猪胸膜肺炎放线杆菌血清1型RTX毒素I的N-端表达多肽具有良好的免疫原性

ApxI外毒素是猪胸膜肺炎放线杆菌(APP)最重要的毒力因子,为了研究其N端多肽的免疫原性,分别将apxIA基因的全长编码区(apxIA,3146bp)及其5′端1140bp的片段(apxIA5)克隆到原核表达载体pET28a,经IPTG诱导后在大肠杆菌中实现了表达,表达产物ApxIA和ApxIAN均以包涵体的形式存在,Westernblot检测证实两种表达产物均具有免疫反应性。将纯化的重组蛋白(rApxIA和rApxIAN)和提取的天然毒素ApxI(nApxI)分别经腹腔免疫BALBc小鼠,于免疫前、免疫2周和4周后分别检测了ELISA抗体和毒素中和抗体水平,结果表明,rApxIAN免疫组的ELISA抗体显著低于rApxIA免疫组和nApxI免疫组,但rApxIAN免疫组血清中和试验中测定的溶血素单位与rApxIA及天然nApxI免疫组没有显著差异。第二次免疫2周后,用1个LD50的APP血清1型J101株和2型标准菌株攻击试验动物,rApxIAN免疫组对血清1型和2型菌株的保护率分别为80%和100%。     

Hemagglutinating properties of Actinobacillus pleuropneumoniae

A total of 26 isolates of Actinobacillus pleuropneumoniae were tested for their ability to agglutinate erythrocytes of different origins. Seven different hemagglutination patterns were found. Ten (38%) isolates did not agglutinate any of the erythrocytes tested. The remaining 16 (62%) isolates agglutinated human erythrocytes, and among these, 12 also agglutinated rat, cat, dog, guinea pig, or bovine erythrocytes. No correlation was found between the seven different hemagglutination patterns observed and the serotypes. Hemagglutination activity was destroyed by heating at 100 degrees C as well as by formaldehyde treatment, but was not affected by heating at 60 degrees C, by treatment with trypsin or pronase, or by homogenization of bacterial cells. No fimbriae were observed on examination of bacterial cells negatively stained with phosphotungstate using electron microscopy. Hydrophobic surface properties of the isolates were evaluated. All the isolates appear to possess a hydrophilic cell surface. The present study provides evidence that certain isolates of A. pleuropneumoniae possess hemagglutinating properties which do not appear to be mediated by fimbriae or to involve hydrophobic interactions.     

Flagella and Motility in Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae has been considered nonmotile and nonflagellate. In this work, it is demonstrated that A. pleuropneumoniae produces flagella composed of a 65-kDa protein with an N-terminal amino acid sequence that shows 100% identity with those of Escherichia coli, Salmonella, and Shigella flagellins. The DNA sequence obtained through PCR of the fliC gene in A. pleuropneumoniae showed considerable identity (93%) in its 5' and 3' ends with the DNA sequences of corresponding genes in E. coli, Salmonella enterica, and Shigella spp. The motility of A. pleuropneumoniae was observed in tryptic soy or brain heart infusion soft agar media, and it is influenced by temperature. Flagella and motility may be involved in the survival and pathogenesis of A. pleuropneumoniae in pigs.     

Isolation and Characterization of LPS Mutants of Actinobacillus pleuropneumoniae Serotype 1

The major adhesin of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, has been previously identified as lipopolysaccharide (LPS). The purpose of the present study was to isolate and characterize A. pleuropneumoniae LPS mutants. Screening of LPS mutants was performed with colony dot and sensitivity to novobiocin. One mutant obtained by colony dot (F19) and one mutant selected for its increased sensitivity to novobiocin (33.1) did not react with a monoclonal antibody against A. pleuropneumoniae serotype 1 O-antigen compared with the parent strain. Mutants F19 and 33.1 did not express high-molecular-mass LPS bands as determined in silver-stained SDS-PAGE gels. The core-lipid A region of mutant 33.1 and of the parent strain had similar relative mobilities and reacted with serum from a pig experimentally infected with the serotype 1 reference strain of A. pleuropneumoniae, while the same region in mutant F19 showed faster migration and did not react with this serum. Use of piglet tracheal frozen sections indicated that mutant F19 was able to adhere to piglet trachea as well as the parent strain, while mutant 33.1 adhered [half as much as] the parent strain. Finally, both LPS mutants were markedly less virulent in mice than the parent strain. Taken together, our observations support the idea that LPS is an important virulence factor of A. pleuropneumoniae. Received: 23 December 1996 / Accepted: 19 February 1997     

Immunoproteomic analysis of bacterial proteins of Actinobacillus pleuropneumoniae serotype 1

Background

Actinobacillus pleuropneumoniae (APP) is one of the most important swine pathogens worldwide. Identification and characterization of novel antigenic APP vaccine candidates are underway. In the present study, we use an immunoproteomic approach to identify APP protein antigens that may elicit an immune response in serotype 1 naturally infected swine and serotype 1 virulent strain S259-immunized rabbits.

Results

Proteins from total cell lysates of serotype 1 APP were separated by two-dimensional electrophoresis (2DE). Western blot analysis revealed 21 immunoreactive protein spots separated in the pH 4-7 range and 4 spots in the pH 7-11 range with the convalescent sera from swine; we found 5 immunoreactive protein spots that separated in the pH 4-7 range and 2 in the pH 7-11 range with hyperimmune sera from S259-immunized rabbits. The proteins included the known antigens ApxIIA, protective surface antigen D15, outer membrane proteins P5, subunit NqrA. The remaining antigens are being reported as immunoreactive proteins in APP for the first time, to our knowledge.

Conclusions

We identified a total of 42 immunoreactive proteins of the APP serotype 1 virulent strain S259 which represented 32 different proteins, including some novel immunoreactive factors which could be researched as vaccine candidates.     

Screening of Actinobacillus pleuropneumoniae LuxS Inhibitors

LuxS, a conserved bacterial enzyme involved in the activated methyl cycle, catalyzes S-ribosylhomocysteine (SRH) into homocysteine and AI-2 (the inter-species quorum-sensing signal molecule). This enzyme has been reported to be essential for the survival of Actinobacillus pleuropneumoniae in its natural host. Therefore, it is a potential drug target against A. pleuropneumoniae, an important swine respiratory pathogen causing great economic losses in the pig industry worldwide. In this study, the enzymatic activity determination method was established using the recombinant LuxS of A. pleuropneumoniae. Thirty-five compounds similar to the shape of SRH were screened from the Specs compound library by the software vROCS and were evaluated for LuxS inhibition. Three compounds could inhibit LuxS activity. Two of them were confirmed to be competitive inhibitors and the third one was uncompetitive. All the three compounds displayed inhibitory effects on the growth of A. pleuropneumoniae and two other important swine pathogens, Haemophilis parasuis and Streptococcus suis, with MIC₅₀ values ranging from 11 to 51 μg/ml. No significant cytotoxic effect of the compounds was detected on porcine PK-15 cells at the concentration which showed inhibitory effect on bacterial growth. These results suggest that LuxS is an ideal target to develop antimicrobials for porcine bacterial pathogens. The three LuxS inhibitors identified in this study can be used as lead compounds for drug design.     

Comparative Genomic Characterization of Actinobacillus pleuropneumoniae

The Gram-negative bacterium Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumoniae, a lethal respiratory infectious disease causing great economic losses in the swine industry worldwide. In order to better interpret the genetic background of serotypic diversity, nine genomes of A. pleuropneumoniae reference strains of serovars 1, 2, 4, 6, 9, 10, 11, 12, and 13 were sequenced by using rapid high-throughput approach. Based on 12 genomes of corresponding serovar reference strains including three publicly available complete genomes (serovars 3, 5b, and 7) of this bacterium, we performed a comprehensive analysis of comparative genomics and first reported a global genomic characterization for this pathogen. Clustering of 26,012 predicted protein-coding genes showed that the pan genome of A. pleuropneumoniae consists of 3,303 gene clusters, which contain 1,709 core genome genes, 822 distributed genes, and 772 strain-specific genes. The genome components involved in the biogenesis of capsular polysaccharide and lipopolysaccharide O antigen relative to serovar diversity were compared, and their genetic diversity was depicted. Our findings shed more light on genomic features associated with serovar diversity of A. pleuropneumoniae and provide broader insight into both pathogenesis research and clinical/epidemiological application against the severe disease caused by this swine pathogen.Actinobacillus pleuropneumoniae, a Gram-negative facultative anaerobic encapsulated coccobacillus, belongs to the Actinobacillus genus of the Pasteurellaceae family (19). A. pleuropneumoniae is a primary bacterial etiologic agent of porcine contagious pleuropneumonia, a severe respiratory disease leading to great economic losses to the global swine industry (7). The cases usually display pleuropneumonia and pulmonary lesions characterized by serious hemorrhage and necrosis. To date, several factors involved in the virulence of A. pleuropneumoniae have been described, including Apx exotoxins, capsular polysaccharides (CPS), lipopolysaccharides (LPS), outer membrane proteins, iron-acquisition proteins and adhesin factors (11, 19, 24). However, the genetic differences of pathogenesis remain poorly characterized and are worth interpreting from the perspective of comparative genomics for this bacterium.Thus far, 15 serovars and two biotypes of A. pleuropneumoniae have been recognized, with great variations in virulence and interlocal distributions (6). The predominant serovar-specific antigens are composed of CPS, which could rigorously define serovars of A. pleuropneumoniae (6, 34). Antigenic differences in the LPS can further determine A. pleuropneumoniae subtypes within a same capsular serovar (13). The metabolic and virulent characteristics of this pathogen have been systematically described based on the prior knowledge and two complete genomes (18, 47), but the molecular basis and evolutionary mechanism of serotypic diversity are still not well explained due to the lack of sequence information. To investigate the associations of serovar diversity with the underlying genetic components, more serovar-related genomic islands involved in the biosynthesis of capsular and lipopolysaccharide antigens should be decoded at the pan-genome level of A. pleuropneumoniae. At present, through the next-generation of sequencing technique (454 GS FLX pyrosequencing platform), more and more bacterial species, subspecies or typical strains have been quickly sequenced, such as eight species in the Yersinia genus (9), 17 strains of Streptococcus pneumoniae (22), and 5 strains from different Francisella tularensis subspecies (8). Multiple genome sequences from different strains of a single species can offer comprehensive information to explore the relationship between genotypes and phenotypes and to further discover additional genetic markers for clinical purpose.In the present study, we sequenced the A. pleuropneumoniae genomes of nine reference strains of serovars 1, 2, 4, 6, 9, 10, 11, 12, and 13. Together with three public complete genome sequences of A. pleuropneumoniae serovars 3, 5b, and 7, the analysis of comparative genomics was performed to report a global genomic characterization of this pathogenic bacterium. The acquisition and loss of genome compositions that contribute to virulence and serovar diversity were identified. The genetic loci involved in the biogenesis of capsule and O-specific polysaccharide were compared, and their vital roles in serotypic diversity were investigated.     

Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection

Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, a highly contagious disease for which there is no effective vaccine. This review considers how adhesins, iron-acquisition factors, capsule and lipopolysaccharide, RTX cytotoxins and other potential future vaccine components contribute to colonisation, to avoidance of host clearance mechanisms and to damage of host tissues.     

Natural competence in strains of Actinobacillus pleuropneumoniae

The fatty acid (FA) profiles of myxobacteria contain FA species with double bonds at the Δ⁵ and Δ¹¹ positions, the latter being rather unusual among bacteria. Despite this knowledge, the mechanism for introduction of these double bonds has never been described before in myxobacteria. Searches for candidate genes in the genome of the model organism Myxococcus xanthus revealed 16 genes, which have been annotated as FA desaturases. However, due to redundant substrate specificity, functional analyses of these enzymes by construction of inactivation mutants did not lead to the identification of their function or substrate specificity. Therefore, we elucidated the regioselectivity of the desaturation reactions by heterologous expression of eight desaturases from M. xanthus in Pseudomonas putida and thus could prove five of them to be indeed active as desaturases, with three (MXAN_1742, MXAN_3495 and MXAN_5461) and two (MXAN_0317 and MXAN_6306) acting as Δ⁵ and Δ¹¹ desaturases, respectively. This is the first report about the heterologous expression and regioselectivity of FA desaturases in myxobacteria.     

Effect of bovine apo-lactoferrin on the growth and virulence of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae (App) is a Gram-negative bacterium that causes porcine pleuropneumonia, leading to economic losses in the swine industry. Due to bacterial resistance to antibiotics, new treatments for this disease are currently being sought. Lactoferrin (Lf) is an innate immune system glycoprotein of mammals that is microbiostatic and microbicidal and affects several bacterial virulence factors. The aim of this study was to investigate whether bovine iron-free Lf (BapoLf) has an effect on the growth and virulence of App. Two serotype 1 strains (reference strain S4074 and the isolate BC52) and a serotype 7 reference strain (WF83) were analyzed. First, the ability of App to grow in iron-charged BLf was discarded because in vivo, BapoLf sequesters iron and could be a potential source of this element favoring the infection. The minimum inhibitory concentration of BapoLf was 14.62, 11.78 and 10.56 µM for the strain BC52, S4074 and WF83, respectively. A subinhibitory concentration (0.8 µM) was tested by assessing App adhesion to porcine buccal epithelial cells, biofilm production, and the secretion and function of toxins and proteases. Decrease in adhesion (24–42 %) was found in the serotype 1 strains. Biofilm production decreased (27 %) for only the strain 4074 of serotype 1. Interestingly, biofilm was decreased (60–70 %) in the three strains by BholoLf. Hemolysis of erythrocytes and toxicity towards HeLa cells were not affected by BapoLf. In contrast, proteolytic activity in all strains was suppressed in the presence of BapoLf. Finally, oxytetracycline produced synergistic effect with BapoLf against App. Our results suggest that BapoLf affects the growth and several of the virulence factors in App.     

Cloning and characterization of the groE locus from Actinobacillus pleuropneumoniae

A 4.4-kb DNA fragment was cloned from Actinobacillus pleuropneumoniae (strain 4074, serotype 1) by genetic complementation with Escherichia coli groES-groEL mutant strains. Sequence analysis of this fragment revealed a purine nucleoside phosphorylase (DeoD)-encoding gene homolog (deoD), heat-shock response-encoding genes for the small (groES) and large subunits (groEL) and a partial open reading frame encoding an alcohol dehydrogenase homolog (adhE). The predicted amino-acid sequence of groES and groEL genes showed extensive sequence identity (80–95%) with other Pasteurellaceae. The gene organization surrounding the groE locus was different from that of Haemophilus infuenzae. When expressed in E. coli, groES-groEL genes were capable of complementing the growth of a λ lytic phage, indicating a structural as well as functional conservation.     

Global effects of catecholamines on Actinobacillus pleuropneumoniae gene expression

Bacteria can use mammalian hormones to modulate pathogenic processes that play essential roles in disease development. Actinobacillus pleuropneumoniae is an important porcine respiratory pathogen causing great economic losses in the pig industry globally. Stress is known to contribute to the outcome of A. pleuropneumoniae infection. To test whether A. pleuropneumoniae could respond to stress hormone catecholamines, gene expression profiles after epinephrine (Epi) and norepinephrine (NE) treatment were compared with those from untreated bacteria. The microarray results showed that 158 and 105 genes were differentially expressed in the presence of Epi and NE, respectively. These genes were assigned to various functional categories including many virulence factors. Only 18 genes were regulated by both hormones. These genes included apxIA (the ApxI toxin structural gene), pgaB (involved in biofilm formation), APL_0443 (an autotransporter adhesin) and genes encoding potential hormone receptors such as tyrP2, the ygiY-ygiX (qseC-qseB) operon and narQ-narP (involved in nitrate metabolism). Further investigations demonstrated that cytotoxic activity was enhanced by Epi but repressed by NE in accordance with apxIA gene expression changes. Biofilm formation was not affected by either of the two hormones despite pgaB expression being affected. Adhesion to host cells was induced by NE but not by Epi, suggesting that the hormones affect other putative adhesins in addition to APL_0443. This study revealed that A. pleuropneumoniae gene expression, including those encoding virulence factors, was altered in response to both catecholamines. The differential regulation of A. pleuropneumoniae gene expression by the two hormones suggests that this pathogen may have multiple responsive systems for the two catecholamines.     

Isolation and characterization of mini-Tn10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1

Lipopolysaccharide (LPS) has previously been identified as the major adhesin of Actinobacillus pleuropneumoniae involved in adherence to porcine respiratory tract cells. The purpose of the present study was to isolate and characterize mutants in LPS biosynthesis by using a mini-Tn10 transposon mutagenesis system. Seven mutants appeared to possess a rough LPS (among which two had similar Southern blot profiles) while one mutant (#5.1) expressed the high-molecular-mass LPS, but as visualized by Tricine SDS-PAGE, showed an additional band in the core-lipid A region. The LPS mutants showed sensitivity to pig serum to various degrees, while the parent strain was serum-resistant. Use of piglet frozen tracheal sections indicated that, surprisingly, the rough LPS mutants adhered similarly or in greater numbers than the parent strain. However, the LPS mutant #5.1 adhered significantly less than the parent strain and was also less virulent in pigs. The gene affected by mini-Tn10 in LPS mutant #5.1 is galU, the structural gene for UTP-alpha-D-glucose-1-phosphate uridylyltransferase, involved in LPS core biosynthesis. Complementation analysis confirmed that the phenotypic characteristics of LPS mutant #5.1 are the result of the inactivation of the galU gene. Our data suggest that although the presence of O-antigen does not seem to be essential, an intact core-lipid A region might be required for adherence of A. pleuropneumoniae to porcine respiratory tract cells. To the best of our knowledge, these mutants represent the first isogenic mutants of A. pleuropneumoniae defective in LPS biosynthetic genes.     

Structural insights into the glycosyltransferase activity of the Actinobacillus pleuropneumoniae HMW1C-like protein

Glycosylation of proteins is a fundamental process that influences protein function. The Haemophilus influenzae HMW1 adhesin is an N-linked glycoprotein that mediates adherence to respiratory epithelium, an essential early step in the pathogenesis of H. influenzae disease. HMW1 is glycosylated by HMW1C, a novel glycosyltransferase in the GT41 family that creates N-glycosidic linkages with glucose and galactose at asparagine residues and di-glucose linkages at sites of glucose modification. Here we report the crystal structure of Actinobacillus pleuropneumoniae HMW1C (ApHMW1C), a functional homolog of HMW1C. The structure of ApHMW1C contains an N-terminal all α-domain (AAD) fold and a C-terminal GT-B fold with two Rossmann-like domains and lacks the tetratricopeptide repeat fold characteristic of the GT41 family. The GT-B fold harbors the binding site for UDP-hexose, and the interface of the AAD fold and the GT-B fold forms a unique groove with potential to accommodate the acceptor protein. Structure-based functional analyses demonstrated that the HMW1C protein shares the same structure as ApHMW1C and provided insights into the unique bi-functional activity of HMW1C and ApHMW1C, suggesting an explanation for the similarities and differences of the HMW1C-like proteins compared with other GT41 family members.     

构建胸膜肺炎放线杆菌apxIC基因插入突变株

目的：构建胸膜肺炎放线杆菌（APP）apxIC基因插入突变菌株,以鉴定ApxⅠ毒素的生物学特性。方法：根据apxⅠ核酸序列（U05042）设计1对引物,用于自APP血清10型参考菌株（D13039）基因组DNA中扩增apxIC基因及其上下游约2．8kb的基因片段,经克隆测序后在apxIC基因下游xbI酶切位点处插入约0．9kb的氯霉素（Chl）抗性基因表达盒,构建用于转化的转移载体pUIC-Chl^r,将转移载体DNA经电转化导入APP血清10型参考菌株中进行同源重组,以获得突变菌株。结果：在含有氯霉素的培养基中经筛选获得2株丧失溶血活性的突变菌株（D13039C-Chl^r）;利用PCR和Southern blot对突变菌株鉴定,显示氯霉素抗性基因已被插入细菌基因组中。结论：利用电转化和同源重组技术构建成功APP apxIC基因插入突变菌株,为分析ApxⅠ毒素的生物学特性,进而研制APP基因工程减毒活疫苗奠定了基础。     

The lipopolysaccharide core of Actinobacillus suis and its relationship to those of Actinobacillus pleuropneumoniae

The Gram-negative bacteria Actinobacillus suis colonizes the upper respiratory and genital tracts of swine. Along with capsular polysaccharides, lipopolysaccharides (O-chain→core→lipid A~cell) are a main cell-surface component of A.?suis. In this study, we determined that A.?suis lipopolysaccharide incorporates a conserved core that shares some structural features with several core types of A.?pleuropneumoniae . These common core structural features likely account for the observed serological cross-reactivity between A.?suis and A.?pleuropneumoniae, and the data suggest that the structural epitopes responsible for immunogenicity are those in the outer core domain.     

Transcriptional profiling of Actinobacillus pleuropneumoniae under iron-restricted conditions

Background

Toll-like receptors (TLRs) play a central role in innate immunity. TLRs are membrane glycoproteins and contain leucine rich repeat (LRR) motif in the ectodomain. TLRs recognize and respond to molecules such as lipopolysaccharide, peptidoglycan, flagellin, and RNA from bacteria or viruses. The LRR domains in TLRs have been inferred to be responsible for molecular recognition. All LRRs include the highly conserved segment, LxxLxLxxNxL, in which "L" is Leu, Ile, Val, or Phe and "N" is Asn, Thr, Ser, or Cys and "x" is any amino acid. There are seven classes of LRRs including "typical" ("T") and "bacterial" ("S"). All known domain structures adopt an arc or horseshoe shape. Vertebrate TLRs form six major families. The repeat numbers of LRRs and their "phasing" in TLRs differ with isoforms and species; they are aligned differently in various databases. We identified and aligned LRRs in TLRs by a new method described here.

Results

The new method utilizes known LRR structures to recognize and align new LRR motifs in TLRs and incorporates multiple sequence alignments and secondary structure predictions. TLRs from thirty-four vertebrate were analyzed. The repeat numbers of the LRRs ranges from 16 to 28. The LRRs found in TLRs frequently consists of LxxLxLxxNxLxxLxxxxF/LxxLxx ("T") and sometimes short motifs including LxxLxLxxNxLxxLPx(x)LPxx ("S"). The TLR7 family (TLR7, TLR8, and TLR9) contain 27 LRRs. The LRRs at the N-terminal part have a super-motif of STT with about 80 residues. The super-repeat is represented by STTSTTSTT or _TTSTTSTT. The LRRs in TLRs form one or two horseshoe domains and are mostly flanked by two cysteine clusters including two or four cysteine residue.

Conclusion

Each of the six major TLR families is characterized by their constituent LRR motifs, their repeat numbers, and their patterns of cysteine clusters. The central parts of the TLR1 and TLR7 families and of TLR4 have more irregular or longer LRR motifs. These central parts are inferred to play a key role in the structure and/or function of their TLRs. Furthermore, the super-repeat in the TLR7 family suggests strongly that "bacterial" and "typical" LRRs evolved from a common precursor.