Mapping Epitopes of a Novel Peptidoglycan Cross-Linking Enzyme Cwp22 Recognized by Human Sera Obtained from Patients with Clostridioides difficile Infection and Cord Blood
Abstract
:1. Introduction
2. Materials and Methods
2.1. Blood Sera
2.1.1. Human Peripheral Blood Sera
2.1.2. Human Umbilical Cord Blood Sera
2.2. Bacterial Strains
2.3. Protein Isolation
2.4. SDS-PAGE Electrophoresis
2.5. Silver Staining
2.6. Western Blotting
2.7. Protein Identification
2.8. Analysis of Sequence Homology
2.9. Protein Modeling
2.10. Predicting Epitopes
2.11. Chemical Synthesis of Peptides
2.12. Pin Enzyme-Linked Immunosorbent Assay
2.13. Epitope Identification
2.14. Epitope Analysis
3. Results
3.1. The Identification of Cwp22 as a New Immunoreactive CD Surface Protein
3.2. In Silico Analysis Shows that Cwp22 Protein is a Good Vaccine Antigen Candidate
3.3. Predicting the Epitopes of Cwp22 Protein
3.4. Epitope Mapping
3.5. Defining the Epitopes of Cwp22 Protein
3.6. Epitopes Characteristics
4. Discussion
Author Contributions
Funding
Conflicts of Interest
References
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NCBI Identification | CD Strain | Characteristics | Sequence Coverage | % of Identical Amino Acids |
---|---|---|---|---|
CBA64897.1 | CD196 | Isolated from an adult, ribotype 027, producing toxins | 100% | 99.84 |
PBF93815.1 | 7468-NonSp/ST97 | Isolated from a child, producing toxins | 100% | 99.69 |
EQG56151.1 | DA00145 | 100% | 99.69 | |
EQH98359.1 | F314 | Acute CDI, producing toxins | 100% | 99.69 |
OFU21376.1 | HMSC19C09 | 100% | 99.69 | |
OFU05949.1 | HMSC19D07 | 100% | 99.53 | |
EHJ35368.1 | 70-100-2010 | 100% | 99.38 | |
PBG98735.1 | 7481-NonSp/ST59 | 100% | 99.22 | |
EQF50178.1 | CD174 | Asymptomatic carriage, no toxins | 100% | 99.07 |
EQE30253.1 | CD34 | Asymptomatic carriage, no toxins | 100% | 99.22 |
AFV69537.1 | C192 | Ribotype 012, hipervirulent | 100% | 99.22 |
EQJ05755.1 | P7 | Reference strain, producing toxins | 100% | 99.07 |
PBE99335.1 | 5573-CF/ST37 | Producing TcdB | 100% | 98.76 |
EQE84199.1 | CD69 | Acute CDI, producing toxins | 100% | 98.91 |
OJT75205.1 | NT422 | Ribotype 231 | 98% | 99.37 |
EFH06251.1 | NAP08 | Hipervirulent | 100% | 96.89 |
PBI20744 | 6615-NonSp/ST11 | Isolated from a child, hipervirulent | 100% | 96.74 |
KPI50551 | RA09-70 | Producing TcdA | 100% | 93.17 |
PBE36357.1 | 5537-D/ST9 | Isolated from a child, producing toxins | 100% | 91.93 |
ERM37167 | P64 | Asymptomatic carriage, producing toxins | 79% | 99.41 |
EQE47488 | CD42 | Acute CDI, producing toxins | 73% | 99.30 |
EQK74493 | CD113 | Relapse, producing toxins | 72% | 99.51 |
NCBI Identification | Protein Name | Bacteria Strain | Sequence Coverage | % of Identical Aminoacids |
---|---|---|---|---|
WP_092726434.1 | l,d-transpeptidase | Romboutsia lituseburensis | 53% | 56.07 |
WP_007287442.1 | l,d-transpeptidase | Clostridium bartletti | 53% | 51.59 |
WP_084157842.1 | Hipothetical protein | Bacillus manliponensis | 58% | 47.59 |
WP_082007829.1 | Hipothetical protein | Terrisporobacter othiniensis | 54% | 52.10 |
WP_052404759.1 | l,d-transpeptidase | Bacillus rubiinfantis | 55% | 48.66 |
WP_066389794.1 | l,d-transpeptidase | Bacillus mesonae | 52% | 41.90 |
KLR55379.1 | Surface protein | Clostridium sordelli | 53% | 45.98 |
54EFRVAT59 | 201KVNGKM207 | 268WQEKNGKKYY277 | |
---|---|---|---|
Molecular mass (g/mol) | 721.8 | 675.84 | 1343.49 |
Water solubility | good | good | good |
pI | 6.86 | 10.69 | 9.9 |
Charge in physiological pH | 0 | +2 | +2 |
Peptide structure |
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Razim, A.; Pacyga, K.; Martirosian, G.; Szuba, A.; Gamian, A.; Myc, A.; Górska, S. Mapping Epitopes of a Novel Peptidoglycan Cross-Linking Enzyme Cwp22 Recognized by Human Sera Obtained from Patients with Clostridioides difficile Infection and Cord Blood. Microorganisms 2019, 7, 565. https://doi.org/10.3390/microorganisms7110565
Razim A, Pacyga K, Martirosian G, Szuba A, Gamian A, Myc A, Górska S. Mapping Epitopes of a Novel Peptidoglycan Cross-Linking Enzyme Cwp22 Recognized by Human Sera Obtained from Patients with Clostridioides difficile Infection and Cord Blood. Microorganisms. 2019; 7(11):565. https://doi.org/10.3390/microorganisms7110565
Chicago/Turabian StyleRazim, Agnieszka, Katarzyna Pacyga, Gajane Martirosian, Andrzej Szuba, Andrzej Gamian, Andrzej Myc, and Sabina Górska. 2019. "Mapping Epitopes of a Novel Peptidoglycan Cross-Linking Enzyme Cwp22 Recognized by Human Sera Obtained from Patients with Clostridioides difficile Infection and Cord Blood" Microorganisms 7, no. 11: 565. https://doi.org/10.3390/microorganisms7110565