| Porcine contagious pleuropneumonia(PCP),caused by Actinobacillus pleuropneumoniae(APP),is a porcine respiratory infectious disease leading to severe economic losses worldwide in the swine industry.Immunization is the major method to prevent and control this disease.At present,researchers have found many virulence factors from APP,including four kinds of RTX toxins,capsular polysaccharide, lipopolysaccharide,outer membrane proteins,transferrin binding proteins,adhesins and proteases.On the base of the characteristics and functional roles of these virulence factors, a lot of vaccine candidates have been designed to prevent and control APP.However,the current applied vaccines are still killed whole cell bacterins and subunit vaccines,which frequently fail to protect pigs from the heterologous serotypes of APP infection.New and effective vaccines are urgently needed to control and eradicate this important disease. Understanding of the molecular biology and pathogenesis of pathogenic bacteria is the base for new vaccine design and development.This study mainly focused on the structural and fuctional characterization of the flp and dam genes of APP.At the same time,a part of signature-tagged mutagenesis(STM) bank has been constructed.1.Construction and characterization of an flp gene deletion mutant of APP.Flp (fimbrial low-molecular-weight protein) operon,consisting of 13-15 genes,commonly exists in many kinds of bacteria.It encodes a novel secretion system for the assembly and release of long,bundled pili which is considered to be associated with tight adherence of bacteria to host cells.Our genomics analysis revealed that there are two copies of flp genes(flp-1 and flp-2) end-to-end in the flp operon of APP.To understand the function of Flp proteins in APP,a recombinant transfer plasmid pEMOC2-flpLR was constructed and transformed into APP serotype 1 by electroporation.An flp-1/flp-2 double deletion mutant without antibiotics marker was obtained by antibiotics/SacB counter-selection strategy.The following study revealed that the flp deletion did not affect the in vitro growth,but failed to formation of Flp pili.Compared to the parental strain,the mutant showed a reduced virulence in a mouse infection model.2.Virulence regulation by DNA methylation in APP.DNA methylation by the DNA adenine methyltransferase(Dam) provides an epigenetic signal that influences and regulates numerous physiological processes in the bacterial cells,including chromosome replication,mismatch repair,transposition,and transcription.The molecular basis for the pleiotropic phenotypes associated with Dam is the differential methylation of DNA resulting in an altered affinity of regulatory DNA-binding proteins.In this study,the dam gene was PCR amplified from the genomic DNA of APP serotype 1,and cloned into the broad-host shuttle plasmid pJN105,resulting in a recombinant plasmid pJN105-dam.The Dam activity was confirmed by transformation of pJN105-dam into a dam-negative E. coli strain,followed by dam-sensitive restriction endonuclease analysis.Then the pJN105-dam was electroporated into APP serotype 1,obtaining a Dam overproduction strain.The following analysis indicated that the Dam overproduction strain grew more slowly that the parental strain.The haemolytic activity in vitro and virulence in vivo were significantly decreased when compared with the parental strain.3.Construction of STM mutants of APP.STM is a powerful approach to identify genes associated with virulence of the pathogenic bacteria through negative selection.In this study,we established a sophisticated biparental mating method for E.coli and APP and acquired 212 STM mutants of APP using four of the 48 STM tags(Tag 1,Tag 2, Tag 15,and Tag 16).A biofilm-forming mutant was identified from the mutant pools. With the collaboration of our colleagues the STM bank has currently been finished.This will contribute to identification of new functional genes from APP.
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