Study On The Construction And Bological Characteristic Of Pasteurella Multocida Quorum-Sensing Gene Deletion Mutants (?qseC And ?qseC?luxS)

Pasteurella multocida(P.multocida)is an important veterinary pathogen that can cause severe diseases in a wide range of mammals and birds.Diseases caused by Pasteurella multocida include enzootic pneumonia and haemorrhagic septicaemia in cattle,lung disease and atrophic rhinitis in pigs,fowl cholera in birds,snuffles in rabbit.It has been reported that Pasteurella multocida can infect and cause local tissue and even lead to systemic disease in humans.This not only brings great challengges to livestock husbandry,but also poses a threat to the safety of human life..In recent years,with the rapid development of cattle industry,the risk of bovine infected with P.multocida is highly increased.However,most of the cattle infected enzootic pneumonia is caused by capsular serotype A.Therefore,the research on the pathogenic mechanism of P.multocida serotype A is very important for both the production of breeding industry and the life and health of human beings.Quorum Sensing(QS)is a communication mechanism widely existed in bacteria.Bacteria produces autoinducer(AI)in the process of growth and reproduction,by simple diffusion modes secreting the agent to the environment and recognized by bacteria receptor.When the concentration in the environment reaches threshold,the signal transduction process of the bacteria will start,resulting in bacterial gene expression changes and bacterial population behavior changes accordingly.It is mainly manifested in the regulation of nutrient uptake,bioluminescence,biofilm formation,virulence factor expression and so on.According to the different autoinducer,the quorum sensing system can be divided into four categories:AHL-mediated quorum sensing system,signal-peptide mediated quorum sensing system,AI-2 mediated quorum sensing system and AI-3 mediated quorum sensing system.qseC and luxS belong to the QS system mediated by AI-3 and AI-2 respectively.As for the function of these two QS systems in Pasteurella multocida,there is no relevant research report at home and abroad.In this study,PmCQ2 strain was selected and its quorum sensing genes qseC and luxS were deleted,to explore the effect of quorum sensing gene deletion on the virulence of PmCQ2 and to provides basis for the pathogenesis of Pasteurella multocida and the development of attenuated vaccine,the following work was carried out in this study:1.The construction of PmCQ2-?qseC strainPmCQ2 genome was used as template to amplify upstream and downstream homologous arm and qseC gene.Plasmid pUC19ori Kan^R was used as the carrier to construct recombinant plasmid pUC19ori Kan^R–?qseC.We successfully constructed PmCQ2-?qseC strain by homologous recombination.2.The basic biological characteristics of PmCQ2-?qseCBy comparing the biological characteristics of PmCQ2 and PmCQ2-?qseC,it was found that there was no significant difference between them on the growth curve;PmCQ2-?qseC formed a smaller colony,whose capsule content was significantly lower than PmCQ2 and biofilm content significantly higher than PmCQ2;The stress resistance test showed that compared with PmCQ2,the high temperature tolerance and oxidative stress tolerance of PmCQ2-?qseC are weakened,but the osmotic stress tolerance is slightly enhanced.3.The virulence and pathogenicity of PmCQ2-?qseCThe virulence of PmCQ2 and PmCQ2-?qseC were compared by LD₅₀ in mice,the LD₅₀ of PmCQ2 was 5.79 CFU and PmCQ2-?qseC was 6.63×10⁷ CFU,it showed that the virulence of PmCQ2-?qseC was far less than PmCQ2.The pathogenicity of PmCQ2 and PmCQ2-?qseC were compared by mouse lung colonization test.The challenge dose of PmCQ2 and PmCQ2-?qseC was 3.48×10⁵ CFU and 3.20×10⁵ CFU,respectively.In the time of colonization for 8h,16h and 24h,PmCQ2-?qseC had a very small amount of colonization amount and was far lower than that of PmCQ2.Pathological sections of the lung of the mouse colonization for 24h were prepared.It was found that PmCQ2-?qseC caused less lung damage in mice compared with PmCQ2.4.The immune protection of PmCQ2-?qseC against wild strainsTo evaluated the potential of PmCQ2-?qseC as a attenuated vaccine,we immunized mice with live bacteria via intramuscular injection.After three times of injection with PmCQ2-?qseC,wild-type strain(PmCQ2),serotype capsular type B strain(PmB)and avian strain(PmQ)were used to challenge these immunized mice.The results showed that PmCQ2-?qseC had complete protection(100%)against PmCQ2,partial protection(62.5%)against PmB,and no protection(0)against PmQ.5.Analysis and verification of the results of transcriptome sequencing of PmCQ2and PmCQ2-?qseCTranscriptome verification results showed that transcriptome sequencing results are accurate.And compared with PmCQ2,most of the genes related to biofilm formation were up-regulated in PmCQ2-?qseC,which was consistent with the results of biofilm test.Most of virulence related genes were down-regulated,which was consistent with the results of virulence reduction.Most of the genes involved in the stress pathway were down-regulated,which was consistent with the results of the stress resistance test.6.The basic biological characteristics of PmCQ2-?qseC-?luxSPmCQ2-?qseC-?luxS gene deletion strain was constructed.Firstly,luxS affected the capsule and biofilm formation of PmCQ2.Compared with PmCQ2 and PmCQ2-?qseC,the capsular formation of PmCQ2-?qseC-?luxS reduced but biofilm formation increased;Secondly,luxS affected the stress resistance of PmCQ2.Compared with PmCQ2 and PmCQ2-?qseC,the high temperature tolerance of PmCQ2-?qseC-?luxS decreased,the oxidative pressure tolerance showed that low concentration of H₂O₂ reduced the oxidative pressure tolerance of PmCQ2-?qseC-?luxS and high concentration of H₂O₂ enhanced it.And osmotic stress tolerance is weakened compared with PmCQ2 and luxS gene had a opposite effect on it with qseC gene.Thirdly,luxS affected the serum sensitivity of PmCQ2.The antiserum killing ability was reduced compared with PmCQ2.