Point Mutations in TbpA Abrogate Human Transferrin Binding in Neisseria gonorrhoeae.

TonB-dependent transporters (TDTs) are essential proteins for metal acquisition, an important step in the growth and pathogenesis of many pathogens, including Neisseria gonorrhoeae, the causative agent of gonorrhea. There is currently no available vaccine for gonorrhea; TDTs are being investigated as vaccine candidates because they are highly conserved and expressed in vivo. Transferrin binding protein A (TbpA) is an essential virulence factor in the initiation of experimental infection in human males and functions by acquiring iron upon binding to host transferrin (human transferrin [hTf]). The loop 3 helix (L3H) is a helix finger that inserts into the hTf C-lobe and is required for hTf binding and subsequent iron acquisition. This study identified and characterized the first TbpA single-point substitutions resulting in significantly decreased hTf binding and iron acquisition, suggesting that the helix structure is more important than charge for hTf binding and utilization. The tbpA D355P ΔtbpB and tbpA A356P ΔtbpB mutants demonstrated significantly reduced hTf binding and impaired iron uptake from Fe-loaded hTf; however, only the tbpA A356P ΔtbpB mutant was able to grow when hTf was the sole source of iron. The expression of tbpB was able to restore function in all tbpA mutants. These results implicate both D355 and A356 in the key binding, extraction, and uptake functions of gonococcal TbpA.

Transferrin Binding Protein B and Transferrin Binding Protein A2 Expand the Transferrin Recognition Range of Histophilus somni.

The bacterial bipartite transferrin receptor is an iron acquisition system that several important human and animal pathogens require for survival. It consists of the TonB-dependent transporter transferrin binding protein A (TbpA) and the surface lipoprotein transferrin binding protein B (TbpB). Curiously, the Tbps are only found in host-specific pathogens and are themselves host specific, meaning that they will bind to the transferrin of their host species but not to the transferrins of other animal species. While this phenomenon has long been established, neither the steps in the evolutionary process that led to this exquisite adaptation for the host nor the steps that could alter it are known. We sought to gain insight into these processes by studying Tbp specificity in Histophilus somni, an economically important pathogen of cattle. A past study showed that whole cells of H. somni specifically bind bovine transferrin but not transferrin from sheep and goats, two bovids whose transferrins share 93% amino acid sequence identity with bovine transferrin. To our surprise, we found that H. somni can use sheep and goat transferrins as iron sources for growth and that HsTbpB, but not HsTbpA, has detectable affinity for sheep and goat transferrins. Furthermore, a third transferrin binding protein found in H. somni, HsTbpA2, also showed affinity for sheep and goat transferrins. Our results suggest that H. somni TbpB and TbpA2 may contribute to broadening the host transferrin recognition range of H. somniIMPORTANCE Host-restricted pathogens infect a single host species or a narrow range of host species. Histophilus somni, a pathogen that incurs severe economic losses for the cattle industry, infects cattle, sheep, and goats but not other mammals. The transferrin binding proteins, TbpA and TbpB, are thought to be a key iron acquisition system in H. somni; however, despite their importance, H. somni TbpA and TbpB were previously shown to be cattle transferrin specific. In our study, we find that H. somni TbpB and another little-studied Tbp, TbpA2, bind sheep and goat transferrins, as well as bovine transferrin. Our results suggest that TbpB and TbpA2 may allow for host range expansion and provide a mechanism for how host specificity in Tbp-encoding pathogens can be altered.

Integrated strategy of differentially expressed genes associated with ulcerative colitis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is associated with both genetic and environmental factors; however, the underlying pathogenesis of UC remains unclear. The present study aimed to further explore 12 microarray datasets from patients with UC obtained from the Gene Expression Omnibus repository, for potential genetic pathogenesis of UC through a global bioinformatics view, which included identification of differentially expressed genes (DEGs), functional enrichments, protein­protein interactions, transcriptional and post­transcriptional regulation and drug­gene associations. This integrated analysis screened 233 DEGs that were compared between UC and normal control tissue samples; these included 173 upregulated and 60 downregulated DEGs. Subsequently, transcription factors, such as TATA­binding protein 1 (TBP1; hsa_TATAAA_V$TATA_01) and nuclear factor-κB (NF-κB; hsa_V$NFKAPPAB_01) and microRNAs (miRNAs; such as miR­516­3p and miR­23a) were revealed to be associated with 233 DEGs. Notably, further analysis indicated that these DEGs were enriched in certain diseases, including inflammation, fibrosis and immune system diseases, and were also associated with some drugs, including prednisone, collagenase and mycophenolate mofetil, which may provide choice for treatment of UC. In conclusion, this study may provide novel insights into discovering potential molecular targets involved in the pathogenesis and treatment of UC.

Iron acquisition through the bacterial transferrin receptor.

Transferrin is one of the sources of iron that is most readily available to colonizing and invading pathogens. In this review, we look at iron uptake by the bacterial transferrin receptor that is found in the families Neisseriaceae, Pasteurellaceae and Moraxellaceae. This bipartite receptor consists of the TonB-dependent transporter, TbpA, and the surface lipoprotein, TbpB. In the past three decades, major advancements have been made in our understanding of the mechanism through which the Tbps take up iron. We summarize these findings and discuss how they relate to the diversity and specificity of the transferrin receptor. We also outline several of the remaining unanswered questions about iron uptake via the bacterial transferrin receptor and suggest directions for future research.

The genes that encode the gonococcal transferrin binding proteins, TbpB and TbpA, are differentially regulated by MisR under iron-replete and iron-depleted conditions.

Neisseria gonorrhoeae produces two transferrin binding proteins, TbpA and TbpB, which together enable efficient iron transport from human transferrin. We demonstrate that expression of the tbp genes is controlled by MisR, a response regulator in the two-component regulatory system that also includes the sensor kinase MisS. The tbp genes were up-regulated in the misR mutant under iron-replete conditions but were conversely down-regulated in the misR mutant under iron-depleted conditions. The misR mutant was capable of transferrin-iron uptake at only 50% of wild-type levels, consistent with decreased tbp expression. We demonstrate that phosphorylated MisR specifically binds to the tbpBA promoter and that MisR interacts with five regions upstream of the tbpB start codon. These analyses confirm that MisR directly regulates tbpBA expression. The MisR binding sites in the gonococcus are only partially conserved in Neisseria meningitidis, which may explain why tbpBA was not MisR-regulated in previous studies using this related pathogen. This is the first report of a trans-acting protein factor other than Fur that can directly contribute to gonococcal tbpBA regulation.

Beyond the Crystal Structure: Insight into the Function and Vaccine Potential of TbpA Expressed by Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, is not preventable by vaccination and is rapidly developing resistance to antibiotics. However, the transferrin (Tf) receptor system, composed of TbpA and TbpB, is an ideal target for novel therapeutics and vaccine development. Using a three-dimensional structure of gonococcal TbpA, we investigated two hypotheses, i.e., that loop-derived antibodies can interrupt ligand-receptor interactions in the native bacterium and that the loop 3 helix is a critical functional domain. Preliminary loop-derived antibodies, as well as optimized second-generation antibodies, demonstrated similar modest ligand-blocking effects on the gonococcal surface but different effects in Escherichia coli. Mutagenesis of loop 3 helix residues was employed, generating 11 mutants. We separately analyzed the mutants' abilities to (i) bind Tf and (ii) internalize Tf-bound iron in the absence of the coreceptor TbpB. Single residue mutations resulted in up to 60% reductions in ligand binding and up to 85% reductions in iron utilization. All strains were capable of growing on Tf as the sole iron source. Interestingly, in the presence of TbpB, only a 30% reduction in Tf-iron utilization was observed, indicating that the coreceptor can compensate for TbpA impairment. Complete deletion of the loop 3 helix of TbpA eliminated the abilities to bind Tf, internalize iron, and grow with Tf as the sole iron source. Our studies demonstrate that while the loop 3 helix is a key functional domain, its function does not exclusively rely on any single residue.

Escape from bacterial iron piracy through rapid evolution of transferrin.

Iron sequestration provides an innate defense, termed nutritional immunity, leading pathogens to scavenge iron from hosts. Although the molecular basis of this battle for iron is established, its potential as a force for evolution at host-pathogen interfaces is unknown. We show that the iron transport protein transferrin is engaged in ancient and ongoing evolutionary conflicts with TbpA, a transferrin surface receptor from bacteria. Single substitutions in transferrin at rapidly evolving sites reverse TbpA binding, providing a mechanism to counteract bacterial iron piracy among great apes. Furthermore, the C2 transferrin polymorphism in humans evades TbpA variants from Haemophilus influenzae, revealing a functional basis for standing genetic variation. These findings identify a central role for nutritional immunity in the persistent evolutionary conflicts between primates and bacterial pathogens.

Identification of regulatory elements that control expression of the tbpBA operon in Neisseria gonorrhoeae.

Iron is an essential nutrient for survival and establishment of infection by Neisseria gonorrhoeae. The neisserial transferrin binding proteins (Tbps) comprise a bipartite system for iron acquisition from human transferrin. TbpA is the TonB-dependent transporter that accomplishes iron internalization. TbpB is a surface-exposed lipoprotein that makes the iron uptake process more efficient. Previous studies have shown that the genes encoding these proteins are arranged in a bicistronic operon, with the tbpB gene located upstream of tbpA and separated from it by an inverted repeat. The operon is under the control of the ferric uptake regulator (Fur); however, promoter elements necessary for regulated expression of the genes have not been experimentally defined. In this study, putative regulatory motifs were identified and confirmed by mutagenesis. Further examination of the sequence upstream of these promoter/operator motifs led to the identification of several novel repeats. We hypothesized that these repeats are involved in additional regulation of the operon. Insertional mutagenesis of regions upstream of the characterized promoter region resulted in decreased tbpB and tbpA transcript levels but increased protein levels for both TbpA and TbpB. Using RNA sequencing (RNA-Seq) technology, we determined that a long RNA was produced from the region upstream of tbpB. We localized the 5' endpoint of this transcript to between the two upstream insertions by qualitative RT-PCR. We propose that expression of this upstream RNA leads to optimized expression of the gene products from within the tbpBA operon.

Cross-protective efficacy of recombinant transferrin-binding protein A of Haemophilus parasuis in guinea pigs.

The causative agent of Glasser's disease in swine is Haemophilus parasuis. Commercial bacterins are widely used for protection of the swine population. However, cross protection is limited because H. parasuis has more than 15 serovars. Transferrin-binding protein A has shown potential as a broad-spectrum vaccine candidate against homologous and heterologous strains. Here we amplified the full-length tbpA gene from an H. parasuis serovar 13 isolate and cloned it into a pET-SUMO expression vector. We then expressed and purified the TbpA protein by Ni affinity chromatography. First, the immunogenicity and protective efficacy of the protein were evaluated in guinea pigs by two subcutaneous immunizations with different doses of Montanide IMS 206 VG adjuvant. The immunized guinea pigs were, respectively, challenged on week 3 after a booster immunization with homologous strain LJ3 (serovar 13) and heterologous strain FX1 (serovar 4), and vaccine-inoculated groups were compared with nonvaccinated controls. All immunized groups showed serum antibody titers higher than those of negative-control groups. Furthermore, the cytokine and chemokine levels were evaluated at the transcriptional level by the real-time PCR analysis of six cytokines and chemokines. Gamma interferon and interleukin-5 in groups immunized with 100 µg were elevated more than 15-fold over those in negative-control groups. The protection rates were 80 and 60% after a challenge with strains LJ3 and FX1, respectively, in the groups vaccinated with 100 µg of recombinant TbpA protein. Subsequently, the data showed that guinea pigs immunized with a single dose (100 µg) were protected at levels of 80, 80, and 60% against LJ3, FX1, and another heterologous strain, SZ (serovar 14), respectively. The results indicate for the first time that TbpA protein cross protects guinea pigs against serovars 13, 4, and 14 of H. parasuis. Taken together, these results suggest that the recombinant TbpA protein is a promising vaccine candidate that needs to be confirmed in a swine population.

Evidence of Fe3+ interaction with the plug domain of the outer membrane transferrin receptor protein of Neisseria gonorrhoeae: implications for Fe transport.

Neisseria gonorrhoeae is an obligate pathogen that hijacks iron from the human iron transport protein, holo-transferrin (Fe(2)-Tf), by expressing TonB-dependent outer membrane receptor proteins, TbpA and TbpB. Homologous to other TonB-dependent outer membrane transporters, TbpA is thought to consist of a ß-barrel with an N-terminal plug domain. Previous reports by our laboratories show that the sequence EIEYE in the plug domain is highly conserved among various bacterial species that express TbpA and plays a crucial role in iron utilization for gonococci. We hypothesize that this highly conserved EIEYE sequence in the TbpA plug, rich in hard oxygen donor groups, binds with Fe(3+) through the transport process across the outer membrane through the ß-barrel. Sequestration of Fe(3+) by the TbpA-plug supports the paradigm that the ferric iron must always remain chelated and controlled throughout the transport process. In order to test this hypothesis here we describe the ability of both the recombinant wild-type plug, and three small peptides that encompass the sequence EIEYE of the plug, to bind Fe(3+). This is the first report of the expression/isolation of the recombinant wild-type TbpA plug. Although CD and SUPREX spectroscopies suggest that a non-native structure is observed for the recombinant plug, fluorescence quenching titrations indicate that the wild-type recombinant TbpA plug binds Fe (3+) with a conditional log K(d) = 7 at pH 7.5, with no evidence of binding at pH 6.3. A recombinant TbpA plug with mutated sequence (NEIEYEN → NEIAAAN) shows no evidence of Fe(3+) binding under our experimental set up. Interestingly, in silico modeling with the wild-type plug also predicts a flexible loop structure for the EIEYE sequence under native conditions which once again supports the Fe(3+) binding hypothesis. These in vitro observations are consistent with the hypothesis that the EIEYE sequence in the wild-type TbpA plug binds Fe(3+) during the outer membrane transport process in vivo.

Anchor peptide of transferrin-binding protein B is required for interaction with transferrin-binding protein A.

Gram-negative bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae, and Neisseriaceae families rely on an iron acquisition system that acquires iron directly from host transferrin (Tf). The process is mediated by a surface receptor composed of transferrin-binding proteins A and B (TbpA and TbpB). TbpA is an integral outer membrane protein that functions as a gated channel for the passage of iron into the periplasm. TbpB is a surface-exposed lipoprotein that facilitates the iron uptake process. In this study, we demonstrate that the region encompassing amino acids 7-40 of Actinobacillus pleuropneumoniae TbpB is required for forming a complex with TbpA and that the formation of the complex requires the presence of porcine Tf. These results are consistent with a model in which TbpB is responsible for the initial capture of iron-loaded Tf and subsequently interacts with TbpA through the anchor peptide. We propose that TonB binding to TbpA initiates the formation of the TbpB-TbpA complex and transfer of Tf to TbpA.

Development and characterization of protective Haemophilus parasuis subunit vaccines based on native proteins with affinity to porcine transferrin and comparison with other subunit and commercial vaccines.

Haemophilus parasuis is the agent responsible for causing Glässer's disease, which is characterized by fibrinous polyserositis, polyarthritis, and meningitis in pigs. In this study, we have characterized native outer membrane proteins with affinity to porcine transferrin (NPAPT) from H. parasuis serovar 5, Nagasaki strain. This pool of proteins was used as antigen to developed two vaccine formulations: one was adjuvanted with a mineral oil (Montanide IMS 2215 VG PR), while the other was potentiated with a bacterial neuraminidase from Clostridium perfringens. The potential protective effect conferred by these two vaccines was compared to that afforded by two other vaccines, consisting of recombinant transferrin-binding protein (rTbp) A or B fragments from H. parasuis, Nagasaki strain, and by a commercially available inactivated vaccine. Five groups of colostrum-deprived piglets immunized with the vaccines described above, one group per each vaccine, and a group of nonvaccinated control animals were challenged intratracheally with a lethal dose (3 × 108 CFU) of H. parasuis, Nagasaki strain. The two vaccines containing rTbps yielded similar results with minimal protection against death, clinical signs, gross and microscopic lesions, and H. parasuis invasion. In contrast, the two vaccines composed of NPAPT antigen and commercial bacterin resulted in a strong protection against challenge (without deaths and clinical signs), mild histopathological changes, and no recovery of H. parasuis, thus suggesting their effectiveness in preventing Glässer's disease outbreaks caused by serovar 5.

Evidence for a common gene pool and frequent recombinational exchange of the tbpBA operon in Mannheimia haemolytica, Mannheimia glucosida and Bibersteinia trehalosi.

The tbpBA operon was sequenced in 42 representative isolates of Mannheimia haemolytica (32), Mannheimia glucosida (6) and Bibersteinia trehalosi (4). A total of 27 tbpB and 20 tbpA alleles were identified whilst the tbpBA operon was represented by 28 unique alleles that could be assigned to seven classes. There were 1566 (34.8% variation) polymorphic nucleotide sites and 482 (32.1% variation) variable inferred amino acid positions among the 42 tbpBA sequences. The tbpBA operons of serotype A2 M. haemolytica isolates are, with one exception, substantially more diverse than those of the other M. haemolytica serotypes and most likely have a different ancestral origin. The tbpBA phylogeny has been severely disrupted by numerous small- and large-scale intragenic recombination events. In addition, assortative (entire gene) recombination events, involving either the entire tbpBA operon or the individual tbpB and tbpA genes, have played a major role in shaping tbpBA structure and it's distribution in the three species. Our findings indicate that a common gene pool exists for tbpBA in M. haemolytica, M. glucosida and B. trehalosi. In particular, B. trehalosi, M. glucosida and ovine M. haemolytica isolates share a large portion of the tbpA gene, and this probably reflects selection for a conserved TbpA protein that provides effective iron uptake in sheep. Bovine and ovine serotype A2 lineages have very different tbpBA alleles. Bovine-like tbpBA alleles have been partially, or completely, replaced by ovine-like tbpBA alleles in ovine serotype A2 isolates, suggesting that different transferrin receptors are required by serotype A2 isolates for optimum iron uptake in cattle and sheep. Conversely, the tbpBA alleles of bovine-pathogenic serotype A1 and A6 isolates are very similar to those of closely related ovine isolates, suggesting a recent and common evolutionary origin.

Characterization of a recombinant transferrin-binding protein A (TbpA) fragment from Haemophilus parasuis serovar 5.

Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, possesses iron acquisition pathways mediated by a surface receptor that specifically bind porcine transferrin. This receptor is composed of transferrin-binding protein A (TbpA) and TbpB. As it has been reported for other gram-negative organisms, H. parasuis TbpA could be useful as a candidate target for H. parasuis vaccination. In this study, a 600-bp tbpA fragment of the gene encoding TbpA from H. parasuis serovar 5, the Nagasaki strain, was amplified by PCR and cloned into a pBAD/Thio-TOPO expression vector, generating the pBAD-Thio-TbpA-V5-His (TbpA-His) construction. Escherichia coli LMG194-competent cells were transformed with this construction, followed by the induction of protein expression with arabinose. A band (38.5 kDa) corresponding to a 200-amino acid recombinant TbpA (rTbpA) fragment was seen on the sodium dodecyl sulfate polyacrylamide gel electrophoresis and confirmed by immunoblotting. Polyclonal antibodies raised against this fragment were specific for H. parasuis and Actinobacillus pleuropneumoniae, reacted at the cell surface with H. parasuis, and a significant bactericidal activity was also detected. Therefore, this rTbpA fragment induces an immunological response and might be useful as an antigen for vaccination against Glässer's disease.

Kinetic analysis of ligand interaction with the gonococcal transferrin-iron acquisition system.

The transferrin iron acquisition system of Neisseria gonorrhoeae consists of two dissimilar transferrin binding proteins (Tbp) A and B. TbpA is a TonB dependent transporter while TbpB is a lipoprotein that makes iron acquisition from transferrin (Tf) more efficient. In an attempt to further define the individual roles of these receptors in the process of Tf-iron acquisition, the kinetics of the receptor proteins in regards to ligand association and dissociation were evaluated. Tf association with TbpB was rapid as compared to TbpA. Tf dissociation from the wild-type receptor occurred in a biphasic manner; an initial rapid release was followed by a slower dissociation over time. Both TbpA and TbpB demonstrated a two-phase release pattern; however, TbpA required both TonB and TbpB for efficient Tf dissociation from the cell surface. The roles of TbpA and TbpB in Tf dissociation were further examined, utilizing previously created HA fusion proteins. Using a Tf-utilization deficient TbpA-HA mutant, we concluded that the slower rate of ligand dissociation demonstrated by the wild-type transporter was a function of successful iron internalization. Insertion into the C-terminus of TbpB decreased the rate of Tf dissociation, while insertion into the N-terminus had no effect on this process. From these studies, we propose that TbpA and TbpB function synergistically during the process of Tf iron acquisition and that TbpB makes the process of Tf-iron acquisition more efficient at least in part by affecting association and dissociation of Tf from the cell surface.

Identification of TbpA residues required for transferrin-iron utilization by Neisseria gonorrhoeae.

Neisseria gonorrhoeae requires iron for survival in the human host and therefore expresses high-affinity receptors for iron acquisition from host iron-binding proteins. The gonococcal transferrin-iron uptake system is composed of two transferrin binding proteins, TbpA and TbpB. TbpA is a TonB-dependent, outer membrane transporter critical for iron acquisition, while TbpB is a surface-exposed lipoprotein that increases the efficiency of iron uptake. The precise mechanism by which TbpA mediates iron acquisition has not been elucidated; however, the process is distinct from those of characterized siderophore transporters. Similar to these TonB-dependent transporters, TbpA is proposed to have two distinct domains, a beta-barrel and a plug domain. We hypothesize that the TbpA plug coordinates iron and therefore potentially functions in multiple steps of transferrin-mediated iron acquisition. To test this hypothesis, we targeted a conserved motif within the TbpA plug domain and generated single, double, and triple alanine substitution mutants. Mutagenized TbpAs were expressed on the gonococcal cell surface and maintained wild-type transferrin binding affinity. Single alanine substitution mutants internalized iron at wild-type levels, while the double and triple mutants showed a significant decrease in iron uptake. Moreover, the triple alanine substitution mutant was unable to grow on transferrin as a sole iron source; however, expression of TbpB compensated for this defect. These data indicate that the conserved motif between residues 120 and 122 of the TbpA plug domain is critical for transferrin-iron utilization, suggesting that this region plays a role in iron acquisition that is shared by both TbpA and TbpB.

Gonococcal transferrin binding protein chimeras induce bactericidal and growth inhibitory antibodies in mice.

We have previously demonstrated the full-length gonococcal transferrin binding proteins (TbpA and TbpB) to be promising antigens in the development of a protective vaccine against Neisseria gonorrhoeae. In the current study we employed a genetic chimera approach fusing domains from TbpA and TbpB to the A2 domain of cholera toxin, which naturally binds in a non-covalent fashion to the B subunit of cholera toxin during assembly. For one construct, the N-terminal half of TbpB (NB) was fused to the A2 subunit of cholera toxin. In a second construct, the loop 2 region (L2) of TbpA was genetically fused between the NB domain and the A2 domain, generating a double chimera. Both chimeras were immunogenic and induced serum bactericidal and vaginal growth-inhibiting antibodies. This study highlights the potential of using protective epitopes instead of full-length proteins in the development of an efficacious gonococcal vaccine.

Cloning and expression of genes encoding transferrin-binding protein A and B from Actinobacillus pleuropneumoniae serotype 5.

Actinobacillus pleuropneumoniae is an important primary pathogen in pigs, which causes a highly contagious pleuropneumonia. As an adaptation to the iron-restricted environment of the host, A. pleuropneumoniae possesses iron acquisition pathways mediated by surface receptors that specifically bind transferrin from the host. The receptor is composed of two receptor proteins, transferrin-binding protein A and B (TbpA and B), which are both capable of binding to transferrin. An impairment of iron uptake mechanisms is likely to reduce virulence. For this reason, these two proteins can be useful as a candidate target for A. pleuropneumoniae vaccination. To do this, genes encoding the TbpA and B from a serotype 5 isolate of A. pleuropneumoniae were amplified from genomic DNA template by PCR and cloned into a pRSET prokaryotic expression vector, generating the pRSET-A.pp-TbpA and B. Escherichia coli BL21(DE3)pLysS competent cells were transformed with each construct followed by the induction of protein expression by the addition of IPTG. Bands corresponding to the predicted sizes (110 and 60 kDa) were seen on the SDS-PAGE. Polyclonal antibodies raised against recombinant TbpA and B from mice were reacted with bacterial proteins. This result indicates that the recombinant proteins can induce immunological responses and might be useful as candidate targets for A. pleuropneumoniae vaccination.