Identification and Functional Study of T6SS Effector Protein PA0423 of Pseudomonas aeruginosa

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

Abstract

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

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.

Figures/Tables 10

References 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