Molecular characterization of a protective outer membrane lipoprotein (OmlA) from Actinobacillus pleuropneumoniae serotype 1.

An expression library was constructed from an Actinobacillus pleuropneumoniae serotype 1 clinical isolate using a plasmid vector. The library was screened with serum raised against the culture supernatant of this strain. One Escherichia coli transformant which also reacted with convalescent serum was isolated and found to express a protein with an electrophoretic mobility of approximately 50,000. The A. pleuropneumoniae-derived DNA encoding the protein was localized and characterized by nucleotide sequence analysis and primer extension mapping. One open reading frame of 1,095 bases was detected and confirmed by TnphoA insertion mutagenesis. It encoded a protein with a calculated molecular mass of 40 kDa which was lipid modified and present in the outer membrane and in membrane blebs of A. pleuropneumoniae. This protein was designated as outer membrane lipoprotein A (OmlA), and the encoding gene as omlA. Southern blotting under low-stringency conditions revealed the presence of hybridizing sequences in all A. pleuropneumoniae type strains, and a specific serum detected a homologous protein in serotypes 2, 8, 9, 11, and 12 type strains. Pigs immunized with this recombinant protein preparation were protected from death in an aerosol challenge experiment with an A. pleuropneumoniae serotype 1 isolate.     

Molecular characterization of a common 48-kilodalton outer membrane protein of Actinobacillus pleuropneumoniae.

Previous studies have shown that a vaccine prepared from outer membranes of Actinobacillus pleuropneumoniae serotype 5 can elicit protective immunity in swine against challenge with either serotype 5 or serotype 1. These results suggest the presence of common subcapsular surface antigens, such as outer membrane proteins, that contribute to cross-protective immunity. We have identified a 48-kDa outer membrane protein that is common to all 12 capsular serotypes of A. pleuropneumoniae but is not present in the outer membranes of related species of gram-negative swine pathogens. This protein is immunogenic in swine infected with either A. pleuropneumoniae serotype 5 or 1A, as well as in swine vaccinated with A. pleuropneumoniae serotype 5 outer membranes. This 48-kDa protein is readily detected in outer membranes produced by sucrose density gradient centrifugation, but it is sarcosyl soluble and therefore not found in outer membranes prepared by detergent treatment. The gene encoding the 48-kDa outer membrane protein has been cloned from A. pleuropneumoniae serotype 5 and and has been designated aopA, for Actinobacillus outer membrane protein A. The gene is 1,347 bp in length and encodes a protein, designated AopA, of 449 amino acids with a predicted molecular weight of 48,603. Southern blot analysis under high-stringency conditions showed that strains of all 12 serotypes of A. pleuropneumoniae contain DNA homologous to this gene, as do strains of two closely related species, Actinobacillus suis and Pasteurella multocida. Whether antibodies against the AopA antigen contribute to cross-protective immunity against A. pleuropneumoniae infection remains to be determined.     

Cytolysins of Actinobacillus pleuropneumoniae serotype 9.

Cytolysin I (ClyI) and cytolysin II (ClyII), which are present in the culture supernatant of Actinobacillus pleuropneumoniae serotype 9, are thought to play an important role in the pathogenesis of pig pleuropneumonia. The purpose of this study was to clone and characterize the genetic determinants of these cytolysins. Cloning was accomplished by the screening of DNA libraries for the presence of cytolytic activity and for the presence of DNA sequences homologous to leukotoxin DNA of Pasteurella haemolytica. Both genetic determinants were found to be members of the RTX cytotoxin family. The ClyII determinant was characterized in more detail. It appeared that ClyII more closely resembled the leukotoxin of P. haemolytica than the alpha-hemolysin of Escherichia coli. The ClyII amino acid sequence was identical to a hemolysin gene sequence of A. pleuropneumoniae serotype 5; this finding indicates that the latter gene also codes for ClyII and not for ClyI, as has previously been suggested. The genetic organization of the ClyII determinant differed from the genetic organization of other RTX determinants. Genes responsible for secretion of ClyII were not contiguous with the toxin gene. Instead, secretion genes were present elsewhere in the genome. These secretion genes, however, belong to the ClyI operon. This indicates that the secretion genes of the ClyI operon are responsible for secretion of ClyI and ClyII.     

Isolation and characterization of a capsule-deficient mutant of Actinobacillus pleuropneumoniae serotype 1

The capsular polysaccharides (CPS) play a major role in pathogenicity of Actinobacillus pleuroIpneumoniae, the causative agent of porcine pleuropneumonia. The purpose of the present study was to isolate a mutant in CPS biosynthesis by using a mini-Tn 10 transposon mutagenesis system and evaluate its adherence to host cells. One mutant apparently did not possess CPS as it did not react with a monoclonal antibody against A. pleuropneumoniae serotype 1 capsular antigen. Absence of capsule was confirmed by flow cytometry and also by transmission electron microscopy after polycationic ferritin labelling. The site of insertion of the mini-Tn 10 was determined and found to be in the cpxC gene. Its gene product, CpxC, is a protein involved in polysaccharide transport across the cytoplasmic membrane during CPS biosynthesis. Use of piglet tracheal frozen sections indicated that the CPS mutant adhered significantly (P=0.0001) more than the parent strain. The non-capsular mutant was less virulent in pigs compared to the parent strain and showed no mortality in experimentally infected pigs. The CPS mutant was however resistant to pig serum. This CPS mutant is the first A. pleuropneumoniae mutant in a CPS transport gene. It is also the first time that adherence of a CPS mutant of A. pleuropneumoniae is evaluated. Our observations indicate that capsular polysaccharides of A. pleuropneumoniae serotype 1 are not involved in adherence to piglet tracheal frozen sections but rather mask, at least in part, the adhesive functions.     

Virulence properties and protective efficacy of the capsular polymer of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5.

The role of the capsule of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5 in bacterial virulence, and the protective efficacy of antibody to serotype 5 capsule was investigated. Encapsulated H. pleuropneumoniae serotype 5 were resistant to killing by complement and antibody to capsule or somatic antigens, whereas a noncapsulated mutant was sensitive to killing by the alternative complement pathway alone. Antiserum to whole H. pleuropneumoniae serotype 5 bacteria or monospecific antiserum to capsule was capable of opsonizing bacteria of the homologous serotype for phagocytosis by swine polymorphonuclear leukocytes but was not opsonic for a heterologous serotype. An immunoglobulin M monoclonal antibody to the serotype 5 capsule was not opsonic for any serotype. Mice were protected against lethal, intranasal challenge with the homologous or heterologous serotype after immunization with live encapsulated or noncapsulated bacteria, but not after immunization with killed bacteria, lipopolysaccharide, or a capsule-protein conjugate vaccine. The protection induced by immunization with live bacteria was transferred to nonimmune, syngeneic mice by serum but not by spleen cells. Nonimmune pigs passively immunized with monospecific swine serum to capsule were protected from lethal infection but not from development of hemorrhagic lung lesions, whereas pigs passively immunized with swine antiserum to live bacteria did not develop severe respiratory lesions. Thus, the capsule of H. pleuropneumoniae serotype 5 was inhibitory to the bactericidal activity of serum and was antiphagocytic. Antibody to the capsule was opsonic but was not fully protective.     

Identification and characterization of a DNA region involved in the export of capsular polysaccharide by Actinobacillus pleuropneumoniae serotype 5a.

Actinobacillus pleuropneumoniae synthesizes a serotype-specific capsular polysaccharide that acts as a protective barrier to phagocytosis and complement-mediated killing. To begin understanding the role of A. pleuropneumoniae capsule in virulence, we sought to identify the genes involved in capsular polysaccharide export and biosynthesis. A 5.3-kb XbaI fragment of A. pleuropneumoniae serotype 5a J45 genomic DNA that hybridized with DNA probes specific for the Haemophilus influenzae type b cap export region was cloned and sequenced. This A. pleuropneumoniae DNA fragment encoded four open reading frames, designated cpxDCBA. The nucleotide and predicted amino acid sequences of cpxDCBA contained a high degree of homology to the capsule export genes of H. influenzae type b bexDCBA, Neisseria meningitidis group B ctrABCD, and, to a lesser extent, Escherichia coli K1 and K5 kpsE and kpsMT. When present in trans, the cpxDCBA gene cluster complemented kpsM::TnphoA or kpsT::TnphoA mutations, determined by enzyme immunoassay and by restored sensitivity to a K5-specific bacteriophage. A cpxCB probe hybridized to genomic DNA from all A. pleuropneumoniae serotypes tested, indicating that this DNA was conserved among serotypes. These data suggest that A. pleuropneumoniae produces a group II family capsule similar to those of related mucosal pathogens.     

Isolation and molecular characterization of spontaneously occurring cytolysin-negative mutants of Actinobacillus pleuropneumoniae serotype 7.

Actinobacillus pleuropneumoniae serotype 7 strains are shown to spontaneously lose cytolytic activity with a frequency of approximately 10(-4). The phenotypic change is associated with the loss of approximately 8.5 kbp of chromosomal DNA. A genomic fragment encoding the cytolysin and its flanking sequences was cloned and characterized. Also, the corresponding truncated fragment was cloned from a spontaneous mutant. Comparison of the two clones allowed the definition of the excision site. The ends of the excised fragment are composed of 1,201 bp long direct identical repeats, possibly facilitating the genotypic change by homologous recombination. In accordance with this hypothesis, one repeat is conserved in the spontaneous mutant. Each repeat contains one open reading frame preceded by a Shine-Dalgarno consensus sequence, and the ends of each repeat contain 26-bp complementary sequences with four mismatches.     

Characterization of immunodominant and potentially protective epitopes of Mannheimia haemolytica serotype 1 outer membrane lipoprotein PlpE

Mannheimia haemolytica serotype 1 (S1) is the most common bacterial isolate found in shipping fever pneumonia in beef cattle. Currently used vaccines against M. haemolytica do not provide complete protection against the disease. Research with M. haemolytica outer membrane proteins (OMPs) has shown that antibodies to one particular OMP from S1, PlpE, may be important in immunity. In a recently published work, members of our laboratory showed that recombinant PlpE (rPlpE) is highly immunogenic when injected subcutaneously into cattle and that the acquired immunity markedly enhanced resistance to experimental challenge (A. W. Confer, S. Ayalew, R. J. Panciera, M. Montelongo, L. C. Whitworth, and J. D. Hammer, Vaccine 21:2821-2829, 2003). The objective of this work was to identify epitopes of PlpE that are responsible for inducing the immune response. Western blot analysis of a series of rPlpE with nested deletions on both termini with bovine anti-PlpE hyperimmune sera showed that the immunodominant region is located close to the N terminus of PlpE. Fine epitope mapping, in which an array of overlapping 13-mer synthetic peptides attached to a derivatized cellulose membrane was probed with various affinity-purified anti-PlpE antibodies, identified eight highly reactive regions, of which region 2 (R2) was identified as the specific epitope. The R2 region is comprised of eight imperfect repeats of a hexapeptide (QAQNAP) and is located between residues 26 and 76. Complement-mediated bactericidal activity of affinity-purified anti-PlpE bovine antibodies confirmed that antibodies directed against the R2 region are effective in killing M. haemolytica.     

Identification of a maltose-inducible major outer membrane protein in Actinobacillus (Haemophilus) pleuropneumoniae

The addition of maltose to the growth media of Actinobacillus pleuropneumonia (serotype 1) resulted in the induction of an outer membrane protein (OMP) with a molecular mass of 42 kDa. This protein had porin-like properties in that it was peptidoglycan-associated and was resistant to proteolysis by trypsin. A pleuropneumoniae expressing the 42 kDa OMP were unable to bind lambda phage. Similar proteins were also induced in A. pleuropneumoniae isolates representing serotypes 2 to 7 with the exception of serotype 4; however, not all isolates of any given serotype expressed a maltose-inducible OMP. Western immunoblotting using convalescent antiserae against the serotype 1 A. pleuropneumoniae indicated that the 42 kDa OMP was expressed in vivo and was cross-reactive with the maltose-inducible OMPs from other serotypes.     

Characterization of porcine plasma ficolins that bind Actinobacillus pleuropneumoniae serotype 5B

Ficolins are collagenous lectins implicated in resistance to infection by some micro-organisms with surfaces containing N-acetylglucosamine residues. Pigs have two known ficolin genes, alpha and beta, but ficolins in pig plasma appear in various electrophoretic forms, the origin and function of which are unclear. In this investigation the forms of ficolin in pig plasma that bind to the pneumonic pathogen Actinobacillus pleuropneumoniae serotype 5b (APP5) were compared with affinity-purified porcine plasma ficolins. APP5-bound ficolins consisted of two distinct multimeric (non-denatured) forms composed of subunits that migrated as multiple 38,40 and 42 kDa forms (apparent MW) with pI range 5.2-6.0. The APPS-binding forms of ficolin were consistent with ficolin alpha and were indistinguishable from affinity-purified plasma ficolins by peptide mass fingerprint analysis. Subunit bands separated by 2D-PAGE were consistent with porcine ficolin alpha. Affinity-purified plasma ficolins had a major subunit mass of 35081 Da by MALDI-TOF MS. Affinity-purified ficolins digested with N-glycosidase F retained APP5-binding activity and migrated faster as a single band of 38 kDa. These studies indicate that ficolin alpha is the major APPS-binding form in porcine plasma and suggest that N-glycosylation contributes to the apparent electrophoretic heterogeneity of porcine ficolins but is not required for APP5-binding activity.     

Cloning and expression of a transferrin-binding protein from Actinobacillus pleuropneumoniae.

An expression library was constructed from Actinobacillus pleuropneumoniae serotype 7. Escherichia coli transformants expressing recombinant proteins were identified by immunoscreening with porcine convalescent serum. One transformant expressing a 60-kDa protein (60K protein) in aggregated form was identified. Serum raised against the recombinant protein recognized a polypeptide with an indistinguishable electrophoretic mobility in the A. pleuropneumoniae wild type after iron-restricted growth only. The recombinant protein bound transferrin after blotting onto nitrocellulose. Using a competitive enzyme-linked immunosorbent assay (ELISA), the specificity of this binding for the amino-terminal half of iron-saturated porcine transferrin was established. Also, the 60K wild-type protein bound hemin as assessed by hemin-agarose chromatography. Hemin could inhibit transferrin binding of the recombinant protein in the competitive ELISA, whereas hemoglobin and synthetic iron chelators failed to do so. Southern blot analysis of several other A. pleuropneumoniae strains indicated that highly homologous sequence is present in eight of eight isolates of serotype 7 and in some isolates of serotypes 2, 3, and 4.     

Cloning and molecular characterization of Cu,Zn superoxide dismutase from Actinobacillus pleuropneumoniae.

Copper-zinc superoxide dismutases (Cu,Zn SODs), until recently considered very unusual in bacteria, are now being found in a wide range of gram-negative bacterial species. Here we report the cloning and characterization of sodC, encoding Cu,Zn SOD in Actinobacillus pleuropneumoniae, a major pathogen of pigs and the causative organism of porcine pleuropneumonia. sodC was shown to lie on a monocistronic operon, at the chromosomal locus between the genes asd (encoding aspartate semialdehyde dehydrogenase) and recF. The primary gene product was shown to have an N-terminal peptide extension functioning as a leader peptide, so that the mature Actinobacillus enzyme, like other bacterial examples, is directed to the periplasm, where it is appropriately located to dismutate exogenously generated superoxide. While the role of these secreted bacterial SODs is unknown, we speculate that in A. pleuropneumoniae the enzyme may confer survival advantage by accelerating dismutation of superoxide derived from neutrophils, a central host defense response in the course of porcine infection.     

Isolation of an atypical strain of Actinobacillus pleuropneumoniae serotype 1 with a truncated lipopolysaccharide outer core and no O-antigen

A field isolate of Actinobacillus pleuropneumoniae, the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia, was sent to the diagnostic laboratory for serotyping. The isolate presented a clear reaction, with both polyclonal antibodies against serotype 1 and monoclonal antibodies against the capsular polysaccharide of serotype 1. It also exhibited a PCR profile of Apx toxins expected for serotype 1. The isolate, however, failed to react with monoclonal antibodies against the O-antigen of serotype 1 lipopolysaccharide (LPS), suggesting a rough phenotype. The lipid A-core region of the isolate migrated faster than the corresponding region of the serotype 1 reference strain S4074 by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the presence of a truncated core. Sugar analysis and mass spectrometry analysis of the O-deacylated LPS from the field isolate were consistent with the absence of O-antigen and truncation of the outer core compared to the wild-type reference strain. Experimental infection of pigs confirmed the virulence of the isolate. This is the first report of an isolate of A. pleuropneumoniae serotype 1 with a truncated outer core and a rough LPS phenotype. Veterinary diagnostic laboratories should be vigilant, since infections caused by such an isolate will not be detected by serological tests based on LPS O-antigen.     

Immunogenicity of Actinobacillus pleuropneumoniae outer membrane proteins and enhancement of phagocytosis by antibodies to the proteins.

To determine the opsonic effect of antibodies to Actinobacillus (Haemophilus) pleuropneumoniae outer membrane proteins on phagocytosis by porcine polymorphonuclear leukocytes (PMN), we separated the integral outer membrane proteins (IOMPs) by Triton X-114 extraction. Four major IOMPs with molecular masses of 76, 50, 39, and 29 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These IOMPs were found to be essentially free of endotoxin in the Limulus amebocyte lysate assay. The 76-kDa protein exhibited a more intensely stained electrophoresis band when isolated from iron-restricted cultures, and a new band at 105 kDa was present in the whole-membrane fraction but not in the integral fraction, indicating that the 105-kDa iron-repressible protein is a peripheral membrane protein. The 76-, 50-, and 39-kDa proteins were shown to be surface exposed, since antibodies to these IOMPs could be absorbed out of convalescent-phase sera by whole cells. Percentages of phagocytosis by porcine PMN of A. pleuropneumoniae opsonized with convalescent-phase sera, convalescent-phase sera absorbed with IOMPs, or convalescent-phase sera absorbed with whole cells were 46.75, 21.81, and 7.96%, respectively. These results demonstrate that antibodies to IOMPs of A. pleuropneumoniae serve as important opsonins in phagocytosis by porcine PMN.     

Effect of iron restriction on the outer membrane proteins of Actinobacillus (Haemophilus) pleuropneumoniae.

The outer membrane protein profile of Actinobacillus (Haemophilus) pleuropneumoniae grown under iron-restricted and iron-replete conditions was studied by polyacrylamide gel electrophoresis and immunoblotting. A virulent serotype 1 isolate synthesized a novel protein with an apparent molecular weight of 105,000 (105K) and increased the synthesis of a 76K protein under iron-restricted conditions. Both proteins were synthesized within 15 min of establishment of iron-restricted conditions. Proteins of equivalent molecular weights could also be induced by iron restriction in serotype 2, 3, 4, 5, and 7 isolates of A. pleuropneumoniae. Convalescent-phase sera from serotype 1-infected pigs contained antibodies which recognized both the 105K and 76K proteins from all six serotypes examined, indicating that these proteins were expressed in vivo and were immunologically conserved. Cells expressing the 105K and 76K proteins also displayed an enhanced ability to bind Congo red and hemin, suggesting that one or both of these proteins functioned to acquire complexed iron during in vivo growth.     

Optimization and standardization of an enzyme-linked immunosorbent assay protocol for serodiagnosis of Actinobacillus pleuropneumoniae serotype 5.

An indirect enzyme-linked immunosorbent assay protocol has been optimized with special emphasis given to assay standardization and quality control. Technical aspects such as choice of a microplate, antigen immobilization, buffer composition, optimal screening dilution of sera, and kinetics of the enzymatic reaction were studied and evaluated in order to design a standard protocol offering maximal analytical sensitivity and specificity, as well as to obtain minimal within- and between-plate variability. Among the 27 plates tested, the Nunc 475-094 and 269-620 immunoplates were found to be the best in terms of high positive-to-negative ratio and low variability. No significant differences in antigen immobilization were found by using buffers of various compositions or pHs; however, the presence of magnesium ions (Mg2+; 0.02 M) resulted in a twofold increase in nonspecific background. An optimal screening dilution of sera was established at 1:200. A 1-h incubation period for test serum was found to be optimal. Maximum enzymatic activity for peroxidase was obtained by adjusting both substrate (H2O2) and hydrogen donor [2,2' -azinobis(3-ethylbenz-thiazoline sulfonic acid)] concentrations to 4 and 1 mM, respectively. To control between-plate variability, a timing protocol was adopted. Within-plate variability was also controlled by using a sample placement configuration pattern. Sliding scales were determined by repeated testing of a cross section of samples to set acceptance limits for both within- and between-plate variability. These limits were used in a quality control program to monitor assay performance. The results obtained suggest that this standardized protocol might be useful in the serodiagnosis of Actinobacillus pleuropneumoniae serotype 5.     

Cloning and expression of a cohemolysin, the CAMP factor of Actinobacillus pleuropneumoniae.

The genetic determinant of the cohemolysin which is responsible for the CAMP phenomenon, a cohemolysis, of Actinobacillus pleuropneumoniae was cloned in Escherichia coli. Total DNA from the A. pleuropneumoniae serotype 1 type strain 4074 was used to construct a gene library in plasmid pUC18 in E. coli JM83. A total of 10,500 clones containing recombinant plasmids have been screened for hemolysis on blood plates. Fifty-five clones which showed a weak hemolytic response after 24 to 48 h of incubation were screened for the CAMP reaction with Staphylococcus aureus. This led to the identification of one clone which showed a positive CAMP reaction. Immunoblot analysis revealed that the recombinant strain expressed a protein with a molecular mass of 27,000 daltons, similar in size to the CAMP protein of the group B streptococci. Rabbit antibodies against the CAMP+ clone neutralized the CAMP reaction mediated by the E. coli strain containing the cloned CAMP gene as well as that of A. pleuropneumoniae. Antibodies raised against the cloned CAMP cohemolysin cross-reacted with Streptococcus agalactiae protein B. We designate the 27,000-dalton molecule CAMP factor protein and name its corresponding gene cfp.     

Influence of Actinobacillus pleuropneumoniae serotype 2 and its cytolysins on porcine neutrophil chemiluminescence.

The effects of Actinobacillus pleuropneumoniae serotype 2 and its metabolites on the oxidative activity of porcine neutrophils were studied by using a chemiluminescence technique. Viable A. pleuropneumoniae stimulated the production of oxygen radicals by neutrophils. After having reached a peak value, the oxidative activity decreased until a total inhibition of the oxidative activity of the neutrophils was achieved. All effects were neutralized with homologous convalescent-phase pig sera which had been adsorbed by heat-inactivated A. pleuropneumoniae. Inactivated bacteria and bacteria in the presence of chloramphenicol each had no influence on the oxidative activity of neutrophils. In contrast, a heat-labile factor in A. pleuropneumoniae culture supernatants stimulated and inhibited the oxidative activity of the neutrophils in a dose-dependent manner. Undiluted and low dilutions of culture supernatants were toxic for the phagocytes, while high dilutions stimulated the oxygen radical production of the neutrophils. These effects were neutralized with homologous convalescent-phase pig sera. In order to investigate whether the heat-labile factors in the culture supernatant could be cytolysins, we repeated the experiments with cytolysin II and cytolysin III produced by recombinant Escherichia coli. It was demonstrated that stimulation and inhibition could be reproduced by both cytolysins. In conclusion, the observations made in this study showed that A. pleuropneumoniae secretes heat-labile metabolites that stimulate neutrophil-oxidative metabolism at relatively low concentrations and kill the neutrophils at higher concentrations. Cytolysins may be responsible, at least in part, for these effects.     

Construction of an Actinobacillus pleuropneumoniae serotype 2 prototype live negative-marker vaccine

Deletions were introduced into the ureC and apxIIA genes of an Actinobacillus pleuropneumoniae serotype 2 strain by homologous recombination and counterselection. The double-mutant contains no foreign DNA, is highly attenuated, protects pigs from homologous challenge upon a single aerosol application, and facilitates the serological discrimination of immunized and infected herds.