Functional Properties Of Crp And PhoP Gene In Acain Paseurella Multocida And Evaluation Of The Effects Of The Deletion On Immunogenicity

Pasteurella multocida, a Gram-negative bacteria, causes serious diseases in a wide range of animal species. It is quite effective to develop vaccines against E.coli, Salmonella or other Gram-negative bacteria by targeting the crp or phoP gene, two global regulators. However, the crp, phoP gene sequence and its function were less described in Pasteurella multocida. So, our results were showed as follows:1. Identification of the potential crp and phoP gene in P. multocida. The cloned the potential crp and phoP (including phoPl and phoP2) genes were inserted into low copy plasmid pYA3332 respectively and then the constructed plasmids were transformed into the Salmonella crp or phoP mutant, respectively. The crp gene has characterized via MacConkey agar assay, and confirmed phoP2 was the ture phoP gene though X-P plates assay and polymyxin B killing assay. Also, crp-specific and phoP specific polycloned antibody were also prepared.2. Construction of the P. multocida deltetion mutans. The P. multocida DY120818 crp and phoP deletion mutants were constructed via the suicide plasmid pYA4278 mediated homologous recombination method, respectively. The mutants had good hereditary stability. As required, the in trans complementary strain of P. multocida DY120818 crp mutant was also been constructed based on the shuttle plasmid pMC-express.3. Identification of biological function. The P. multocida DY120818 crp mutant strain grow slower, change some carbon source and amino acids metabolic capability and was more sensitive to serum compared with parent strain. The virulence reduced 22 times and 86 times after ducks inoculation via oral and intranasal routes, respectively. The P. multocida DY120818 phoP mutant strain has unobserved significant difference in these biological function compared with parent strain except that the virulence reduced 32 times and 154 times after ducks inoculation via oral and intranasal routes, respectively. There were no obviously difference of the phenotypic character between this two mutant strains and parent strain.4. Immunological efficacy test. Duckings immunized with P. multocida DY 120818 crp mutant strain intranasally, or immunized with P. multocida DY120818 phoP mutant strain orally, provided 60% and 54.5% protection after challenged with parent strain, respectively. Immunized ducks produced significantly high level of serum IgY and bile IgA responses against whole-cell antigen or OMPs.5. Transcriptome sequencing analysis. Comparing the transcriptome sequencing data between crp, phoP mutant strain and P. multocida DY120818 wide-type,186 and 334 significant differentially expressed genes were related to crp and phoP in P. multocida DY120818 crp and phoP mutant strain respectively. Also, some bioinformatics analysis (including GO function and KEGG pathway analysis) had been applied to these differentially expressed genes, which it promotes our understanding of the crp, phoP regulatory function in Pasteurella multocida.In summary, the sequence and functions of crp or phoP gene in Pasteurella multocida is less described. In this study, the crp and phoP gene were first cloned from Pasteurella multocida. The non-polar crp and phoP deletion mutations of Pasteurella multocida DY120818 was then constructed and the roles of crp and phoP in bacterial growth, LPS and OMPs profiles and virulence were systemically analyzed. More over, the immunogenicity and protection efficacy of the Acrp and AphoP strain were determined. Also, at last, we compared the significant differentially expressed genes between Acrp or AphoP strain and wide-type via RNA-sequencing.