system（T6SS）.【Method】 The secretion mode and function of PA0423 were explored by gene knockout, immunoblotting, co-immunoprecipitation, intraspecific and interspecific competition, Escherichia coli toxicity assays and crystal violet biofilm assays.【Result】Western blotting and co-immunoprecipitation experiments showed that PA0423 interacted with PA0262, and the secretion of PA0423 depended on H2-T6SS. PA0423 is a H2-T6SS-dependent antimicrobial effector protein with interspecific competitive advantages. PA0423 is toxic to Escherichia coli and PA0422 is its immune protein. PA0423 affected the formation of bacterial biofilms.【Conclusion】PA0423 is a H2-T6SS-dependent antimicrobial effector protein, which has bactericidal function and affects the formation of bacterial biofilms.

Key words: Pseudomonas aeruginosa, type VI secretion system, PA0423, effector proteins

王彩虹, 蒋梦媛, 邵煜涵. 铜绿假单胞菌潜在VI型分泌系统效应蛋白PA0423的鉴定及功能研究[J]. 生物技术通报, 2024, 40(4): 297-305.

WANG Cai-hong, JIANG Meng-yuan, SHAO Yu-han. Identification and Functional Study of T6SS Effector Protein PA0423 of Pseudomonas aeruginosa[J]. Biotechnology Bulletin, 2024, 40(4): 297-305.

图/表 10

参考文献 30

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菌株 Strain	特点 Characteristic	来源 Source
DH5a	大肠杆菌基因克隆菌株	全式金
BL21（DE3）pLysS	大肠杆菌基因表达菌株	全式金
PAO1	野生型	本实验室
S17-1	接合菌株	本实验室
PAO1△rets	PAO1的rets敲除突变	本实验室
PAO1△rets△clpV1	PAO1的rets和clpV1敲除突变	本实验室
PAO1△rets△clpV2	PAO1的rets和clpV2敲除突变	本实验室
PAO1△rets△clpV3	PAO1的rets和clpV3敲除突变	本实验室
PAO1△PA0262	PAO1的PA0262敲除突变	本实验室
PAO1△PA0423	PAO1的PA0423敲除突变	本实验室
PAO1△PA0423+PME-PA0423	包含PME6032-PA0423重组质粒的PAO1的PA0423敲除菌	本实验室
PAO1△PA0422-0423	PAO1的PA0422和PA0423敲除突变	本实验室

菌株 Strain	特点 Characteristic	来源 Source
DH5a	大肠杆菌基因克隆菌株	全式金
BL21（DE3）pLysS	大肠杆菌基因表达菌株	全式金
PAO1	野生型	本实验室
S17-1	接合菌株	本实验室
PAO1△rets	PAO1的rets敲除突变	本实验室
PAO1△rets△clpV1	PAO1的rets和clpV1敲除突变	本实验室
PAO1△rets△clpV2	PAO1的rets和clpV2敲除突变	本实验室
PAO1△rets△clpV3	PAO1的rets和clpV3敲除突变	本实验室
PAO1△PA0262	PAO1的PA0262敲除突变	本实验室
PAO1△PA0423	PAO1的PA0423敲除突变	本实验室
PAO1△PA0423+PME-PA0423	包含PME6032-PA0423重组质粒的PAO1的PA0423敲除菌	本实验室
PAO1△PA0422-0423	PAO1的PA0422和PA0423敲除突变	本实验室

引物 Primer	序列 Sequence	作用 Function
PA0423-up-F	ACAGCTATGACATGATTAC GAATTCGCGGGTCGCCTTCGCCGGCG AGCAC	构建pk18-PA0423
PA0423-up-R	GCGTTTTTCGTTTCAGGTATCCGCGGACATTCTCCTTAACGTGGTTGTTG	构建pk18-PA0423
PA0423-down-F	CAACAACCACGTTAAGGAGAATGTCCGCGGATACCTGAAACGAAAAACGC	构建pk18-PA0423
PA0423-down-R	CTGCAGGTCGACTCTAGA GGATCCGGCGCTTGAGGATGATGCCGTTCAC	构建pk18-PA0423
PA0422-0423-up-F	ACAGCTATGACATGATTAC GAATTCGGTGCTGGCGGAAGCCTTCG	构建pk18-PA0422-0423
PA0422-0423-up-R	TTCGTTTCAGGTATCCGCGAAAAACTTCCTTGAAAAGC	构建pk18-PA0422-0423
PA0422-0423-down-F	GCTTTTCAAGGAAGTTTTTCGCGGATACCTGAAACGAA	构建pk18-PA0422-0423
PA0422-0423-down-R	CTGCAGGTCGACTCTAGA GGATCCTGCCGTTCACCTGGGGACGC	构建pk18-PA0422-0423
PME-0423-F	ATTTCACACAGGAAACA GAATTCATGCTGAAGAAGACCCTTGC	构建PME6032-PA0423-FLAG
PME-0423-flag-R	ACTGATCCGCTAGTCCGAGGCCTCGAGCTTATCGTCGTCATCCTTGTAAT C	构建PME6032-PA0423-FLAG
PME-flag-R	ACTGATCCGCTAGTCCGAGGC CTCGAGCTTATCGTCGTCATCCTTGTAAT C	构建PME6032-PA0423-FLAG
RSF-PA0423-F	CATCACCACAGCCAGGATCC GAATTCGATGCTGAAGAAGACCCTTGCCG	构建RSFduet-PA0423-myc
RSF-PA0423-R	GTCGAAGGCATTCGCCAGGAGCAGAAACTCATCTCTGAAGAGGATCTG AAGCTTGCGGCCGCATAATGCT	构建RSFduet-PA0423-myc
pET-PA0262-F	CATATGGCAGATCTCAATTG GATATCATGCGTCAAA GGGACCTGAA ATTC	构建pETduet-PA0262
pET-PA0262-R	GCAGCGGTTTCTTTACCAGA CTCGAGGTATCCCGTTGGGAAGTTTTTCAG	构建pETduet-PA0262
pET28-PA0423-F	ATGCTGAAGAAGACCCTTGC	构建pET28-PA423
pET28-PA0423-R	CTTATCGTCGTCATCCTTG	构建pET28-PA423

引物 Primer	序列 Sequence	作用 Function
PA0423-up-F	ACAGCTATGACATGATTAC GAATTCGCGGGTCGCCTTCGCCGGCG AGCAC	构建pk18-PA0423
PA0423-up-R	GCGTTTTTCGTTTCAGGTATCCGCGGACATTCTCCTTAACGTGGTTGTTG	构建pk18-PA0423
PA0423-down-F	CAACAACCACGTTAAGGAGAATGTCCGCGGATACCTGAAACGAAAAACGC	构建pk18-PA0423
PA0423-down-R	CTGCAGGTCGACTCTAGA GGATCCGGCGCTTGAGGATGATGCCGTTCAC	构建pk18-PA0423
PA0422-0423-up-F	ACAGCTATGACATGATTAC GAATTCGGTGCTGGCGGAAGCCTTCG	构建pk18-PA0422-0423
PA0422-0423-up-R	TTCGTTTCAGGTATCCGCGAAAAACTTCCTTGAAAAGC	构建pk18-PA0422-0423
PA0422-0423-down-F	GCTTTTCAAGGAAGTTTTTCGCGGATACCTGAAACGAA	构建pk18-PA0422-0423
PA0422-0423-down-R	CTGCAGGTCGACTCTAGA GGATCCTGCCGTTCACCTGGGGACGC	构建pk18-PA0422-0423
PME-0423-F	ATTTCACACAGGAAACA GAATTCATGCTGAAGAAGACCCTTGC	构建PME6032-PA0423-FLAG
PME-0423-flag-R	ACTGATCCGCTAGTCCGAGGCCTCGAGCTTATCGTCGTCATCCTTGTAAT C	构建PME6032-PA0423-FLAG
PME-flag-R	ACTGATCCGCTAGTCCGAGGC CTCGAGCTTATCGTCGTCATCCTTGTAAT C	构建PME6032-PA0423-FLAG
RSF-PA0423-F	CATCACCACAGCCAGGATCC GAATTCGATGCTGAAGAAGACCCTTGCCG	构建RSFduet-PA0423-myc
RSF-PA0423-R	GTCGAAGGCATTCGCCAGGAGCAGAAACTCATCTCTGAAGAGGATCTG AAGCTTGCGGCCGCATAATGCT	构建RSFduet-PA0423-myc
pET-PA0262-F	CATATGGCAGATCTCAATTG GATATCATGCGTCAAA GGGACCTGAA ATTC	构建pETduet-PA0262
pET-PA0262-R	GCAGCGGTTTCTTTACCAGA CTCGAGGTATCCCGTTGGGAAGTTTTTCAG	构建pETduet-PA0262
pET28-PA0423-F	ATGCTGAAGAAGACCCTTGC	构建pET28-PA423
pET28-PA0423-R	CTTATCGTCGTCATCCTTG	构建pET28-PA423

分析 Analysis	编号 Accession No.	描述 Description	Interpro 编号 Interpro accession	Interpro 描述 Interpro description	氨基酸起始Amino acid start	氨基酸终止 Amino acid stop	误差 E-value
Hamap	MF_00780	Protein YceI	IPR023480	Uncharacterised protein family UPF0312/YceI	2	191	45.339172
PANTHER	PTHR34406	Protein YCEI	-	-	2	190	6.6E-62
SUPERFAMILY	SSF101874	YceI-like	IPR036761	Lipid/polyisoprenoid-binding,Yce-like superfamily	22	191	2.88E-56
Gene3D	G3DSA:2.40128.110	-	IPR036761	Lipid/polyisoprenoid-binding,Yce-like superfamily	24	191	1.7E-58
FunFam	G3DSA:2.40128.110:FF:000001	UPF0312 protein HMPREF3014_17255	-	-	24	191	3.7E-118
SMART	SM00867	YceI_2	IPR007372	Lipid/polyisoprenoid-binding,Yce-like	25	189	1.3E-71
Pfam	PF04264	YceI-like domain	IPR007372	Lipid/polyisoprenoid-binding,Yce-like	26	187	2.9E-41