Peptidoglycan-Free Bacterial Ghosts Confer Enhanced Protection against Yersinia pestis Infection
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains, Plasmids, and Culture Conditions
2.2. Animals
2.3. Construction of Hypo-Endotoxic Y. pestis Strain
2.4. Generation of BGs
2.5. Transmission Electron Microscopy (TEM)
2.6. Ethics Statement
2.7. Animal Immunization and Exposure to Virulent Y. pestis Challenge
2.8. Immune Response Assays
2.8.1. ELISA
2.8.2. Cellular Responses: Analysis of Stimulated Splenocytes
2.9. Statistics
3. Results
3.1. Generation and Characterization of Y. pestis Bacterial Ghosts
3.2. Humoral Immune Responses
3.3. IFN-γ Analysis
3.4. Analysis of Inflammatory Response
3.5. Protection of Immunized Animals against Y. pestis Challenge
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Financial Disclosure
References
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Strains/Plasmid/Bacteriophage | Relevant Genotype or Annotation | Source |
---|---|---|
E. coli | ||
DH5α | F−, gyrA96(Nalr), recA1, relA1, endA1, thi-1, hsdR17(rk−, mk+), glnV44, deoR, Δ(lacZYA-argF)U169, [φ80dΔ(lacZ)M15], supE44 | SCPM-O * |
Y. pestis | ||
231 | wild type strain; universally virulent (LD50 for mice ≤ 10 CFU, for guinea pigs ≤ 10 CFU); Pgm+, pMT1, pPCP1, pCD− | SCPM-O [23] |
KM260(12) | avirulent derivative of 231; Pgm+, pMT1−, pPCP1−, pCD− | SCPM-O |
KM260(12)ΔlpxM | ΔlpxM derivative of KM260(12) | SCPM-O |
KM260(12)ΔlpxM/pEYR’ | KM260(12)ΔlpxM containing pEYR’ | This study |
KM260(12)ΔlpxM/pEYR’-E | KM260(12)ΔlpxM containing pEYR’-E | This study |
KM260(12)ΔlpxM/pEYR’-E-Y-K | KM260(12)ΔlpxM containing pEYR’-E-Y-K | This study |
KM260(12)ΔlpxM/pEYR’-Y-K | KM260(12)ΔlpxM containing pEYR’-Y-K | This study |
KM260(12)ΔlpxM/pEYR’-E-S-R-Rz | KM260(12)ΔlpxM containing pEYR’-E-S-R-Rz | This study |
KM260(12)ΔlpxM/pEYR’-S-R-Rz | KM260(12)ΔlpxM containing pEYR’-S-R-Rz | This study |
Plasmid | ||
pACYC184 | Source of p15A ori and cat gene | [24] |
pBAD/myc-HisA | Source of rrnB transcription terminator | Invitrogen |
pET32b (+) | Source of multiple cloning site | Novagene |
pEYR’ | Expression vector, phage Lambda modified right promoter (pR’) (Cmr) | SCPM-O |
pEYR’-E | Lysis plasmid, pEYR’-lysis E (Cmr) | SCPM-O |
pEYR’-E-Y-K | Lysis plasmid, pEYR’-lysis E, Y, K (Cmr) | SCPM-O |
pEYR’-Y-K | Lysis plasmid, pEYR’-lysis Y, K (Cmr) | SCPM-O |
pEYR’-E-S-R-Rz | Lysis plasmid, pEYR’-lysis E, S, R, Rz (Cmr) | SCPM-O |
pEYR’-S-R-Rz | Lysis plasmid, pEYR’-lysis S, R, Rz (Cmr) | SCPM-O |
Bacteriophage | ||
λCE6 | Source of pR promoter, holin (S) and endolysins (R-Rz) genes (cI857Sam7) | Thermo Scientific |
φX174 | Source of E protein gene | Thermo Scientific |
L-413C | Source of holin (Y) and endolysin (K) genes | Russian Research Anti-Plague Institute Microbe |
Name | Sequence |
---|---|
pEY-1 (ClaI) | 5′CCCATCGATCATATCGTCAATTATTAC3′ * |
pEY-2 (SphI) | 5′ATATTGCATGCTGTCAAACATGAGAATTAC3′ |
pEY-5 (NdeI) | 5′GGCCATATGCACCATCATCATC3′ |
pEY-6б | 5′TCAGTGGTGGTGGTGGTGGTG3′ ** |
pEY-7б | 5′CACCACCACCACCACCACTGATGAGTTTAAACGGTCTCCAG3′ |
pEY-8 (ClaI) | 5′CCCATCGATTTGCTTCGCAACGTTCAAATC3′ |
pR-For (SphI) | 5′CACAAAGCATGCGGAGTGAAAATTCCCCTAATTCG3′ |
pR-Rev (NdeI) | 5′GATACCATATGAACCTCCTTAGTACATGCAACCATT3′ |
pR (T > C) (NdeI) | 5′GTGCATATGAACCTCCTTAGTACATGCAACCATTATCACCGCCAGAGGTAAAATAGTCAACACGCGCGGTGTTAG3′ |
E1 (NdeI) | 5′AGGCATATGGTACGCTGGACTTTGTG3′ |
E2 (XhoI) | 5′AATCTCGAGTCACTCCTTCCGCACGTAA3′ |
Y-NdeI (XhoI) | 5′GGTGGCATATGACAGCAGAAGAAAAAAGC3′ |
Y-SalI (SalI) | 5′GCCGTCGACAACAGGAGGAATTAACCATGACAGCAGAAGAAAAAAGC3′ |
K-XhoI (XhoI) | 5′ATTCTCGAGTTAAGCCGGTACGCCGCCAG3′ |
S-R-Rz-For (SalI) | 5′AAAGTCGACAACAGGAGGAATTAACCATGAAGATGCCAGAAAAACATG3′ |
S-R-Rz-Rev (XhoI) | 5′ATTCTCGAGCTATCTGCACTGCTCATTAAT3′ |
Animals Immunized with BGs/Antigen | Guinea Pigs | Mice | ||
---|---|---|---|---|
LD50, CFU * | II ** | LD50, CFU | II | |
…. | 147 37 ÷ 584 | 4.9 × 101 | 32 8 ÷ 126 | 4.6 × 100 |
Y-K | 6813 2154 ÷ 27123 | 2.3 × 103 | 22 5 ÷ 86 | 3 × 100 |
E-Y-K | 146,780 36,869 ÷ 926,119 | 4.9 × 104 | 22 5 ÷ 86 | 3 × 100 |
S-R-Rz | 68 17 ÷ 271 | 2.3 × 101 | 15 4 ÷ 58 | 2 × 100 |
E-S-R-Rz | 147 37 ÷ 584 | 4.9 × 101 | 46 12 ÷ 233 | 6.6 × 100 |
F1 ** | 316 79 ÷ 1259 | 1.0 × 102 | 100,000 25,119 ÷ 630,957 | 1.4 × 104 |
PBS | 3 1 ÷ 13 | 1 × 100 | 7 2 ÷ 27 | 1 × 100 |
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Dentovskaya, S.V.; Vagaiskaya, A.S.; Platonov, M.E.; Trunyakova, A.S.; Kotov, S.A.; Krasil’nikova, E.A.; Titareva, G.M.; Mazurina, E.M.; Gapel’chenkova, T.V.; Shaikhutdinova, R.Z.; et al. Peptidoglycan-Free Bacterial Ghosts Confer Enhanced Protection against Yersinia pestis Infection. Vaccines 2022, 10, 51. https://doi.org/10.3390/vaccines10010051
Dentovskaya SV, Vagaiskaya AS, Platonov ME, Trunyakova AS, Kotov SA, Krasil’nikova EA, Titareva GM, Mazurina EM, Gapel’chenkova TV, Shaikhutdinova RZ, et al. Peptidoglycan-Free Bacterial Ghosts Confer Enhanced Protection against Yersinia pestis Infection. Vaccines. 2022; 10(1):51. https://doi.org/10.3390/vaccines10010051
Chicago/Turabian StyleDentovskaya, Svetlana V., Anastasia S. Vagaiskaya, Mikhail E. Platonov, Alexandra S. Trunyakova, Sergei A. Kotov, Ekaterina A. Krasil’nikova, Galina M. Titareva, Elizaveta M. Mazurina, Tat’yana V. Gapel’chenkova, Rima Z. Shaikhutdinova, and et al. 2022. "Peptidoglycan-Free Bacterial Ghosts Confer Enhanced Protection against Yersinia pestis Infection" Vaccines 10, no. 1: 51. https://doi.org/10.3390/vaccines10010051
APA StyleDentovskaya, S. V., Vagaiskaya, A. S., Platonov, M. E., Trunyakova, A. S., Kotov, S. A., Krasil’nikova, E. A., Titareva, G. M., Mazurina, E. M., Gapel’chenkova, T. V., Shaikhutdinova, R. Z., Ivanov, S. A., Kombarova, T. I., Gerasimov, V. N., Uversky, V. N., & Anisimov, A. P. (2022). Peptidoglycan-Free Bacterial Ghosts Confer Enhanced Protection against Yersinia pestis Infection. Vaccines, 10(1), 51. https://doi.org/10.3390/vaccines10010051