Identification of the Avian Pasteurella multocida phoP Gene and Evaluation of the Effects of phoP Deletion on Virulence and Immunogenicity
Abstract
:1. Introduction
2. Results and Discussion
2.1. Results
2.1.1. Cloning and Characterization of the phoP Gene of P. multocida
2.1.2. Construction of the Non-Polar ΔphoP Mutant in P. multocida 0818
2.1.3. Phenotype of the ΔphoP Mutant Strain
2.1.4. Virulence of Wild-Type P. multocida and the ΔphoP Mutant
Route | Strains | Infection Dose (CFU) and Survival | LD50 | ||||||
---|---|---|---|---|---|---|---|---|---|
103 | 104 | 105 | 106 | 107 | 108 | 109 | |||
Intranasal | P. multocida 0818 | 5/5 | 7/8 | 8/16 | 1/16 | 0/16 | 0/8 | 0/8 | 8.66 × 104 |
S413 (ΔphoP) | 8/8 | 8/8 | 7/8 | 12/16 | 9/16 | 5/16 | 0/8 | 1.33 × 107 | |
Oral | P. multocida 0818 | – | 8/8 | 8/8 | 12/16 | 5/16 | 0/13 | 0/8 | 4.87 × 106 |
S413 (ΔphoP) | – | 8/8 | 8/8 | 8/8 | 11/13 | 7/13 | 1/13 | 1.56 × 108 |
2.1.5. Identification of Genes Regulated by phoP in P. multocida
GeneID a | Locus | Description | Fold Change (log2) |
---|---|---|---|
Genes up-regulated in strain S413 (ΔphoP) | |||
1244903 | comF | competence protein ComF | 18.25 |
1243560 | PM0213 | membrane protein | 11.79 |
1244002 | cspA | cold-shock protein | 5.98 |
1244528 | lexA | repressor LexA | 5.73 |
1245238 | PM1891 | hypothetical protein | 5.39 |
1244663 | PM1316 | hypothetical protein | 4.66 |
1244906 | def | peptide deformylase, partial | 4.66 |
1244239 | impA | type VI secretion protein ImpA | 4.29 |
1244515 | rraA | ribonuclease activity regulator protein RraA | 4.06 |
1245264 | acpP | MULTISPECIES: acyl carrier protein | 4.00 |
Genes down-regulated in strain S413 (ΔphoP) | |||
1244450 | aspA | aspartate ammonia-lyase | 9.92 |
1244459 | deaD | RNA helicase | 7.62 |
1244098 | PM0751 | DNA glycosylase | 7.16 |
1244150 | PM0803 | TonB-dependent receptor | 5.28 |
1243408 | rnc | ribonuclease III | 5.24 |
1243794 | resA | Pasteurella ResA protein | 5.03 |
1244644 | trmD | tRNA (guanine-N1)-methyltransferase | 4.96 |
1244642 | PM1295 | hypothetical protein | 4.66 |
1244511 | PM1164 | membrane protein | 4.66 |
1244107 | nusA | peptidase M54 | 4.53 |
1244535 | tonB | cell envelope protein TonB | 4.47 |
1244106 | infB | translation initiation factor IF-2 | 4.29 |
1244083 | dnaK | molecular chaperone DnaK | 4.14 |
1244389 | eptA | sulfatase | 4.11 |
2.1.6. Evaluation of the Immune Responses and Protection Rate Conferred by ΔphoP Mutant
Group | Immunization | Challenge | Survival | Protection |
---|---|---|---|---|
Immune group | 105 CFU of S413 (ΔphoP) | 109 CFU of P. multocida 0818 | 6/11 | 54.5% |
Control | PBS | 109 CFU of P. multocida 0818 | 0/8 | 0 |
2.2. Discussion
3. Experimental Section
3.1. Materials and Methods
3.1.1. Bacterial Strains, Plasmids, Media and Growth Conditions
Strain or Plasmid | Description | Source |
---|---|---|
Strains | ||
S184 | S. Typhimurium Δasd66 | Lab collection |
S412 | S. Typhimurium Δasd66 ΔphoP85 | Lab collection |
P. multocida 0818 | Wild type P. multocida serotype A strain. Capsulated and virulent | Lab collection |
S413 | P. multocida 0818 ΔphoP::kanR | This work |
χ7232 | E. coli K-12, endA1 hsdR17 (rK-, mK+) glnV44 thi-1 recA1 gyrA relA1 Δ(lacZYA-argF)U169 λpir deoR (φ80dlac Δ(lacZ)M15) | [39] |
χ7213 | E. coli K-12, thi-1 thr-1 leuB6 glnV44 fhuA21 lacY1 recA1 RP4-2-Tc::Mu λpir ∆asdA4 ∆zhf-2::Tn10 | [39] |
Plasmids | ||
pQK664 | Asd+ Ptrc pSC101 origin replicationp15a, asd+, spec r | Derived from pYA3337 [36] |
pQK167 | Insertion of phoP1 into pQK664 | This work |
pQK168 | Insertion of phoP2 into pQK664 | This work |
pYA4278 | pRE112 derivative, sacB mobRP4 R6K ori Cm+ | [38] |
pQK171 | pYA4278-ΔphoP | This work |
pQK173 | pYA4278-ΔphoP::kanR, for deletion of phoP in P. multocida | This work |
pMC-Express | A broad host-range shuttle vector derived from pMIDG100, chloramphenicol r | [40] |
pQK180 | Insertion of phoP into pMC-Express | This work |
3.1.2. Molecular and Genetic Procedures
Primers | Sequence 5’-3’ |
---|---|
CphoP1-F | GCATGCCATGGAGGTACAAATGACCAAAAT |
CphoP1-R | CGCGGATCCATACTTAATTGAAAAGGTTC |
CphoP2-F | GCATGCCATGGCGGTCAAAAAAATGCGAAT |
CphoP2-R | CGCGGATCCACGTGAAAATTAAACGAACC |
CphoP-F | cgcGGATCCatgcgaattttattaatagaag |
CphoP-R | ataagaatGCGGCCGCccgtaaacttgtttgcttaagc |
DphoP-1F | TGCCAGTTTTCAATGGTGTC |
DphoP-1R | CCTGCAGGGATGCGGCCGCTTTTTTGACCGCACTTTTTTC |
DphoP-2F | GCGGCCGCATCCCTGCAGGGCTAGGAAAAAATGATGAAATG |
DphoP-2R | ATTTTCCTTGATTGACTGGC |
Kan-F | ATAAGAATGCGGCCGCTCAGTGGAACGAAAACTC |
Kan-R | CCTGCAGGTTAGAAAAACTCATCGAGCATC |
Primer 1 | TTAATTGGCAATGGCTTACAG |
Primer 2 | TACCCTACACCATGCACAGT |
Primer 3 | TATTACACACGTTATAACCCG |
Primer 4 | CTGATTCAGGTGAAAATATTG |
Primer 5 | CATTTGTGCAACTTCAGTTTG |
Primer 6 | CAATATTTTCACCTGAATCAG |
16sRNA-F | TAATACCGCGTATTCTCTGAGG |
16sRNA-R | CCCTCCCTAAAGTACTCTAGAC |
3.1.3. Plasmid and Mutant Strain Construction
3.1.4. Phosphatase Activity and Polymyxin B Resistance Assays
3.1.5. Phenotype Determinations
3.1.6. Determination of LD50 in Ducks
3.1.7. Transcriptome Sequencing
3.1.8. Immunization and Challenge
3.1.9. Enzyme-Linked Immunosorbent Assay (ELISA)
3.1.10. Statistical Analyses
4. Conclusions
Supplementary Materials
Acknowledgments
Authors Contributions
Conflicts of Interest
References
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Xiao, K.; Liu, Q.; Liu, X.; Hu, Y.; Zhao, X.; Kong, Q. Identification of the Avian Pasteurella multocida phoP Gene and Evaluation of the Effects of phoP Deletion on Virulence and Immunogenicity. Int. J. Mol. Sci. 2016, 17, 12. https://doi.org/10.3390/ijms17010012
Xiao K, Liu Q, Liu X, Hu Y, Zhao X, Kong Q. Identification of the Avian Pasteurella multocida phoP Gene and Evaluation of the Effects of phoP Deletion on Virulence and Immunogenicity. International Journal of Molecular Sciences. 2016; 17(1):12. https://doi.org/10.3390/ijms17010012
Chicago/Turabian StyleXiao, Kangpeng, Qing Liu, Xueyan Liu, Yunlong Hu, Xinxin Zhao, and Qingke Kong. 2016. "Identification of the Avian Pasteurella multocida phoP Gene and Evaluation of the Effects of phoP Deletion on Virulence and Immunogenicity" International Journal of Molecular Sciences 17, no. 1: 12. https://doi.org/10.3390/ijms17010012
APA StyleXiao, K., Liu, Q., Liu, X., Hu, Y., Zhao, X., & Kong, Q. (2016). Identification of the Avian Pasteurella multocida phoP Gene and Evaluation of the Effects of phoP Deletion on Virulence and Immunogenicity. International Journal of Molecular Sciences, 17(1), 12. https://doi.org/10.3390/ijms17010012