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Contagious porcine pleuropneumonia (CPP), caused by Actinobacillus pleuropneumoniae (APP), is a highly transmissible and fatal respiratory illness that causes tremendous economic losses for the pig breeding industry worldwide. Propionibacterium acnes (PA) has a strong cross-reaction with anti-APP1 and anti-APP5 serum and can efficiently prevent APP infection, which was fortuitously found in researching the differential gene between the different APP serotypes. There seems to be some natural cross-protection between PA and APP. To identify the common epitope, the phage display library of a PA whole genome was constructed, whose size is 10 5 . The DNA sequence of the positive clone was determined after three rounds of biopanning, and ten common protein types were identified and the epitope was predicted by computer software. Six peptide epitopes were selected and synthesized for further analysis. Among these epitopes, Ba1, Bb5 and C1 could bind to anti-PA serum and anti-APP1 serum and vice versa. Furthermore, the IgG and IL-4 levels and CD4 + /CD8 + T cell ratios in the Ba1, Bb5 and C1 groups were significantly higher than that in the control group, indicating that the epitopes could trigger an immune response, which was mainly humoral immunity. Moreover, Ba1 and Bb5 equally protected 80% of mice from a fatal dose of APP1 infection compared with the control group. Mice could resist APP1 and APP5 challenge after being treated with the combination of Ba1 and Bb5, with survival rates of 80% and 90%, respectively. These findings suggest that the PA epitope confers antigenicity and can heterologously resist to the APP infection. This finding provides a novel strategy for preventing APP infection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Members of the Trimeric Autotransporter Adhesin (TAA) family play a crucial role in the adhesion of Gram-negative pathogens to host cells, but the immunopathogenesis of TAAs remains unknown. Our previous studies demonstrated that Adh from Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is required for full bacterial pathogenicity. Alveolar macrophages are the first line of defense against respiratory infections. This study compared the interactions between porcine alveolar macrophages (PAMs) and wild-type A. pleuropneumoniae (5b WT) or an Adh-deletion strain (5b ΔAdh) via gene microarray, immunoprecipitation and other technologies. We found that Adh was shown to interact with the PAMs membrane protein OR5M11, an olfactory receptor, resulting in the high-level secretion of IL-8 by activation of p38 MAPK signaling pathway. Subsequently, PAMs apoptosis via the activation of the Fax and Bax signaling pathways was observed, followed by activation of caspases 8, 9, and 3. The immunological pathogenic roles of Adh were also confirmed in both murine and piglets infectious models in vivo. These results identify a novel immunological strategy for TAAs to boost the pathogenicity of A. pleuropneumoniae. Together, these datas reveal the high versatility of the Adh protein as a virulence factor and provide novel insight into the immunological pathogenic role of TAAs.

Actinobacillus pleuropneumoniae is an important pathogen that causes respiratory disease in pigs. Trimeric autotransporter adhesin (TAA) is a recently discovered bacterial virulence factor that mediates bacterial adhesion and colonization. Two TAA coding genes have been found in the genome of A. pleuropneumoniae strain 5b L20, but whether they contribute to bacterial pathogenicity is unclear. In this study, we used homologous recombination to construct a double-gene deletion mutant, ΔTAA, in which both TAA coding genes were deleted and used it in in vivo and in vitro studies to confirm that TAAs participate in bacterial auto-aggregation, biofilm formation, cell adhesion and virulence in mice. A microarray analysis was used to determine whether TAAs can regulate other A. pleuropneumoniae genes during interactions with porcine primary alveolar macrophages. The results showed that deletion of both TAA coding genes up-regulated 36 genes, including ene1514, hofB and tbpB2, and simultaneously down-regulated 36 genes, including lgt, murF and ftsY. These data illustrate that TAAs help to maintain full bacterial virulence both directly, through their bioactivity, and indirectly by regulating the bacterial type II and IV secretion systems and regulating the synthesis or secretion of virulence factors. This study not only enhances our understanding of the role of TAAs but also has significance for those studying A. pleuropneumoniae pathogenesis.

Actinobacillus pleuropneumoniae is a causative agent of porcine pleuropneumonia, which is a highly contagious endemic disease of pigs. Adhesion is a critical first step in the infection process. Trimeric autotransporter adhesions (TAAs) have been identified as novel virulence factors; however, little is known on their roles in A. pleuropneumoniae pathogenicity. Here, our data show that YadA-like head region (Adh) of Apa1 was the optimal adhesion functional domain via segment expression and adhesion assays in vitro. Additionally, Adh induced partial protection against A. pleuropneumoniae 5b L20 and serotypes 1, 3, and 5a in mice. The deletion of Adh gene significantly decreased autoaggregation, biofilm formation and adherence to host cells in vitro. Furthermore, with delaying of clinical symptoms, reducing production of pro-inflammatory cytokines and lessening the lung injury after infection, Adh deletion strain (5bϕAdh) significantly reduced the pathogenicity to piglets. To elucidate the mechanism of lung injury, the differentially expressed genes in the lung tissues of piglets infected with the 5b L20 or 5bϕAdh strains were investigated using microarray analysis and validated by qRT-PCR. Compared with the 5b L20 infected piglets, 495 genes were differentially expressed in 5bϕAdh infected lung tissue (221 upregulated and 274 downregulated). Especially, the antigen processing and presentation gene IFI30 was increased following infection with the 5bϕAdh strain. Thus, Adh may enhance pathogenicity by depressing host immune recognition. We conclude that the head domain of the A. pleuropneumoniae trimeric autotransporter Apa1 regulates autoagglutination, biofilm formation, adhesion to host cells and pathogenicity., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Actinobacillus pleuropneumoniae is the causative agent of acute and chronic pleuropneumonia. Propionibacterium acnes is a facultative anaerobic gram-positive corynebacterium. We have previously found that anti-P. acnes antibodies can prevent A. pleuropneumoniae infections in mice. To investigate the role of macrophages in this process, affinity-purified anti-P. acnes IgG and anti-A. pleuropneumoniae IgG were used in opsonophagocytosis assays. Additionally, the efficacy of passive immunization with P. acnes serum against A. pleuropneumoniae was tested in macrophage-depleted mice. It was found that anti-P. acnes IgG had an effect similar to that of anti-A. pleuropneumoniae IgG (P > 0.05), which significantly promotes phagocytosis of A. pleuropneumoniae by macrophages (P < 0.01). It was also demonstrated that, after passive immunization with anti-P. acnes serum, macrophage-replete mice had the highest survival rate (90%), whereas the survival rate of macrophage-depleted mice was only 40% (P < 0.05). However, macrophage-depleted mice that had been passively immunized with naïve serum had the lowest survival rate (20%), this rate being lower than that of macrophage-replete mice that had been passively immunized with naïve serum. Overall, anti-P. acnes antibodies did not prevent A. pleuropneumoniae infection under conditions of macrophage depletion (P > 0.05). Furthermore, in mice that had been passively immunized with anti-P. acnes serum, macrophage depletion resulted in a greater A. pleuropneumoniae burden and more severe pathological features of pneumonia in lung tissues than occurred in macrophage-replete mice. It was concluded that macrophages are essential for the process by which anti-P. acnes antibody prevents A. pleuropneumoniae infection in mice., (© 2015 The Societies and Wiley Publishing Asia Pty Ltd.)

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the causative agent of porcine pleuropneumonia, a disease that causes serious problems for the swine industry. Successful infection by this bacterium requires breaking the first line of defence in the lungs, the primary alveolar macrophages (PAMs). Therefore, exploring A. pleuropneumoniae-PAM interactions will provide vital groundwork for the scientific control of this infectious disease, which has been little studied up to now. In this work, PAMs were isolated from piglets and co-incubated with A. pleuropneumoniae serovar 5b strain L20 in vitro, and their interaction, PAM cell death, and differential gene expression of A. pleuropneumoniae in response to PAM cell death were observed and analysed using confocal microscopy, electron microscopy, RT-PCR, Western blot, flow cytometry and the use of a gene expression profile chip. A. pleuropneumoniae quickly adhered to and invaded PAMs, inducing apoptosis, which was confirmed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The highest percentage of apoptosis in cells was confirmed using flow cytometry when the cells were infected at a multiplicity of infection (MOI) of 10 and incubated for 5 h, with higher expression of activated caspase-3 as measured by Western blot. Using microarray gene chips with 2868 probes containing nearly all of the genomic sequence of A. pleuropneumoniae serotype 5b strain L20, a total of 185 bacterial genes were found to be differentially expressed (including 92 up-regulated and 93 down-regulated genes) and involved in the process of apoptosis, as compared with the expression of control bacteria cultured without PAMs in BHI medium (mean expression ratios >1.5-fold, p < 0.05). The up-regulated genes are involved in energy metabolism, gene transcription and translation, virulence related gene such as LPS, Trimeric Autotransporter Adhesin, RTX and similar genes. The down-regulated genes are involved in amino acid, cofactor, and vitamin metabolism, and also include ABC transporters. These data demonstrate that A. pleuropneumoniae induces apoptosis of PAMs and undergoes complex changes in gene transcription, including expression changes in known and potential virulence factors. Some potentially novel virulence targets have been identified, suggesting new strategies for the development of vaccines and medicines for both preventive and clinical use., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, and adherence to host cells is a key step in the pathogenic process. Although trimeric autotransporter adhesins (TAAs) were identified in many pathogenic bacteria in recent years, none in A. pleuropneumoniae have been characterized. In this study, we identified a TAA from A. pleuropneumoniae, Apa, and characterized the contribution of its amino acid residues to the adhesion process. Sequence analysis of the C-terminal amino acid residues of Apa revealed the presence of a putative translocator domain and six conserved HsfBD1-like or HsfBD2-like binding domains. Western blot analysis revealed that the 126 C-terminal amino acids of Apa could form trimeric molecules. By confocal laser scanning microscopy, one of these six domains (ApaBD3) was determined to mediate adherence to epithelial cells. Adherence assays and adherence inhibition assays using a recombinant E. coli- ApaBD3 strain which expressed ApaBD3 on the surface of E. coli confirmed that this domain was responsible for the adhesion activity. Moreover, cellular enzyme-linked immunosorbent assays demonstrated that ApaBD3 mediated high-level adherence to epithelial cell lines. Intriguingly, autoagglutination was observed with the E. coli- ApaBD3 strain, and this phenomenon was dependent upon the association of the expressed ApaBD3 with the C-terminal translocator domain., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The limited information on differential gene expression in the different serotypes of Actinobacillus pleuropneumoniae has significantly hampered the research on the pathogenic mechanisms of this organism and the development of multivalent vaccines against A. pleuropneumoniae infection. To compare the gene expressions in the A. pleuropneumoniae strains CVCC259 (serotype 1) and CVCC261 (serotype 3), we screened the differentially expressed genes in the 2 strains by performing representational difference analysis (RDA). Northern blot analyses were used to confirm the results of RDA. We identified 22 differentially expressed genes in the CVCC259 strain and 20 differentially expressed genes in the CVCC261 strain, and these genes were classified into 11 groups: (1) genes encoding APX toxins; (2) genes encoding transferrin-binding protein; (3) genes involved in lipopolysaccharide (LPS) biosynthesis; (4) genes encoding autotransporter adhesin; (5) genes involved in metabolism; (6) genes involved in the ATP-binding cassette (ABC) transporter system; (7) genes encoding molecular chaperones; (8) genes involved in bacterial transcription and nucleic acid metabolism; (9) a gene encoding protease; (10) genes encoding lipoprotein/membrane protein; and (11) genes encoding various hypothetical proteins. This is the first report on the systematic application of RDA for the analysis of differential gene expression in A. pleuropneumoniae serotypes 1 and 3. The determination of these differentially expressed genes will serve as an indicator for future research on the pathogenic mechanisms of A. pleuropneumoniae and the development of a multivalent vaccine against A. pleuropneumoniae infection.

The limited information on the genetic differences among the 15 currently known serotypes of Actinobacillus pleuropneumoniae has significantly hampered the development of typing-based diagnostic methods and multivalent vaccines. In this study, we compared the genomic differences between A. pleuropneumoniae strains CVCC259 (serotype 1) and CVCC261 (serotype 3) by representational difference analysis. Of the eight differential DNA sequences in the CVCC259 strain and 11 differential DNA sequences in the CVCC261 strain that we identified, seven represent known virulent genes, 10 encode putative proteins, and two encode hypothetical proteins. We also investigated the distribution of these 19 sequences among the 15 serotypes, and each serotype showed a different distribution pattern. The autotransporter adhesin occurred as a novel putative virulence factor in serotypes 1, 5, 7, 8, 9, and 11. Notably, the presence of wzm and wzt in serotypes 1, 9, and 11 and the diverse distribution of wzz and wzy in the other serotypes suggest the presence of different O-antigen biosynthesis pathways among serotypes. The information on the differential distribution of these DNA sequences in the 15 serotypes of A. pleuropneumoniae may contribute to future research on the pathogenic mechanisms of different serotypes, typing-based diagnosis methods, and multivalent vaccines.

Actinobacillus pleuropneumoniae is the causative pathogen of porcine pleuropneumonia, which results in large economic losses in the pig industry worldwide. There are, however, no effective subunit vaccines are available in the market owing to the various serotypes and the absence of cross-protection against this pathogen. Therefore, the selection of protective components is of great significance for vaccine development. We previously showed that trimeric autotransporter adhesins are important virulence factors of A. pleuropneumoniae . To determine the potential role in vaccine development of the functional head domain (Apa2H1) of Apa2, a trimeric autotransporter adhesin found in A. pleuropneumoniae , we obtained nature-like trimeric Apa2H1 using a prokaryotic expression system and co-culture of Apa2H1 with bone marrow derived dendritic cells (BMDCs) in vitro resulted in maturation of BMDCs, characterised by the up-regulation of CD83, MHC-II, CCR7, ICAM-I and the increased expression of factors related to B lymphoid cells stimulation, such as proliferation-inducing ligand (APRIL), B lymphocyte stimulator (BLyS) and B cell activating factor (BAFF). The in vivo results showed that vaccination with Apa2H1 resulted in the robust production of antigen-specific antibodies, modestly induced mixed Th1 and Th2 immunity, impaired bacterial colonization and dissemination, and improved mouse survival rates. This study is the first to show that Apa2H1 is antigenic and can be used as a component of a subunit vaccine against A. pleuropneumoniae infection, providing valuable reference material for the development of an effective vaccine against A. pleuropneumoniae. [ABSTRACT FROM AUTHOR]