Identification by Reverse Vaccinology of Three Virulence Factors in Burkholderia cenocepacia That May Represent Ideal Vaccine Antigens
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bioinformatic Analysis
2.2. Bacterial Strains and Plasmids
2.3. General Molecular Biology Techniques
2.4. Proteomic Analysis
2.5. Antimicrobial Susceptibility Testing for Planktonic Cells
2.6. Bacterial Autoaggregation Assay
2.7. In Vitro Biofilm Formation Test in 96-Well Microtiter Plates
2.8. Biofilm Evaluation by Confocal Laser Scanning Microscopy
2.9. Swimming Motility Assay
2.10. Infection in Galleria Mellonella
2.11. Lipase Activity Assay
2.12. Rhamnolipid Analysis
2.13. Statistical Methods
3. Results and Discussion
3.1. In Silico Identification of Antigen Candidates
3.2. Construction of Deletion Mutants of the Selected Antigen Candidates
3.3. Analysis of Protein Localization
3.4. Antibiotic Susceptibility
3.5. Phenotypic Characterization of the Deleted Mutant
3.5.1. Biofilm Formation
3.5.2. Bacterial Autoaggregation
3.5.3. Swimming Motility
3.6. Infection in Galleria mellonella
3.7. Characterization of the Lipase BCAM0949
3.7.1. Lipolytic Activity
3.7.2. Rhamnolipid Production
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Strain | Source |
---|---|
B. ambifaria AMMD | cell culture |
B. anthina AZ-4-10-S1-D7 | soil |
B. cepacia ATCC25416 | wash glove |
B. cenocepacia J2315 | CF sputum |
B. cenocepacia DDS-22E-1 | aerosol sample |
B. cenocepacia H111 | CF patient |
B. cenocepacia HI12424 | cell culture |
B. cenocepacia MSMB384WGS | water |
B. cenocepacia VC12308 | CF sputum |
B. cenocepacia YG-3 | cell culture |
B. contaminans SK875 | pig with swine respiratory disease |
B. diffusa RF2-nonBP9 | soil |
B. dolosa AU0158 | CF patient |
B. lata A05 | blood of patient with cepacia syndrome |
B. latens AU17928 | CF Maxillary Sinus |
B. gladioli ATCC10248 | plant |
B. glumae 257SH-1 | rice panicles |
B. metallica FL-6-5-30-S1-D7 | soil |
B. multivorans ATCCBAA-247 | CF patient |
B. pyrrocinia DSM10685 | soil |
B. seminalis FL-5-4-10S1-D7 | soil |
B. stabilis ATCC BAA-67 | CF sputum |
B. stagnalis MSMB735WGS | soil |
B. territorii RF8-non-BP5 | soil |
B. ubonensis MSMB1471WGS | soil |
B. vietnamiensis AU1233 | CF blood |
Time | Voltage (V) |
---|---|
1 h | 0 |
8 h | 30 |
1 h | 12 |
30 min | 300 |
3 h | Up to 3500 |
10 min | 500 |
Overnight | 7950 |
Protein Name | Functional Prediction | Protein Type | Length aa | Cellular Localization Prediction | N° of Predicted TMD | VaxiJen Score | Vaxign-ML Score |
---|---|---|---|---|---|---|---|
BCAL0151 | ABC-type branched-chain amino acid transport systems, periplasmic component | Extracellular ligand binding protein | 381 | unknown | Signal peptide | 0.5863 | 98.9 |
BCAL0198 | Outer membrane protein, OmpW | Putative outer membrane protein | 278 | unknown | 8 | 0.6727 | 90.9 |
BCAL0199 | DUF2957 domain-containing protein, putatative lipoprotein | Lipoprotein | 414 | unknown | Signal peptide | 0.6030 | 95.2 |
BCAL0200 | DUF2957 domain-containing protein, putative lipoprotein | Lipoprotein | 477 | extracellular | ≤1 | 0.7576 | 90.9 |
BCAL0358 | peptidase M1 family M1 metalopeptidase | Enzyme | 723 | extracellular | ≤1 | 0.4835 | 99.7 |
BCAL1524 | Collagen-like triple helix repeat-containing protein | Lipoprotein | 558 | extracellular | ≤1 | 0.9966 | 90.9 |
BCAL2229 | Beta-propeller fold lactonase family protein, surface antigen | Enzyme | 330 | extracellular | ≤1 | 0.3297 | 90.9 |
BCAL2615 | Putative exported outer membrane porin protein | Putative outer membrane protein | 359 | outermembrane | 16 | 0.6883 | 91.0 |
BCAL3279 | DUF3971 domain-containing protein, Possible exported protein | Hypothetical protein | 1400 | extracellular | 1 | 0.5901 | 90.9 |
BCAL3353 | Putative outer membrane autotransporter | Autotransporter/adhesin protein | 1772 | outermembrane/extracellular | ≤1 | 0.8135 | 94.9 |
BCAM0949 | LipA triacylglycerol lipase | Enzyme | 365 | extracellular | 1 | 0.4937 | 90.9 |
BCAM1514 | Outer membrane protein | Other protein | 294 | outermembrane | ≥1 | 0.6875 | 90.9 |
BCAM1737 | Alpha-2-macroglobulin | Other protein | 2021 | outermembrane | 1 | 0.6400 | 97.7 |
BCAM1740 | Adhesin | Lipoprotein | 233 | extracellular | ≥1 | 0.6559 | 90.9 |
BCAM1931 | Outer membrane porin | Putative outer membrane protein | 360 | outermembrane | 16 | 0.6868 | 92.5 |
BCAM2311 | Outer membrane porin OmpC | Putative outer membrane protein | 380 | outermembrane | 16 | 0.5846 | 91.4 |
BCAM2328 | Coagulation factor 5/8 type-like protein | Other protein | 470 | extracellular | 1 | 0.5850 | 95.4 |
BCAM2418 | Cell surface protein putative haemagglutinin-related autotransporter/adhesin protein | Autotransporter/ adhesin protein | 558 | extracellular | 1 | 0.9141 | 93.5 |
BCAM2444 | NHL-superfamily, Six-bladed beta-propeller, TolB-like | Other protein | 643 | extracellular | ≤1 | 0.5015 | 95.0 |
BCAS0147 | YncE super family, beta-propeller fold lactonase family protein | Enzyme | 397 | outermembrane/extracellular | 1 | 0.6027 | 90.9 |
BCAS0236 | putative haemagglutinin-related autotransporter/adhesin protein | Autotransporter/ adhesin protein | 1497 | outermembrane/extracellular | ≤1 | 0.8682 | 92.2 |
BCAS0321 | Autotransporter | Autotransporter/ adhesin protein | 4250 | outermembrane | ≥1 | 1.115 | 90.9 |
BCAS0335 | putative haemagglutinin-related autotransporter/adhesin protein | Autotransporter/ adhesin protein | 1198 | extracellular | ≤1 | 0.8436 | 98.9 |
BCAS0409 | M4 family metallopeptidase | Enzyme | 566 | extracellular | ≤1 | 0.6513 | 95.0 |
BCAS0641 | phosphatase PAP2 family protein | Enzyme | 464 | unknown | ≤1 | 0.6073 | 93.9 |
Antibiotics | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|
Strain | AMK | AZT | CIP | LVX | MEM | MIN | NAL | PIP | SPX | TOB |
K56-2 | ≥256 | ≥256 | 2 | 4 | 8 | 8 | 16 | 128 | 4 | ≥256 |
∆BCAL1524 | ≥256 | 256 | 2 | 4 | 8 | 4 | 8 | 128 | 4 | ≥256 |
∆BCAM0949 | ≥256 | 256 | 2 | 4 | 4 | 16 | 16 | 32 | 4 | ≥256 |
∆BCAS0335 | ≥256 | 256 | 4 | 4 | 8 | 64 | 16 | 128 | 4 | ≥256 |
∆BCAL1524 pSCrhaB2BCAM1524 | ≥256 | ≥256 | ≤2 | 4 | 16 | ≤2 | 16 | 64 | 4 | ≥256 |
∆BCAM0949 pSCrhaB2BCAM0949 | ≥256 | ≥256 | ≤2 | 4 | 4 | ≤2 | 4 | 128 | 4 | ≥256 |
∆BCAS0335 pSCrhaB2BCAS0335 | ≥256 | ≥256 | ≤2 | 4 | 8 | 4 | 8 | 128 | 4 | ≥256 |
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Irudal, S.; Scoffone, V.C.; Trespidi, G.; Barbieri, G.; D’Amato, M.; Viglio, S.; Pizza, M.; Scarselli, M.; Riccardi, G.; Buroni, S. Identification by Reverse Vaccinology of Three Virulence Factors in Burkholderia cenocepacia That May Represent Ideal Vaccine Antigens. Vaccines 2023, 11, 1039. https://doi.org/10.3390/vaccines11061039
Irudal S, Scoffone VC, Trespidi G, Barbieri G, D’Amato M, Viglio S, Pizza M, Scarselli M, Riccardi G, Buroni S. Identification by Reverse Vaccinology of Three Virulence Factors in Burkholderia cenocepacia That May Represent Ideal Vaccine Antigens. Vaccines. 2023; 11(6):1039. https://doi.org/10.3390/vaccines11061039
Chicago/Turabian StyleIrudal, Samuele, Viola Camilla Scoffone, Gabriele Trespidi, Giulia Barbieri, Maura D’Amato, Simona Viglio, Mariagrazia Pizza, Maria Scarselli, Giovanna Riccardi, and Silvia Buroni. 2023. "Identification by Reverse Vaccinology of Three Virulence Factors in Burkholderia cenocepacia That May Represent Ideal Vaccine Antigens" Vaccines 11, no. 6: 1039. https://doi.org/10.3390/vaccines11061039
APA StyleIrudal, S., Scoffone, V. C., Trespidi, G., Barbieri, G., D’Amato, M., Viglio, S., Pizza, M., Scarselli, M., Riccardi, G., & Buroni, S. (2023). Identification by Reverse Vaccinology of Three Virulence Factors in Burkholderia cenocepacia That May Represent Ideal Vaccine Antigens. Vaccines, 11(6), 1039. https://doi.org/10.3390/vaccines11061039