Deciphering Additional Roles for the EF-Tu, l-Asparaginase II and OmpT Proteins of Shiga Toxin-Producing Escherichia coli
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains
2.2. Construction of Isogenic Mutants
2.3. Cloning and Expression of the ansB and ompT Genes
2.4. Western Blotting
2.5. Transmission Electron Microscopy (TEM) and Immunogold Labeling
2.6. Proteomic Analysis of Outer Membrane Vesicles (OMVs)
2.7. Bacterial Adhesion Assays
2.8. Proliferation Assay
2.9. Statistical Analysis
3. Results
3.1. EF-Tu and l-Asparaginase II Are Secreted and Associated with the Surface of STEC O157:H7
3.2. The Monoclonal Anti-EF-Tu Antibody (mAb 900) Does Not Affect the Adhesion of STEC O157:H7 str. EDL933 to HT-29 Epithelial Cells, Fibronectin and Laminin
3.3. l-Asparaginase II Produced by STEC O157:H7 str. EDL933 Inhibits T Cell Proliferation
3.4. Deletion of The ompT Gene Affects The Adhesion of STEC O157:H7 str. EDL933 to HT-29 Cells
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Primer Name | Sequence 5′ to 3′ * | Protocol | Source |
---|---|---|---|
d-ompT_F | ATCATTAAAACGATTGAATGGAGACCTTTTATGCGGGCGAAACTT CTGGGAATAGTCCTGACAACCCCTAGTGTAGGCTGGAGCTGCTTC | Deletion of the ompT gene | This work |
d-ompT _R | CCAACCGGGGAGAAAATCTGGTTAACTTCGTTAAAAGGTGTACTT AAGACCAGCAGTAGTGATGAAGTTACATATGAATATCCTCCTTAG | Deletion of the ompT gene | This work |
ompT-e _F | TCCAGCATATTAAACCACAACAATAATA | Confirmation of the ompT deletion | This work |
ompTc_F | CATATGCGGGCGAAACTTCT | Cloning of the ompT gene | This work |
ompTc_R | GGATCCTTAAAAGGTGTACTTA | Cloning of the ompT gene | This work |
d-ansB _F | AAGGGATAATGCGTAGCGTTCACGTAACTGGAGGAATGAAATGGA GTTTTTCAAAAAGACGGTGTAGGCTGGAGCTGCTTC | Deletion of the ansB gene | This work |
d-ansB _R | TGAAGTGAAAAAGCCCCGGCGCGATACCGGGGCGAAATGATTAGT ACTGATTGAAGATCTGCATATGAATATCCTCCTTAG | Deletion of the ansB gene | This work |
ansB-e _F | CCTCAATCCAAACCGAGTGGAAA | Confirmation of the ansB deletion | This work |
ansBc_F | CATATGATGGAGTTTTTCAAAAAGACGGCAC | Cloning of the ompT gene | This work |
ansBC_R | GGATCCTTAGTACTGATTGAAGATCTGCTGG | Cloning of the ompT gene | This work |
kanR_R | GCCATGATGGATACTTTCT | Confirmation of allelic replacement | [50] |
Protein Name | Description | Coverage Percentage | No. of Peptides | Protein Score * | Protein Intensity Values | ||
---|---|---|---|---|---|---|---|
STEC O157:H7 | STEC O91:H21 | AIEC NRG857c | |||||
A0A0H3EMH4 | EF-Tu | 69.04 | 27 | 1184.8 | 4.5 × 1014 | 1.6 × 1015 | 1.0 × 1015 |
A0A0H3EPM6 | EF-Tu | 69.04 | 27 | 1184.8 | 4.5 × 1014 | 1.6 × 1015 | 1.0 × 1015 |
A0A0H3ERC0 | OmpT | 14.20 | 6 | 124 | 1.6 × 1013 | 0 | 3.8 × 1013 |
Experiment | HT-29 Cells | Fibronectin | Laminin | |||
---|---|---|---|---|---|---|
% Bacteria ± SD | p Value * | % Bacteria ± SD | p Value * | % Bacteria ± SD | p Value * | |
No antibody | 100 ± 9.9 | - | 100 ± 7.3 | - | 100 ± 10.4 | |
Anti-EF-Tu (1:10) | 107.7 ± 6.0 | >0.05 | 118 ± 23.3 | >0.05 | 135.2 ± 3.9 | >0.05 |
Anti-EF-Tu (1:100) | 105.4 ± 17.2 | >0.05 | Not evaluated | - | Not evaluated | - |
Mouse IgG (1:10) ** | 113.6 ± 5.6 | >0.05 | 131.9 ± 10.1 | >0.05 | 133.8 ± 20.6 | >0.05 |
Mouse IgG (1:100) ** | 121.1 ± 10.6 | >0.05 | Not evaluated | - | Not evaluated | - |
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Torres, A.N.; Chamorro-Veloso, N.; Costa, P.; Cádiz, L.; Del Canto, F.; Venegas, S.A.; López Nitsche, M.; Coloma-Rivero, R.F.; Montero, D.A.; Vidal, R.M. Deciphering Additional Roles for the EF-Tu, l-Asparaginase II and OmpT Proteins of Shiga Toxin-Producing Escherichia coli. Microorganisms 2020, 8, 1184. https://doi.org/10.3390/microorganisms8081184
Torres AN, Chamorro-Veloso N, Costa P, Cádiz L, Del Canto F, Venegas SA, López Nitsche M, Coloma-Rivero RF, Montero DA, Vidal RM. Deciphering Additional Roles for the EF-Tu, l-Asparaginase II and OmpT Proteins of Shiga Toxin-Producing Escherichia coli. Microorganisms. 2020; 8(8):1184. https://doi.org/10.3390/microorganisms8081184
Chicago/Turabian StyleTorres, Alexia N., Nayaret Chamorro-Veloso, Priscila Costa, Leandro Cádiz, Felipe Del Canto, Sebastián A. Venegas, Mercedes López Nitsche, Roberto F. Coloma-Rivero, David A. Montero, and Roberto M. Vidal. 2020. "Deciphering Additional Roles for the EF-Tu, l-Asparaginase II and OmpT Proteins of Shiga Toxin-Producing Escherichia coli" Microorganisms 8, no. 8: 1184. https://doi.org/10.3390/microorganisms8081184