The Impact of Vp-Porin, an Outer Membrane Protein, on the Biological Characteristics and Virulence of Vibrio Parahaemolyticus
Abstract
Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Strains, Media and Experimental Animals
2.2. Protein Domain and Structure Analysis
2.3. Construction of ∆Vp-porin Deletion Mutant and Complement Strain
2.4. Proteolysis Activity Assay
2.5. Outer Membrane Permeabilization Assay
2.6. Morphological Observation
2.7. Antimicrobial Susceptibility Testing and Survival Assay
2.8. Motility Assay
2.9. qRT-PCR Analysis
2.10. Assessment of Strains’ Virulence Using Tetrahymena thermophila
2.11. Statistical Analysis
3. Results
3.1. Vp-porin Sequence and Structural Analysis
3.2. Construction and Characterization of the Deletion Mutant of Vp-porin Gene and Complement Strain
3.3. Permeabilization of Outer Membranes
3.4. Vp-porin Gene Deletion Affects Antimicrobial Susceptibility of V. parahaemolyticus
3.5. The Vp-porin Mutant Exhibits Lower Motility and Decreases Transcription of Polar Flagellar Genes and Lateral Flagellar Genes in V. parahaemolyticus
3.6. Assessment of Virulence of ∆Vp-porin Using Tetrahymena
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Strains | Genotype and Characteristics | Source |
---|---|---|
V. parahaemolyticus 17802 | Cms, Kms, Ampr, wild-type strain, | ATCC |
∆Vp-porin | V. parahaemolyticus strain in-frame deletion in Vp-porin | This study |
∆Vp-porin-C | The complement of ∆Vp-porin | This study |
Escherichia coli | ||
CC118 | λpir lysogen of CC118 (Δ(ara-leu) araD ΔlacX74galEgalKphoA20 thi-1rpsE rpoB argE (Am) recA1 | Our lab |
CC118/pHelper | CC118 λpir harboring plasmid pHelper | Our lab |
Plasmids | ||
pSR47S | Bacterial allelic exchange vector with sacB, KanR | Our lab |
pSR47S-∆Vp-porin | A 1689 bp fragment containing the upstream and downstream sequences of the ∆Vp-porin gene in pSR47S, KanR | This study |
pSR47S-Vp-porin-C | A 2634 bp fragment containing the Vp-porin sequences in pSR47S, KanR | This study |
Primer Name | Primer Sequence (5′ to 3′) | Purpose |
---|---|---|
UP-F | CGAGCTCCTTGATGGACTTCGCCAAC | Creation of ∆Vp-porin deletion fusion fragment |
UP-R | CAACATTCGGTACTCAAGCAGCACTTGGTGCACGTTACTAC | |
DOWN-F | GTAGTAACGTGCACCAAGTGCTGCTTGAGTACCGAATGTTG | |
DOWN-R | GACTAGTGTACACACCGAATGCAGAC | |
Vp-porin-T1 | GAACAACACTAGAACGCGC | Confirmation of ∆Vp-porin deletion |
Vp-porin-T2 | TCGGTTACCGAAGAGTCTTC |
Primer Name | Primer Sequence (5′ to 3′) | Target |
---|---|---|
flgB-F | ACAAGGCACTAGGCATCC | polar flagellar cluster I genes |
flgB-R | GACCATCTGTTCGGCTAAG | |
flgC-F | GCGTCATGCTGTATTTGGTG | |
flgC-R | AACCTGCACATTCGTTTGGT | |
flgM-F | ATTCAAGTGCGACATCAAG | |
flgM-R | CGGAGAAGCTGCCATATC | |
flgK-F | GCCGTCAGTCAGTGATTC | |
flgK-R | GTAGAGGACAGGTTGAGTTC | |
fliE-F | CACTGTGCCCGTTTGCTTAC | polar flagellar cluster II genes |
fliE-R | TCCGGCGGATGCTTCTATTC | |
fliK-F | GTCGAGAAGAATGGCGAGAG | |
fliK-R | CCAACTGAGCCTCTGACTCC | |
flgA-F | TACCGACTGGCAAAGGTTGG | |
flgA-R | TACCGACTGGCAAAGGTTGG | lateral flagellar cluster I genes |
flgB-F | GCAGGTTCAGGCCCAGTATT | |
flgB-R | TCATGTTGAGAAACGTCAGGCT | |
flgG-F | AGATCTAGCGGTAATGGGGC | |
flgG-R | GAGAAAGAGGTCGCGTTGTC | |
lafA-F | GCTGGTGGCCTTATCGAAGA | |
lafA-R | TACTGCGAAGTCTGCATCCAT | |
motY-F | ATTAGTGAGGGTGCGCCTTT | |
motY-R | GGTGAAGGGAAGGAATGGCA | |
fliE-F | CGCTTGAGAAAACGACAGTGG | lateral flagellar cluster I genes |
fliE-R | CCTACTAATGCGGTCTCGGC | |
VPA1043-F | TCGAACAGCACGTAGAATCG | T6SS2 genes |
VPA1043-R | GTGGCACTTCAGTTTCGTGA | |
VPA1044-F | TCCTCAACCAAATCCTCGAC | |
VPA1044-R | GCGTAGTTAGGCGTGTAGCC | |
VPA1045-F | CCGATGCTCAATGGCTTAAT | |
VPA1045-R | GCTGCTCTTTACCCAACTGC | |
rpoN-F | GAGTGCACGGATTGCTGTTG | rpoN gene |
rpoN-R | CGGTGGACATGCATGAATCC | |
16s rRNA-F | TTAAGTAGACCGCCTGGGGA | qPCR of 16s rRNA |
16s rRNA-R | GCAGCACCTGTCTCAGAGTT |
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Che, J.; Fang, Q.; Hu, S.; Liu, B.; Wang, L.; Fang, X.; Li, L.; Luo, T.; Bao, B. The Impact of Vp-Porin, an Outer Membrane Protein, on the Biological Characteristics and Virulence of Vibrio Parahaemolyticus. Biology 2024, 13, 485. https://doi.org/10.3390/biology13070485
Che J, Fang Q, Hu S, Liu B, Wang L, Fang X, Li L, Luo T, Bao B. The Impact of Vp-Porin, an Outer Membrane Protein, on the Biological Characteristics and Virulence of Vibrio Parahaemolyticus. Biology. 2024; 13(7):485. https://doi.org/10.3390/biology13070485
Chicago/Turabian StyleChe, Jinyuan, Qitong Fang, Shaojie Hu, Binghong Liu, Lei Wang, Xiu Fang, Lekang Li, Tuyan Luo, and Baolong Bao. 2024. "The Impact of Vp-Porin, an Outer Membrane Protein, on the Biological Characteristics and Virulence of Vibrio Parahaemolyticus" Biology 13, no. 7: 485. https://doi.org/10.3390/biology13070485
APA StyleChe, J., Fang, Q., Hu, S., Liu, B., Wang, L., Fang, X., Li, L., Luo, T., & Bao, B. (2024). The Impact of Vp-Porin, an Outer Membrane Protein, on the Biological Characteristics and Virulence of Vibrio Parahaemolyticus. Biology, 13(7), 485. https://doi.org/10.3390/biology13070485