Research On Identification And Function Of Type ? Toxin-Antitoxin System In Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae is a highly-contact respiratory disease and the pathogenic bacteria of porcine contagious pleuropneumonia.The major characteristic of APP includes acute haemorrhage and necrotic,chronic fibfinous pleuropneumoniae in clinic.Once the disease,it will cause huge economic losses in the swine industry at home and abroad.Toxin-antitoxin?TA?systems are small genetic elements that are widely prevalent in the genomes of bacteria and archaea.These modules have been identified in various bacteria and proposed to play an important role in bacteria physiology and virulence.However,their presence in the genomes of Actinobacillus species has received no attention.In this study,we want to describe the identification of type ? TA systems in Actinobacillus pleuropneumoniae,the causative agent of porcine pleuropneumonia,so that we can in-depth knowledge biological characteristics about APP.The putative TA modules in A.pleuropneumoniae serover 3 str.JL03 were predicted and identified.We make an in-depth research for the identified TA systems.We expect to screen out TA systems related to virulence and to provide reference for the research and development of attenuated vaccine in clinic.The main contents are as follows:1.Identification of type ? TA systems in Actinobacillus pleuropneumoniaeThe putative type ? TA systems in A.pleuropneumoniae serovar 3 str.JL03 were predicted with RASTA-Bacteria and TADB2.0.Seven modules simultaneously identified by the two tools were considered as putative type ? TA systems and picked for further study.RT-PCR analysis showed that the genes encoding the antitoxin and toxin are co-transcribed.Overexpression of each toxin inhibited the growth of E.coli,and the toxic effect could be countered by its cognate antitoxin.The pull-down experiments revealed that each toxin interacts with its cognate antitoxin in vivo.The promoter activity assays demonstrated that each antitoxin could autoregulate either negatively or positively the TA operon transcription.Besides,the APJL₀660/0659 TA system is shown in half of the tested serovars of APP,but the others are shown in all.At last,we identified four type ? TA systems in APP,and this research has laid the foundation for further functional research of these TA systems.2.The functional research about four type ? toxin-antitoxin systems in Actinobacillus pleuropneumoniaeFirstly,the plasmids of pE?TA were constructed by the recombination suicide plasmid of pEMOC2 and transformed into E.coli?2155 competent cells.E.coli?2155 cells harboring the respective pE?TA plasmids were regarded as donor cells and mixed with APP5?acceptor cells?.Then the bacteria were selected single exchange strains,and the mutant strains of?TA were further obtained by single exchange.Secondly,the recombination plasmids of pJFTA were constructed by E.coli-APP shuttle vector pJFF224-XN and transformed into mutant strains of?TA competent cells by electroporation.A Cm^R transformant was selected,exactly the complementation strains of C?TA were picked.At last,the growth characteristic,the stability and the virulence in mouse were researched about the mutant and complementation strains.Results show that the growth characteristic of TA3 has a little difference compared with APP5,while the others have no significant effect;Four TA systems have no significant effect between mutant strains and APP5 in virulence.