| Actinobacillus pleuropneumoniae (APP) is the etiological agent of porcine contagious pleuropneumonia, a highly contagious respiratory disease of pig which causes serious economic losses in the pig farming industry worldwide. The A. pleuropneumoniae organism is transmitted via respiratory droplets or through direct contact with infected animals. It colonizes the epithelial cells of the lower respiratory tract of pigs. A total of15serotypes of A. pleuropneumoniae have been identified on the base of surface polysaccharide antigens. Serotype prevalence varies in different countries and regions. Strains of serovar1,2,3,4,5,7, and8have been isolated in China, among which serovar7is the most prevalent serotype and followed by serovar1,2and3.APP enrolls multiple virulence factors during the infection process. Currently, a lot of study focuses on its pathogenesis. However, the mechanism of colonization in early infection phase is still unclear. Type Ⅳ pili of different pathogenic bacteria play important roles during infection process, including mediating adherence, breaking immunity barrier, affecting biofilm formation, twitching motility and DNA uptaking. Consequently, to better prevent and control the diseases caused by APP, study of the APP pathogenesis, especially the colonization mechanism in early infection phase is meaningful. In this study we investigate the role of APP type Ⅳ pili play in the infection process and also evaluate the pilus structural protein ApfA as a protective antigen.1. Type Ⅳ pili of APP involve in adherence and colonizationAdherence is the first step to establish infection, which plays a critical role in the infection process. Therefore, in this study, we examined if type Ⅳ pili are involved in APP colonization to host. The results are as follows:First, we analyzed the expression level of type Ⅳ pili operon after contacting with St. Jude porcine lung cells (SJPLC) via RT-PCR analysis. The results showed that after cell contacting, the expression level ofapf operon genes significantly increase. Second, type Ⅳ fimbrial protein mutant strain4074△apfA and complementary strain C4074△apfA are constructed in this study. With adherence cell models of porcine iliac artery endothelial cell (PIEC) and SJPLC, we certified type Ⅳ pili involve in APP adherence to host cells by adherence and adherence inhibition assay. Meanwhile, we observed significantly reduced adherence capacity of4074△apfA mutant strain by confocal microscopy, while the apfA complementary strain had this capability restored, which proved the involvement of type Ⅳ pili in adherence process. Third, in mice infection model, type Ⅳ pili were proved to be involved in colonization and virulence in early infection stage. So far, these results showed that type IV pili of APP are cell-contact induced and play an important role in colonization during infection process, which contribute critically to the pathogenicity.2. APP type Ⅳ pili involve in negative regulation of biofilm formationType IV pili are involved in multiple pathogenic mechanisms. In this study, we further explored other roles type Ⅳ pili may play in the pathogenesis during APP infection. To determine whether type Ⅳ pili involve in the regulation of biofilm formation, the capacity of parent and mutant strains to form biofilm is quantificationally compared by crystal violet staining assay. The mutation of apfA significantly enhanced the biofilm formation ability of APP, implying that type Ⅳ pili take part in the negative regulation of biofilm formation. We speculated that type Ⅳ pili of APP could involve in the negative regulation of biofilm formation by LuxS, a quorum sensing regulator. Furthermore, we compared4074△apfA with parent strain in hemolytic and cytotoxicity assays. The results showed no differences between the two strains in hemolysis and cytotoxicity, which indicates that the absence of type Ⅳ pili didn’t affect the secretion of hemolysins and cytotoxins.3. Searching for the potential receptors for APP type Ⅳ piliType IV pili are proved to mediate colonization in early stage of APP infection. In Neisseria, CD46is proved to be the specific receptor of type IV pili. To verify whether CD46is the specific receptor of APP type IV pili, we constructed pcDNA-CD46eukaryotic expression vector and transfected it into baby hamster kidney (BHK) cells (CD46negative). CD46is verified to be expressed in transfected BHK cells by flow cytometry analysis. However, APP didn’t show enhanced adherence to BHK cells with CD46expression. In adherence inhibition assay, blocking SJPLC and PIEC cells (CD46positive) with CD46specific monoclone antibody didn’t reduce the adherence by APP. These results indicated that CD46is not a specific receptor for APP adherence.We further applied yeast two-hybrid system to look for potential receptors for APP type IV pili. Bait vector pGBKT7-apfA was constructed and transformed into yeast strain Y187. Four positive clones were captured in porcine lung cDNA yeast library by yeast mating. Sequencing analysis found that only one of the four positive clones could be the potential receptor of type IV pili. The protein is homologous with Talinl, a cytoskeletal protein. The interaction between type Ⅳ pili and Talinl remains to be further verified. 5. Type Ⅳ pilius structural protein ApfA is a good protective antigenWe analyzed the homology of type Ⅳ cluster genes in13APP strains of different serotypes and found that ApfA is conserved in12serotypes and type Ⅳ pili operon is conserved in11serotypes. To investigate the immunogenicity of ApfA, recombinant ApfA (rApfA) is expressed in E. coli and purified. Then mice immunized with purified rApfA were induced an elevated humoral immune response and produced high levels of specific antibody. Then pig convalescent serum samples and control serum samples were examined with a rApfA-based ELISA. The results showed that the titers of anti-rApfA antibody in convalescent serum are significantly higher than that in control serum. These results suggest that structural protein ApfA of type Ⅳ pili, which exists in all A. pleuropneumoniae serotypes, is not only highly conserved in sequences, but also highly immunogenic.APP type Ⅳ pilus structural protein ApfA, with good immunogenicity and conserved identity is a promising protective antigen. In next study, we evaluated the protection provided by ApfA in mice. The rApfA showed a90%protective efficacy against lethal infection of serovar1strain4074. For serovar7strain WF83, rApfA conferred80%protection. Antibody typing found that rApfA mainly induces Th2type immune response. Then, naive mice were passively immunized with anti-rApfA serum via i.v. injection and the results showed that the anti-rApfA serum provided protection against both strain WF83(serovar7,60%) and strain4074(serovar1,40%). So far, the results suggested that type Ⅳ pilus structural protein ApfA is a highly effective protective antigen, mainly induce Th2type humoral immune response, which confer significant protection against APP infection of Chinese prevalent serotypes. In addition, the antibody raised against ApfA is effective in passive immunization, which could also provide protection against APP infection of different serotypes. |
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