Construction and Characterization of an <i>Actinobacillus pleuropneumoniae</i> Serotype 2 Mutant Lacking the Apx Toxin Secretion Protein Genes <i>apxIIIB</i> and <i>apxIIID</i>

PARK, Changbo,HA, Yooncheol,KIM, Soohee,CHAE, Chanhee,RYU, Doug-Young Japanese Society of Veterinary Science 2009 The Journal of veterinary medical science Vol.71 No.10

<P>Apx toxins have been identified as important virulence factors of <I>Actinobacillus pleuropneumoniae</I>, the etiologic agent of porcine pleuropneumonia. In some <I>A. pleuropneumoniae</I> serotypes, Apx toxins are secreted by the cell membrane proteins encoded by <I>apxIIIB</I> and <I>apxIIID</I> genes. In an effort to develop a live vaccine strain against <I>A. pleuropneumoniae</I>, we inactivated the <I>apxIIIB</I> and <I>apxIIID</I> genes in <I>A. pleuropneumoniae</I> 1536, a serotype 2 strain, resulting in the Δ<I>apxIIIB/DapxIIID</I> mutant strain (1536ΔBΔD). Immunization of pigs with live 1536ΔBΔD <I>A. pleuropneumoniae</I> conferred protection against homologous challenge with wild-type <I>A. pleuropneumoniae</I> 1536. Thus, impaired Apx toxin secretion may decrease the virulence of <I>A. pleuropneumoniae</I> and may be an effective strategy for the development of a live-attenuated <I>A. pleuropneumoniae</I> vaccine.</P>

Identification of drug target candidates of the swine pathogen Actinobacillus pleuropneumoniae by construction of protein–protein interaction network

Siqi Li,Zhipeng Su,Chengjun Zhang,Zhuofei Xu,Xiaoping Chang,Jiawen Zhu,Ran Xiao,Lu Li,Rui Zhou 한국유전학회 2018 Genes & Genomics Vol.40 No.8

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae has led to severe economic losses in the pig industry worldwide. A. pleuropneumoniae displays various levels of antimicrobial resistance, leading to the dire need to identify new drug targets. Protein–protein interaction (PPI) network can aid the identification of drug targets by discovering essential proteins during the life of bacteria. The aim of this study is to identify drug target candidates of A. pleuropneumoniae from essential proteins in PPI network. The homologous protein mapping method (HPM) was utilized to construct A. pleuropneumoniae PPI network. Afterwards, the subnetwork centered with H-NS was selected to verify the PPI network using bacterial two-hybrid assays. Drug target candidates were identified from the hub proteins by analyzing the topology of the network using interaction degree and homologous comparison with the pig proteome. An A. pleuropneumoniae PPI network containing 2737 non-redundant interaction pairs among 533 proteins was constructed. These proteins were distributed in 21 COG functional categories and 28 KEGG metabolic pathways. The A. pleuropneumoniae PPI network was scale free and the similar topological tendencies were found when compared with other bacteria PPI network. Furthermore, 56.3% of the H-NS subnetwork interactions were validated. 57 highly connected proteins (hub proteins) were identified from the A. pleuropneumoniae PPI network. Finally, 9 potential drug targets were identified from the hub proteins, with no homologs in swine. This study provides drug target candidates, which are promising for further investigations to explore lead compounds against A. pleuropneumoniae.

Predicting Genetic Traits and Epitope Analysis of apxIVA in Actinobacillus pleuropneumoniae

신민경,차승빈,Won-Jung Lee,유한상 한국미생물학회 2011 The journal of microbiology Vol.49 No.3

Actinobacillus pleuropneumoniae causes a severe hemorrhagic pneumonia in pigs. Fifteen serotypes of A. pleuropneumoniae express four different Apx toxins that belong to the pore-forming repeats-in-toxin (RTX)group of toxins. ApxIV, which is conserved and up-regulated in vivo, could be an excellent candidate for the development of a protective cross-serotype immunity vaccine, and could aid in the differential diagnosis of diseases caused by A. pleuropneumoniae. We identified and sequenced apxIVA from A. pleuropneumoniae serotype 2 isolated in Korea (Kor-ApxIVA). The Kor-ApxIVA was closely related to Switzerland (AF021919),China (CP000687), and China (GQ332268), showing 98.6%, 98.4%, and 97.2% amino acid homology,respectively. The level of amino acid homology, however, was higher than the nucleotide homology. The structural characteristics of ApxIVA showed RTX proteins, including N-terminal hydrophobic domains,signature sequences for potential acylation sites, and repeated glycine-rich nonapeptides in the C-terminal region of the protein. Thirty glycine-rich nonapeptides with the consensus sequence, L/V-X-G-G-X-G-N/D-D-X,were found in the C-terminus of the Kor-ApxIVA. In addition, the Kor-ApxIVA was predicted for the linear B-cell epitopes and conserved domains with determined peptide sequences. This genetic analysis of the Kor-ApxIVA might be an important foundation for future biological and functional research on ApxIVA.

국내 분리 흉막폐렴균의 apxIA, IIA, IIIA 유전자 Cloning, 염기서열 분석 및 단백질 발현

신성재,조영욱,유한상,Shin, Sung-jae,Cho, Young-wook,Yoo, Han-sang 대한수의학회 2003 大韓獸醫學會誌 Vol.43 No.2

Actinobacillus pleuropneumoniae causes a highly contagious pleuropneumoniae in swine. The bacterium produces several virulence factors such as exotoxin, LPS, capsular polysaccharide, etc. Among them, the exotoxin, called Apx, has been focused as the major virulence factor, and the toxin consists of 4 gene cluster. apx CABD. apxA is the structural gene of toxin and has four different types, I, II, III, and IV. As the first step of development of a new subunit vaccine, the three different types of apxA gene were amplified from A. pleuropneumoniae isolated from Korea by PCR with primer designed based on the N- and C-terminal of the toxin. The sizes of apxIA, IIA and IIIA were 3,073, 2,971 and 3,159bps, respectively. The comparison of whole DNA sequences of apxIA, IIA and IIIA genes with those of the reference strain demonstrated 98%, 99% and 98% homology, respectively. In addition, the phylogenetic analysis was performed based on the amino acid sequences compared with 12 different RTX toxin family using the neighbor-joining method. ApxA proteins of Korean isolates were identical with reference strains in this study. All ApxA proteins were expressed in E. coli with pQE expression vector and identified using Western blot with polyclonal antibodies against culture supernatants of A. pleuropneumoniae serotype 2 or 5. The sizes of each expressed ApxA protein were about 120, 110, 125 kDa (M.W.), respectively. The results obtained in this study could be used for the future study to develop a new vaccine to porcine pleuropneumoniae.

충남지성(忠南地成) 폐병변(肺病變)으로 분리(分離)한 Actinob acillus Pleuropneumoniae의 생물학적(生物學的) 및 면역학적(免疫學的) 특성(特性)

이종훈 ( Jong Hoon Lee ),안신욱 ( Shin Uk An ),정영재 ( Young Jae Jung ),장경수 ( Kyung Soo Chang ),전무형 ( Moo Hyung Jun ) 한국가축위생학회 1997 韓國家畜衛生學會誌 Vol.20 No.1

An epidemiologic study on pleuropneumonia in the slaughter pigs(Chonan and Asan area, Chungnam province, Korea) during the period of January 1994 through December 1995 was conducted. Isolation of A pleuropneumoniae was attempted in 425 pigs with pneumonic lesions. Biochemical properties, antimicrobial susceptibility, serotypes and pathogenicity of isolated A pleuropneumoniae were investigated. In addition, outer membrane protein(OMP) of the isolates were extracted to determine its properties and immunogenicity in both mice and piglets The results obtained through this study were summarized as followed; 1. Of 3,395 slaughter pigs, pleuropneumonia was observed in 425 pigs(10.6%). A pleuropneumoniae was isolated from 22 pigs(5.2%) out of 425 pigs with pneumonic lesions. The biochemical properties of all isolates were same as those of reference A Dleuropneumoniae strain. Among 22 isolates, 9, 1 and 12 isolates were serovar 2, 3 and 5, respectively. 2. The results of antimicrobial susceptibility test revealed that the isolates showed high susceptibility to ciprofloxacin and cephalothin, moderate susceptibility to amikacin, gentamicin, kanamycin and streptomycin, and low susceptibility to erythromycin, tylosin and sulfadimethoxin. 3. The isolates were varied in pathogenicity to mice. Median lethal dose of LE9402(serovar 2) and LE9511(serovar 5) were 9.2X107CFU and 2.8X10CFU, respectively. Specific pneumonic lesions were observed from the infected mice with clinical signs. Bacteria recovery rate was high in the lung, but low in heart blood and tracheas. 4. Serovar 2 was found to be more pathogenic than serovar 5 in guinea pig. Mortality on guinea pigs inoculated with serovar 2(5.4X 108-5.4X 1O6CFU) and serovar 5(2.8X 108-2.8X 1O6CFU) was 20-40% and 40-80%, respectively. A severe hemorrhagic lesions and focal pneumonic lesions were observed from dead guinea pigs. Bacteria recovery rate was relatively higher in the lung than that of other organs. 5. In the SDS-PAGE analysis, OMP-enriched fractions of both isolates and reference strains contain common peptide bands equivalent to molecular weight of 17, 27, 42, 52 and 95Kd. In addition to common peptide bands, the bands which are specific to each isolate were also observed. The profiles of Sephadex G25 fractions showed 3 major peaks. The common peptide bands which were observed by SDS-PAGE of the crude OMPs were found in the peaks 1 and 2. 6. The OMPs extracted from serovar 2(LE9402) and serovar 5(LE9511) provided high level of protection in mice(70-80%) and pigs(100%). All animals inoculated with OMPs were seroconverted, showing micro-agglutination titer of 640 to 1280.

충남지역 도축돈의 폐병변으로 부터 분리한 Actinob acillus pleuropneumoniae의 생물학적 및 면역학적 특성

이종훈,안신욱,정영재,장경수,전무형 한국동물위생학회 1997 韓國家畜衛生學會誌 Vol.20 No.1

An epidemiologic study on pleuropneumonia in the slaughter pigs(Chonan and Asan area, Chungnam province, Korea) during the period of January 1994 through December 1995 was conducted. Isolation of A pleuropneumoniae was attempted in 425 pigs with pneumonic lesions. Biochemical properties, antimicrobial susceptibility, serotypes and pathogenicity of isolated A pleuropneumoniae were investigated. In addition, outer membrane protein(OMP) of the Isolates were extracted to determine its properties and immunogenicity in both mice and piglets The results obtained through this study were summarized as followed ; 1. Of 3, 395 slaughter pigs, pleuropneumonia was observed in 425 pigs(10.6%). A pleuropneumoniae was isolated from 22 pigs(5.2%) out of 425 pigs with pneumonic lesions. The biochemical properties of all isolates were same as those of reference A pleuropneumoniae strain. Among 22 isolates, 9, 1 and 12 isolates were serovar 2, 3 and 5, respectively. 2. The results of antimicrobial susceptibility test revealed that the isolates showed high susceptibility to ciprofloxacin and cephalothin, moderate susceptibility to amikacin, gentamicin, kanamycin and streptomycin, and low susceptibility to erythromycin, tylosin and sulfadimethoxin. 3. The isolates were varied in pathogenicity to mice. Median lethal dose of LE9402(serovar 2) and LE9511(serovar 5) were $9.2{\times}10^7$ CFU and $2.8{\times}10^7$%CFU, respectively. Specific pneumonic lesions were observed from the infected mice with clinical signs. Bacteria recovery rate was high in the lung, but low In heart blood and tracheas. 4. Serovar 2 was found to be more pathogenic than serovar 5 in guinea pig. Mortality on guinea pigs inoculated with serovar 2($5.4{\times}10^8-5.4{\times}10^6$CFU) and serovar 5($2.8{\times}10^8-2.8{\times}10^6$ CFU) was 20~40% and 40~80%, respectively. A severe hemorrhagic lesions and focal pneumonic lesions were observed from dead guinea pigs. Bacteria recovery rate was relatively higher in the lung than that of other organs. 5. In the SDS-PAGE analysis, OMP-enriched fractions of both isolates and reference strains contain common peptide bands equivalent to molecular weight of 17, 27, 42, 52 and 95Kd. In addition to common peptide bands, the bands which are specific to each isolate were also observed. The profiles of Sephadex G25 fractions showed 3 major peaks. The common peptide bands which were observed by SDS-PAGE of the crude OMPs were found in the peaks 1 and 2. 6. The OMPs extracted from serovar 2(LE9402) and serovar 5(LE9511) provided high level of protection in mice(70~80%) and pigs(100%). All animals inoculated with OMPs were seroconverted, showing micro-agglutination titer of 640 to 1280.

돼지 폐렴병소에서 분리한 Actinobacillus pleuropneumoniae의 특성에 관한 연구

정병열,조길재,김봉환,조광현,Jung, Byeong-yeal,Cho, Gil-jae,Kim, Bong-hwan,Cho, Kwang-hyun 대한수의학회 1996 大韓獸醫學會誌 Vol.36 No.1

The present study was conducted to investigate the biochemical and serologic characteristic of Actinobacillus pleuropneumoniae isolated from pneumonic lungs of pigs during the period from January 1992 to April 1993. A pleuropneumoniae was isolated from 17(27.0%) of 63 growing pigs with respiratory signs and 21(6.4%) of 330 pneumonic lungs of slaughtered pigs. The seasonal isolation frequency of A pleuropneumoniae was higher in winter and spring than that in summer or fall. The biochemical and cultural properties of A pleluropneumoniae isolated from the pneumonic lungs of pigs were identical to those of the reference strains used. The isolates were highly susceptible to ampicillin, cephalothin, ceftiofur, ciprofloxacin(MIC : ${\leq}0.39{\mu}g/ml$) and moderately susceptible to amikacin, chloramphenicol, erythromycin, kanamycin, methicillin, penicillin-G, streptomycin(MIC : 0.78~25IU or ${\mu}g/ml$), respectively. Sulfadimethoxine, sulfamerazine, tylosine showed no response to the isolates(MIC : ${\geq}100{\mu}g/ml$). Among the 38 isolates, 21(55.3%) and 13(34.2%) were resistant to oxytetracycline aid lincomydn, respectively(MIC : ${\geq}50IU$ or ${\mu}g/ml$). The majority of 38 A pleuropneumoniae isolates were turned out as serotype 2(47.4%) or serotype 5(54.7%) and the remaining 3 isolates were evenly classified to serotype 7, 10 or 12. It was noted A pleuropneumonine serotype 5 isolates were more resistant to oxytetracycline than serotype 2 isolates.

돼지에서 Actinobacillus pleuropneumoniae의 혈청학적 진단법에 대한 비교연구

심항섭 ( Hang Sub Shim ),우종태 ( Jong Tae Woo ),조중현 ( Jung Huin Cho ),전무형 ( Moo Hyung Jun ) 한국가축위생학회 1994 韓國家畜衛生學會誌 Vol.17 No.2

To establish an effective diagnostic measure for detection of the antibodies against Actinobacillus pleuropneumoniae, the methods for tube agglutination test(TAT), plate agglutination test(PAT), micro-agglutination test(MAT) and agar-gel immunodiffusion test(ID) were improved and standarized, and the comparative studies were carried out. The results obtained through the experiments were summarized as follows. 1. The rabbit hyperimmune sera to reference serotypes 1 to 6 were cross-tested with TAT, PAT, MAT and ID. In the homologous systems, the range of antibody titers in TAT was 80 to 640, showing the cross-reaction in serotypes 3, 4, 5 and 6. The range of antibody titers in PAT was 4 to 64, showing the cross-reaction in serotypes 3, 4, 5 and 6. In ID, the range of antigen titers was 8 to 32, and cross-reaction was observed in serotype 5. 2. The optimal concentration of antigen in PAT and MAT were 100mg/ml and l.25mg /ml respectively. The most sensitive reaction in MAT was observed in 52°C for 18hrs. 3. In ID, the most promising antigen and the buffer for agar-gel were EDTA-treated antigen and 0.05M tris buffer(pH 7.2), respectively. 4. By the tests for 200 swine sera, it was found that the frequency of positive reaction were 203 in TAT, 240 in PAT and 163 in ID. 5. When compared the titers of TAT with those of MAT for 200 swine sera, MAT showed the higher titer than TAT being increased by relative correlation. Int was found that the titer for positive readings were 20 in TAT and 40 in MAT. 6. when compared the results of ID with those of TAT for 200 swine sera, all sera with TAT titer under 10 were negative in ID. Of the sera with TAT titer 20 and 40, 55.1% nd 91.8% were positive in ID, respectively. All sera with TAT titer above 80 were positive in ID. In comparison of ID and MAT, all sera with MAT titer under 20 were negative in ID. Of the sera with MAT titer 40 and 80, 24.7% and 93.9% were positive in ID, respectively. All sera with MAT titer over 160 showed positive in ID. 7. In conclusion, the established MAT showed high sensitivity but low specificity, wherease ID revealed low sensitivity but high specificity.

Enhancement of Apx Toxin Production in Actinobacillus pleuropneumoniae Serotypes 1, 2, and 5 by Optimizing Culture Conditions

( Hoai Thu Dao ),( Van Tan Do ),( Quang Lam Truong ),( Tae-wook Hahn ) 한국미생물 · 생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.7

Actinobacillus pleuropneumoniae (APP) is a causative agent of porcine pleuropneumonia. Therefore, the development of an effective vaccine for APP is necessary. Here, we optimized the culture medium and conditions to enhance the production yields of Apx toxins in APP serotype 1, 2, and 5 cultures. The use of Mycoplasma Broth Base (PPLO) medium improved both the quantity and quality of the harvested Apx toxins compared with Columbia Broth medium. Calcium chloride (CaCl₂) was first demonstrated as a stimulation factor for the production of Apx toxins in APP serotype 2 cultures. Cultivation of APP serotype 2 in PPLO medium supplemented with 10 μg/ml of nicotinamide adenine dinucleotide (NAD) and 20 mM CaCl₂ yielded the highest levels of Apx toxins. These findings suggest that the optimization of the culture medium and conditions increases the concentration of Apx toxins in the supernatants of APP serotype 1, 2, and 5 cultures and may be applied for the development of vaccines against APP infection.

Escherichia coli-Derived Outer Membrane Vesicles Deliver Galactose- 1-Phosphate Uridyltransferase and Yield Partial Protection against Actinobacillus pleuropneumoniae in Mice

( Keji Quan ),( Zhuang Zhu ),( Sanjie Cao ),( Fei Zhang ),( Chang Miao ),( Xintian Wen ),( Xiaobo Huang ),( Yiping Wen ),( Rui Wu ),( Qigui Yan ),( Yong Huang ),( Xiaoping Ma ),( Xinfeng Han ),( Qin Z 한국미생물 · 생명공학회 2018 Journal of microbiology and biotechnology Vol.28 No.12

In our previous studies, we have identified several in vivo-induced antigens and evaluated their potential as subunit vaccine candidates in a murine model, in which the recombinant protein GalT showed the most potent immunogenicity and immunoprotective efficacy against Actinobacillus pleuropneumoniae. To exploit a more efficient way of delivering GalT proteins, in this study, we employed the widely studied E. coli outer membrane vesicles (OMVs) as a platform to deliver GalT protein and performed the vaccine trial using the recombinant GalTOMVs in the murine model. Results revealed that GalT-OMVs could elicit a highly-specific, IgG antibody titer that was comparable with the adjuvant GalT group. Significantly higher lymphocyte proliferation and cytokines secretion levels were observed in the GalT-OMVs group. 87.5% and 50% of mice were protected from a lethal dose challenge using A. pleuropneumoniae in active or passive immunization, respectively. Histopathologic and immunohistochemical analyses showed remarkably reduced pathological changes and infiltration of neutrophils in the lungs of mice immunized with GalT-OMVs after the challenge. Taken together, these findings confirm that OMVs can be used as a platform to deliver GalT protein and enhance its immunogenicity to induce both humoral and cellular immune responses in mice.