Comparative Genomics Analysis Of Pasteurella Multocida And The Identification Of Protective Antigens

Pasteurella multocida（Pm）is an opportunistic zoonoses pathogenic bacteria and can threat to livestock and poultry,wild animals and humans.The different hosts will be infected with different diseases,such as swine plague,bovine hemorrhagic septicemia,bovine pneumonia,cholera fowl and swine atrophic rhinitis,etc.Pm can be divided into 5 capsular serotypes as A,B,D,E and F according to different capsular antigens.In the past,capsular serotype B that mainly causes hemorrhagic septicemia was the main epidemic pathogenic bacterium among the cattle,however,capsular serotype A that mainly causes pulmonary infection,has been found mostly in recent years.Because Pm has more serotypes（5 capsular serotypes and 16 antigen serotypes）,the cross protection effect for heterogenetic or heterologous Pm is low,the protection period is short,it is easy to become resistant to drugs and the virulence and epidemiology has large differences.The vaccine used in domestic currently is HSC vaccine strain,a traditional vaccine that is mainly specific to bovine hemorrhagic septicemia for capsular serotype B,and there is no vaccine for serotype A.Therefore,carrying out screening and identification for protective antigens of bovine Pm serotype A has important theoretical significance and practical significance in developing new vaccines（especially the one has cross protection for different serotypes）.The research group has successively separated and identified 6 strains for the sick beef cattle include serotype A Pm（PmCQ1-PmCQ6）and 1 strain of serotype F Pm and has purchased 1 strain of serotype B Pm,we found that PmCQ1-PmCQ5 are high virulent strains and PmCQ6 is low virulent strain.The PmCQ6 and PmB have been sequenced with whole genome sequencing using the second-generation sequencing technology in this study.In combination with PmCQ2 and PmF sequenced previously in our laboratory and other Pm whole genome published in the NCBI,through comparative genomics analysis,the differences of the genome structures and functional genes of different strains are revealed,some new virulence genes are excavated and the molecular basis is provided to deeply analyze the pathogenic mechanism of Pm.Meanwhile,some main antigen proteins of Pm are analyzed comprehensively to provide candidate molecules for obtaining new antigen targets and developing the new vaccine（especially the one has cross protection effect for different serotypes）.Almost all candidate antigen molecules from the whole genome are screened through bioinformatics analysis,the reactogenicity of the candidate antigen molecules is analyzed through cloning and expression and indirect ELISA or protein immunoblotting and the vaccine efficacy of the candidate molecules is further evaluated in infective animal models and cross protective antigens can be obtained in the end.Based on above analyses,the research result of this study comprises the following four aspects:1.Sequencing whole genome and assembling of bovine PmThere were the significant differences in the colony morphologies of different Pm strains（PmB,PmCQ2,PmCQ6 and PmF）under the same culture and imaging conditions,especially the colony morphology of PmCQ6 strain significantly less than the other strains.The mice and rabbits pathogenic experiment showed that there were significant differences among four strains of pathogenic,PmB is the high virulent strain,but PmCQ6 for the low virulent strain.The four bovine Pm was not the pathogen to poultry.The former study of our laboratory has sequence the whole genome sequence of serotype A PmCQ2（high virulent strain）and serotype F PmF（low virulent strain）.However,in order to more comprehensive analysis of the virulence differences of Pm and the screening of the cross-protective antigens,we selected serotype A PmCQ6 and serotype B PmB for genome sequencing through the second-generation sequencing technology,and used the SOAPdenovo method to complete spliced assembly.Through basic comparison,the sizes of Pm genomes of four strains are approximately 2.2-2.3M,the average number of predicted ORF is 1,977,the GC content is approximately 39.1-40.39%,the number of tRNA is between 43-50 and 16S-23S-5S rRNA elements are contained.Meanwhile,though the circle diagram of the four Pm genomes,the function of the genome annotation and forecasting of phage related genes,the result showed that the four Pm genomes have certain differences in genome sizes,predicted gene number and functional genes,which may affect the virulence of the bacterial strain.2.Comparative genomics analysis of Pm strainsThe phylogenetic relationship between the 4 Pm strains in our lab and the 24 Pm strains from NCBI was rebuilt from sequences of all conserved core genes,we found PmCQ2,PmCQ6 and 36950 belong to serotype A and get together in the same branch,PmB &2213 and PmF & Pm70 belong to other branchs,respectively.Based on the phylogeny,PmCQ2,PmCQ6,PmB,PmF,Pm70,HN06,36950,3480,RIIF and 2213 were select as representatives of different serotype and host pathogenic Pm strains to perform a comparative genome analysis.It is known from the whole genome collinearity analysis that,the Pm genome collinearity is good and the genome is conservative.The difference is that some strains will be inserted with different segments.To the prediction of the insertion sequence,we found that insertion sequences were present in PmCQ2,PmCQ6 and 36950,but no insertion sequence is found in PmF strain.We hypothesized that the difference of the virulence between the high and low virulent strains is associated with the insertion and deletion of the large fragment transposons.Through the prediction analysis of specific genes,we filtered to some unique genes of different serotype and different host strains,those genes were used as the candidate genes for diagnosed targets and host specificity.To further study the differences between the high and low virulent strains,we analyzed and compared through the pathogenicity islands,capsule gene and LPS-related genes.We found that transposase,prophageintegrase,phage terminase,transcriptional regulation proteins may contribute to the differences between the high and low virulent strains.In order to speculate that the virulence gene exists in Pm,we compared the genome of Pm from P2 CS database,and found that there are 18 two-component regulatory elements in PmB and PmF and 16 two-component regulatory elements in PmCQ2 and PmCQ6,belonging to 7 types of two-component regulatory systems.At last,we analyzed 458 membrane proteins and 162 secreted proteins from genomes of different Pm serotype strains by bioinformatics analysis,these results provided the foundation for the development of identification of candidate antigens against Pm.3.Screening of candidate antigen moleculesTo screen new candidate antigens,through prediction of the secretory proteins and membrane protein with comparative genomics analysis at first,then through further prediction for the analysis result by Signal and TMMHM website,48 candidate antigens are finally obtained.Through the immune proteomics analysis,15 candidate molecules are screened.Combination with these two methods,we shifted 42 candidate molecules.To identify whether these candidate molecules can be as the vaccine,we analyzed reactogenicity of these proteins through indirect ELISA with four bovine serums in laboratory after purification: serotype A artificial infected serum,serotype A inactivated vaccine immune serum,serotype B artificial infected serum and serotype F artificial infected serum.The result showed that there were 27 recombinant proteins with higher reactogenicity,and they contain 5 higher reactogenicity with four bovine serums;1 higher reactogenicity with serotype A,B and F artificial infected serum;10 higher reactogenicity with serotype A and B artificial infected serum;6 higher reactogenicity with serotype A artificial infected serum and 5 higher reactogenicity with serotype B artificial infected serum.In order to build a mice model and select a proper approach for challenging,the mice infected with PmCQ2 through three approaches as intranasal infection,intraperitoneal injection and intramuscular injection,we analyzed the surviving condition,colonization in vivo and pathological changes of the mice.The result showed that the three approaches can infect the mice,and be colonized in visceral organs as liver,spleen and lung of the mice,and be able to induce lung pathological changes.These results indicated that the mice model is proved as being built successfully.Because PmB could not cause death in mice by intranasal infection,so we chose intramuscular injection as the best way to evaluate candidate protective antigen molecules.Finally,we evaluated the efficacy of 10 recombination proteins against homologousand heterologous strains by challenging immunized mice.The results indicated that the protective rates for rPmCQ2₁g0524 and rPmCQ2₁g0311 were 30% and 30% against 10LD50 of PmCQ2,but not against PmB and PmF;80%/20%/10%（rPmCQ2₄g0132）and 50%/40%/30%（rPmCQ2₂g0128）against 10 LD50 of the PmCQ2,PmB and PmF in mice model,respectively.Meanwhile,we also found that the protection rates of rPmCQ2₁g0327 to serotypes A and B are 50% and 40% and the protection rates of rPmCQ2₁g0376 to serotypes A and B are 50% and 30%,respectively.It is the first to demonatrate that the candidate molecule with cross protection effect is screened in the bovine serotype A strain.4.The research on cross protection of rPmCQ2₁g0327Based on bioinformatics analysis,PmCQ2₁g0327 only exists in Pm serotype A,but PmCQ2₁g0376,PmCQ2₄g0132 and PmCQ2₂g0128 were present in Pm serotype A,B,D and F.Through homology analysis between the remain three genomic data and Nr database in NCBI and qPCR analysis of bacteria in vitro,it is found that PmCQ2₁g0327 is only exists in Pm serotype A,none exists in other serotype strains as PmB and PmF,and it has low expressions in the low virulent PmCQ6 strain.We used the DNAstar software and IEDB on-line to predict potential advantage antigen epitopes of PmCQ2₁g0327 sequences.And the antigen epitope more focused on the N-terminal sequence.Conduct cloning and expression to the seven sections of PmCQ2₁g0327 sequences and purify.The indirect ELISA result showed that the 75-87AA（TIDTAPKQNPVPT）had the higher reactionogenicity between PmCQ2 and PmB and can resist the attracting from10 LD50 of PmCQ2 and PmB in mice model,wherein,the protection rate were 40% and 10%,respectively.It is thus speculated that antigen epitope（TIDTAPKQNPVPT）may be played a major role in protection of host resistance to PmB.Through homology analysis in the PmB whole genome sequence,we found the PmB₁759g0022 sequence has higher homology with the PmCQ2₁g0327 sequence.Then,rPmB₁759g0022 immunized mice can resist the attracting from PmB and the protection rate was 40%.This result surport our hypothesis thst TIDTAPKQNPVPT sequence was played a key role in the resistance to PmB.In addition,the polyclonal antibody（1:102400）of rPmCQ2₁g0327 is prepared.The rPmB₁759g0022 have the higher reactogenicity throughWB analysis with the polyclonal antibody of rPmCQ2₁g0327.Morever,it is thus clear from the IFA result that the antibody of rPmCQ2₁g0327 can particularly recognize the antigens on the PmCQ2 cell wall and cell membrane and have immune reaction with the antigens.Therefore it is speculated that the distribution of protein on the surface of the PmCQ2.To detect adhesion ability,we used antibody blocking tests,and found that rPmCQ2₁g0327 does not play a major role when bacteria adhering cells.In conclusion,we analyzed the genome of the high and low virulent strains for screening cross candidate antigen molecules,combine with proteomics analysis,we screened four cross candidate protective antigen molecules in bovine Pm strains.We also found that PmCQ2₁g0327 which was only present at Pm serotype A strains can resist the challenging from homologous and heterologous strains,was truncated into seven sections after the prediction of antigen epitope.The results showed that the multiple antigen epitopes of rPmCQ2₁g0327 were played common role in cross protection,and the antigen epitope of TIDTAPKQNPVPT sequence was played especially a key role in the resistance to PmB.Thus,PmCQ2₁g0327 can be used as Pm new vaccine candidate antigen molecules.