The Study Of QseBC Two-component System Affecting The Virulence Of Actinobacillus Pleuropneumoniae By Regulating The Apf Gene Cluster

Actinobacillus pleuropneumoniae(APP)is the main pathogen of Porcine contagious pleuropneumoniae(PCP),which is widely prevalent all over the world.It has brought serious threats to Chinese biosafity and great losses to intensive pig industry.It is recognized internationally as one of the most important diseases harmful to modern pig industry.QseBC two-component regulation system is one of the five pairs of two-component APP,which has the function of quorum sensing and can regulate a variety of bacterial behavior changes.In this study,RNA-seq,q-PCR and EMSA were used to determine that QseBC two-component system can directly regulate the downstream gene apf gene cluster(apf ABCD).Furthermore,the functions of the QseBC two-component system were verified by using the downstream gene knockout mutant strain ?apf A,?apf B,?apf C and?apf D as well as the corresponding complementary strains,and the effects of the QseBC two-component system on APP virulence and other related biological functions by regulating the apf gene cluster were further clarified.Then,the virulence related factor apf A was continued to be deleted to construct the knockout mutant strain DBZ08.Specific research contents are as follows:1.RNA-seq data analysisIn this study,51 genes with Fold Change ? 1.5 and p ? 0.001 were selected as differentially expressed genes between the knockout mutant strain ?qse BC and the wild strain sample.Among them,24 genes were up-regulated and 27 genes were down-regulated.We analyzed the main characteristics of these genes and described them as follows: Most down-regulated genes are related to virulence,encoding biological proteins of adhesion factors,encoding proteins related to iron metabolism,participating in glycerol metabolism,transportation and DNA uptake;The up-regulated genes are mainly involved in amino acid metabolism,inorganic ion metabolism,biofilm formation,signal transduction,transcriptional activation,DNA recombination and unknown functions.2.The QseBC two-component system of APP can directly regulate the apfgene clusterIn this study,RNA-seq was used to preliminarily screen the differentially expressed genes between the knockout mutant strain ?qse BC and the wild strain samples,and q RT-PCR was used to verify the reliability of the RNA-seq results.By EMSA technique,we found that Qse B can bind to the promoter fragment of apf gene cluster,it was further confirmed that QseBC two-component system could directly regulate apf gene cluster.3.QseBC two-component system affects APP virulence and other related biological functions by regulating apf gene clusterThe functions of QseBC two-component system were verified by biological characteristics analysis,cell test,animal test,etc.,using the downstream gene knockout mutant strains ?apf A,?apf B,?apf C and ?apf D as well as the corresponding complementary strains.We found that when the above genes were deleted,the survival ability of the knockout mutant strains under in vitro stress conditions decreased by34.65% compared with the wild strain,and the biofilm formation ability decreased by29.68%.The percentage of being engulfed by cells increased by 42.85%,the ability to adhere to cells decreased by 39.58%,and the ability to invade cells decreased by 45.69%.The survival rate of mice inoculated with the knockout mutant strains increased by279.93%,and the bacterial load of lung tissue decreased by 79.95-fold,and that of blood tissue decreased by 319.45-fold.These results indicate that QseBC two-component system regulation of apf gene cluster can significantly reduce the resistance to in vitro stress,biofilm formation ability,anti-phagocytosis ability,adhesion ability,invasion ability,virulence and colonization ability of APP.Therefore,the QseBC two-component system can affect the expression of apf gene clusters,mediating APP adhesion and invasion,and thus establishing infection.