Cytokine expression by CD163+ monocytes in healthy and Actinobacillus pleuropneumoniae-infected pigs.

Distinct monocyte subpopulations have been previously described in healthy pigs and pigs experimentally infected with Actinobacillus pleuropneumoniae (APP). The CD163+ subpopulation of bone marrow (BM), peripheral blood (PB) and lung monocytes was found to play an important role in the inflammatory process. The inflammation is accompanied by elevation of inflammatory cytokines. The aim of the study was to evaluate the contribution of CD163+ monocytes and macrophages to cytokine production during APP-induced lung inflammation. Cytokine production was assessed by flow cytometry (FC) and quantitative PCR (qPCR) in CD163+ monocytes and by qPCR, immunohistochemistry/fluorescence in lungs and tracheobronchial lymph nodes (TBLN). Despite the systemic inflammatory response after APP infection, BM and PB CD163+ monocytes did not express elevated levels of a wide range of cytokines compared to control pigs. In contrast, significant amounts of IL-1ß, IL-6, IL-8 and TNF-α were produced in lung lesions and IL-1ß in the TBLN. At the protein level, TNF-α was expressed by both CD163+ monocytes and macrophages in lung lesions, whereas IL-1ß, IL-6 and IL-8 expression was found only in CD163+ monocytes; no CD163+ macrophages were found to produce these cytokines. Furthermore, the quantification of CD163+ monocytes expressing the two cytokines IL-1ß and IL-8 that were most elevated was performed. In lung lesions, 36.5% IL-1ß positive CD163+ monocytes but only 18.3% IL-8 positive CD163+ monocytes were found. In conclusion, PB and BM CD163+ monocytes do not appear to contribute to the elevated cytokine levels in plasma. On the other hand, CD163+ monocytes contribute to inflammatory cytokine expression, especially IL-1ß at the site of inflammation during the inflammatory process.

Effects of Actinobacillus pleuropneumoniae on barrier function and inflammatory response of pig tracheal epithelial cells.

Actinobacillus pleuropneumoniae is a respiratory pathogen that causes porcine pleuropneumonia, a fatal respiratory disease responsible for high economic losses in the swine industry worldwide. With the objective to better understand the interactions between A. pleuropneumoniae and the porcine respiratory epithelium, we investigated the capacity of this pathogen to damage the epithelial barrier and induce an inflammatory response. We showed that A. pleuropneumoniae, even at a multiplicity of infection of 10, is able to break the tracheal epithelial barrier integrity as determined by monitoring the transepithelial electrical resistance and fluorescein-isothiocyanate-dextran transport. Immunofluorescence staining analysis suggested that A. pleuropneumoniae is affecting two important tight junction proteins (occludin, zonula occludens-1). As a consequence of the breakdown of the epithelial barrier integrity, A. pleuropneumoniae can translocate across a cell monolayer. We also showed that tracheal epithelial cells secrete pro-inflammatory cytokines (IL-8, IL-6, TNF-α) in response to a stimulation with this pathogen. In summary, A. pleuropneumoniae is able to induce damage to the porcine respiratory epithelial barrier. Challenging the epithelial cells with A. pleuropneumoniae was also associated with the secretion of pro-inflammatory cytokines. This better knowledge of the interactions between A. pleuropneumoniae and the epithelial cells may help to design novel strategies to prevent epithelium invasion by this bacterium along with other swine respiratory pathogens.

Activation of Porcine Alveolar Macrophages by Actinobacillus pleuropneumoniae Lipopolysaccharide via the Toll-Like Receptor 4/NF-κB-Mediated Pathway.

Actinobacillus pleuropneumoniae is the causative agent of porcine contagious pleuropneumonia. Overproduction of proinflammatory cytokines, like interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor alpha, and resistin, in the lung is an important feature of A. pleuropneumoniae infection. These proinflammatory cytokines enhance inflammatory and immunological responses. However, the mechanism that leads to cytokine production remains unclear. As a major virulence factor of A. pleuropneumoniae, lipopolysaccharide (LPS) may act as a potent stimulator of Toll-like receptor 4 (TLR4), triggering a number of intracellular signaling pathways that lead to the synthesis of proinflammatory cytokines. Porcine alveolar macrophages (PAMs) are the first line of defense against pathogenic microbes during pathogen invasion. The results of the present study demonstrate that A. pleuropneumoniae LPS induces PAMs to produce inflammatory cytokines in time- and dose-dependent manners. Moreover, PAMs were activated by A. pleuropneumoniae LPS, resulting in upregulation of signaling molecules, including TLR4, MyD88, TRIF-related adaptor molecule, and NF-κB. In contrast, the activation effects of A. pleuropneumoniae LPS on PAMs could be suppressed by specific inhibitors, like small interfering RNA and Bay11-7082. Taken together, our data indicate that A. pleuropneumoniae LPS can induce PAMs to produce proinflammatory cytokines via the TLR4/NF-κB-mediated pathway. These findings partially reveal the mechanism of the overproduction of proinflammatory cytokines in the lungs of swine with A. pleuropneumoniae infection and may provide targets for the prevention of A. pleuropneumoniae-induced pneumonia. All the data could be used as a reference for the pathogenesis of respiratory infection.

Role of (p)ppGpp in Viability and Biofilm Formation of Actinobacillus pleuropneumoniae S8.

Actinobacillus pleuropneumoniae is a Gram-negative bacterium and the cause of porcine pleuropneumonia. When the bacterium encounters nutritional starvation, the relA-dependent (p)ppGpp-mediated stringent response is activated. The modified nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) are known to be signaling molecules in other prokaryotes. Here, to investigate the role of (p)ppGpp in A. pleuropneumoniae, we created a mutant A. pleuropneumoniae strain, S8ΔrelA, which lacks the (p)ppGpp-synthesizing enzyme RelA, and investigated its phenotype in vitro. S8ΔrelA did not survive after stationary phase (starvation condition) and grew exclusively as non-extended cells. Compared to the wild-type (WT) strain, the S8ΔrelA mutant had an increased ability to form a biofilm. Transcriptional profiles of early stationary phase cultures revealed that a total of 405 bacterial genes were differentially expressed (including 380 up-regulated and 25 down-regulated genes) in S8ΔrelA as compared with the WT strain. Most of the up-regulated genes are involved in ribosomal structure and biogenesis, amino acid transport and metabolism, translation cell wall/membrane/envelope biogenesis. The data indicate that (p)ppGpp coordinates the growth, viability, morphology, biofilm formation and metabolic ability of A. pleuropneumoniae in starvation conditions. Furthermore, S8ΔrelA could not use certain sugars nor produce urease which has been associated with the virulence of A. pleuropneumoniae, suggesting that (p)ppGpp may directly or indirectly affect the pathogenesis of A. pleuropneumoniae during the infection process. In summary, (p)ppGpp signaling represents an essential component of the regulatory network governing stress adaptation and virulence in A. pleuropneumoniae.

[TLR-4 involvement in pyroptosis of mice with pulmonary inflammation infected by Actinobacillus pleuropneumoniae].

OBJECTIVE: Pyroptosis is a caspase-1 dependent programmed cell death and involves pathogenesis of infectious diseases by releasing many pro-inflammatory cytokines to induced inflammation. TLR-4 plays an important role in mediating pathogenesis of some infectious diseases. In this study, we detected the expression of TLR-4 and some molecules (e. g caspase-1, TNF-α, IL-1ß, IL-6, IL-18 ) related with pyroptosis to determine its involvement and mechanisms of pulmonary inflammation in mice infected by A. pleuropneumoniae. METHODS: Mice were intranasally infected by A. pleuropneumoniae and killed 48 hours post infection. Pulmonary gross lesion and histological pathology by H-E were observed. Expression levels of caspase-1 , caspase-3, TNF-α, IL-1ß, IL-6, IL-18, and TLR-4 in lung of mice were detected by RT-PCR and qPCR. RESULTS: Serious pulmonary hemorrhage and inflammation in infected mice were observed. Expression levels of caspase-1, caspase-3, TNF-α, IL-1ß, IL-6, IL-18 and TLR-4 increased, and expression levels of caspase-3 were not changed in lung of infected mice. CONCLUSION: TLR-4 might be involved in pulmonary inflammation of mice infected by A. pleuropneumoniae. After induced by activated TLR-4 some cells in this lesion expressed pro-inflammatory cytokines. These cytokines would induce pulmonary inflammation. This lesion might involve pyroptosis with caspase-1 expression.

Macrophages largely contribute to heterologous anti-Propionibacterium acnes antibody-mediated protection from Actinobacillus pleuropneumoniae infection in mice.

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

Acute and subacute response of iron, zinc, copper and selenium in pigs experimentally infected with Actinobacillus pleuropneumoniae.

This study was performed to characterise the response of iron (Fe), zinc (Zn), copper (Cu) and selenium (Se) in bacterial-induced porcine acute phase reaction (APR). Twenty piglets were challenged by aerosolic infection with Actinobacillus pleuropneumoniae (A.pp.) serotype 2, ten piglets serving as controls. Blood sampling was done initially and at day 4 and 21 after infection, collection of liver tissue was done at day 21 (autopsy). A.pp.-infection caused fever and respiratory symptoms. APR at day 4 after infection was marked by an increase in total white blood cells, granulocytes and monocytes in whole blood samples and an increase in globulin/albumin ratio (G/A), α2-globulins, C-reactive protein, haptoglobin, ceruloplasmin (Cp), Cu and Se in serum. Concurrently, there was a decrease in haemoglobin (Hb) and packed cell volume (PCV) in whole blood as well as a decrease in albumin, transferrin, total iron binding capacity and Fe in serum and Zn in plasma. The subacute stage at day 21 was characterised by progressively increased concentrations of G/A, ß-globulins and γ-globulins reflecting the specific immune reaction. Hb and PCV showed further decreases, all other parameters returned to the initial concentrations. Glutathione peroxidase activity in plasma and liver tissue remained unaffected by A.pp.-infection. The liver concentration (day 21) of Zn was found to be higher, that of Se was lower in the A.pp.-group, whereas hepatic concentrations of Cu and Fe were not affected by A.pp.-infection. In summary, the acute and subacute stages of A.pp.-infection were accurately characterised by the APR-related parameters. Se was only marginally affected by the A.pp.-infection. The elevated plasma Cu concentration may be a side effect of the transient hepatic induction of Cp synthesis. Zn responded, being distinctly reduced in plasma and probably having been sequestered in the liver tissue. Reduction in serum Fe can be regarded as an unspecific defence mechanism in A.pp.-infection to withdraw Fe from bacterial acquisition systems.

The RNA chaperone Hfq promotes fitness of Actinobacillus pleuropneumoniae during porcine pleuropneumonia.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, an economically important disease of pigs. The hfq gene in A. pleuropneumoniae, encoding the RNA chaperone and posttranscriptional regulator Hfq, is upregulated during infection of porcine lungs. To investigate the role of this in vivo-induced gene in A. pleuropneumoniae, an hfq mutant strain was constructed. The hfq mutant was defective in biofilm formation on abiotic surfaces. The level of pgaC transcript, encoding the biosynthesis of poly-ß-1,6-N-acetylglucosamine (PNAG), a major biofilm matrix component, was lower and PNAG content was 10-fold lower in the hfq mutant than in the wild-type strain. When outer membrane proteins were examined, cysteine synthase, implicated in resistance to oxidative stress and tellurite, was not found at detectable levels in the absence of Hfq. The hfq mutant displayed enhanced sensitivity to superoxide generated by methyl viologen and tellurite. These phenotypes were readily reversed by complementation with the hfq gene expressed from its native promoter. The role of Hfq in the fitness of A. pleuropneumoniae was assessed in a natural host infection model. The hfq mutant failed to colonize porcine lungs and was outcompeted by the wild-type strain (median competitive index of 2 × 10(-5)). Our data demonstrate that the in vivo-induced gene hfq is involved in the regulation of PNAG-dependent biofilm formation, resistance to superoxide stress, and the fitness and virulence of A. pleuropneumoniae in pigs and begin to elucidate the role of an in vivo-induced gene in the pathogenesis of pleuropneumonia.

Apa is a trimeric autotransporter adhesin of Actinobacillus pleuropneumoniae responsible for autoagglutination and host cell adherence.

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

Mechanisms underlying Actinobacillus pleuropneumoniae exotoxin ApxI induced expression of IL-1ß, IL-8 and TNF-α in porcine alveolar macrophages.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes fibrino-hemorrhagic necrotizing pleuropneumonia in pigs. Production of proinflammatory mediators in the lungs is an important feature of A. pleuropneumoniae infection. However, bacterial components other than lipopolysaccharide involved in this process remain unidentified. The goals of this study were to determine the role of A. pleuropneumoniae exotoxin ApxI in cytokine induction and to delineate the underlying mechanisms. Using real-time quantitative PCR analysis, we found native ApxI stimulated porcine alveolar macrophages (PAMs) to transcribe mRNAs of IL-1ß, IL-8 and TNF-α in a concentration- and time-dependent manner. Heat-inactivation or pre-incubation of ApxI with a neutralizing antiserum attenuated ApxI bioactivity to induce cytokine gene expression. The secretion of IL-1ß, IL-8 and TNF-α protein from PAMs stimulated with ApxI was also confirmed by quantitative ELISA. In delineating the underlying signaling pathways contributing to cytokine expression, we observed mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK) were activated upon ApxI stimulation. Administration of an inhibitor specific to p38 or JNK resulted in varying degrees of attenuation on ApxI-induced cytokine expression, suggesting the differential regulatory roles of p38 and JNK in IL-1ß, IL-8 and TNF-α production. Further, pre-incubation of PAMs with a CD18-blocking antibody prior to ApxI stimulation significantly reduced the activation of p38 and JNK, and subsequent expression of IL-1ß, IL-8 or TNF-α gene, indicating a pivotal role of ß2 integrins in the ApxI-mediated effect. Collectively, this study demonstrated ApxI induces gene expression of IL-1ß, IL-8 and TNF-α in PAMs that involves ß2 integrins and downstream MAPKs.

Analysis on Actinobacillus pleuropneumoniae LuxS regulated genes reveals pleiotropic roles of LuxS/AI-2 on biofilm formation, adhesion ability and iron metabolism.

LuxS is an enzyme involved in the activated methyl cycle and the by-product autoinducer-2 (AI-2) was a quorum sensing signal in some species. In our previous study, the functional LuxS in AI-2 production was verified in the porcine respiratory pathogen Actinobacillus pleuropneumoniae. Enhanced biofilm formation and reduced virulence were observed in the luxS mutant. To comprehensively understand the luxS function, in this study, the transcriptional profiles were compared between the A. pleuropneumoniae luxS mutant and its parental strain in four different growth phases using microarray. Many genes associated with infection were differentially expressed. The biofilm formation genes pgaABC in the luxS mutant were up-regulated in early exponential phase, while 9 genes associated with adhesion were down-regulated in late exponential phase. A group of genes involved in iron acquisition and metabolism were regulated in four growth phases. Phenotypic investigations using luxS mutant and both genetic and chemical (AI-2) complementation on these virulence traits were performed. The results demonstrated that the luxS mutant showed enhanced biofilm formation and reduced adhesion ability and these effects were not due to lack of AI-2. But AI-2 could increase biofilm formation and adhesion of A. pleuropneumoniae independent of LuxS. Growth under iron restricted condition could be controlled by LuxS through AI-2 production. These results revealed pleiotropic roles of LuxS and AI-2 on A. pleuropneumoniae virulence traits.

Analysis of the Actinobacillus pleuropneumoniae HlyX (FNR) regulon and identification of iron-regulated protein B as an essential virulence factor.

The Gram-negative rod Actinobacillus pleuropneumoniae is a facultative anaerobic pathogen of the porcine respiratory tract, and HlyX, the A. pleuropneumoniae homologue of fumarate and nitrate reduction regulator (FNR), has been shown to be important for persistence. An A. pleuropneumoniae hlyX deletion mutant has a decreased generation time but highly prolonged survival in comparison to its wild type parent strain when grown anaerobically in glucose-supplemented medium. Applying a combination of proteomic and transcriptomic approaches as well as in silico analyses, we identified 23 different proteins and 418 genes to be modulated by HlyX (> or = twofold up- or down-regulated). A putative HlyX-box was identified upstream of 54 of these genes implying direct control by HlyX. Consistent with its role as a strong positive regulator, HlyX induced the expression of genes for anaerobic metabolism encoding alternative terminal reductases and hydrogenases. In addition, expression of virulence-associated genes encoding iron uptake systems, a putative DNA adenine modification system, and an autotransporter serine protease were induced by HlyX under anaerobic growth conditions. With respect to virulence-associated genes, we focused on the iron-regulated protein B (FrpB) as it is the outer membrane protein most strongly up-regulated by HlyX. An frpB deletion mutant of A. pleuropneumoniae had the same growth characteristics as wild type grown aerobically and anaerobically. In contrast, A. pleuropneumoniae DeltafrpB did not cause any disease and could not be re-isolated from experimentally infected pigs, thereby identifying FrpB as a previously unknown virulence factor.

Host-pathogen interactions of Actinobacillus pleuropneumoniae with porcine lung and tracheal epithelial cells.

Host-pathogen interactions are of great importance in understanding the pathogenesis of infectious microorganisms. We developed in vitro models to study the host-pathogen interactions of porcine respiratory tract pathogens using two immortalized epithelial cell lines, namely, the newborn pig trachea (NPTr) and St. Jude porcine lung (SJPL) cell lines. We first studied the interactions of Actinobacillus pleuropneumoniae, an important swine pathogen, using these models. Under conditions where cytotoxicity was absent or low, we showed that A. pleuropneumoniae adheres to both cell lines, stimulating the induction of NF-kappaB. The NPTr cells consequently secrete interleukin 8, while the SJPL cells do not, since they are deprived of the NF-kappaB p65 subunit. Cell death ultimately occurs by necrosis, not apoptosis. The transcriptomic profile of A. pleuropneumoniae was determined after contact with the porcine lung epithelial cells by using DNA microarrays. Genes such as tadB and rcpA, members of a putative adhesin locus, and a gene whose product has high homology to the Hsf autotransporter adhesin of Haemophilus influenzae were upregulated, as were the genes pgaBC, involved in biofilm biosynthesis, while capsular polysaccharide-associated genes were downregulated. The in vitro models also proved to be efficient with other swine pathogens, such as Actinobacillus suis, Haemophilus parasuis, and Pasteurella multocida. Our results demonstrate that interactions of A. pleuropneumoniae with host epithelial cells seem to involve complex cross talk which results in regulation of various bacterial genes, including some coding for putative adhesins. Furthermore, our data demonstrate the potential of these in vitro models in studying the host-pathogen interactions of other porcine respiratory tract pathogens.

Hemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae: homology modelling reveals regions of potential interactions with hemoglobin and heme.

Analyses of the primary sequence of hemoglobin-binding protein HgbA from Actinobacillus pleuropneumoniae by comparative modelling and by a Hidden Markov Model identified its topological similarities to bacterial outer membrane receptors BtuB, FepA, FhuA, and FecA of Escherichia coli. The HgbA model has a globular N-terminal cork domain contained within a 22-stranded beta barrel domain, its folds being similar to the structures of outer membrane receptors that have been solved by X-ray crystallography. The barrel domain of the HgbA model superimposes onto the barrel domains of the four outer membrane receptors with rmsd values less than 1.0 A. This feature is consistent with a phylogenetic tree which indicated clustering of polypeptide sequences for three barrel domains. Furthermore, the HgbA model shares the highest structural similarity to BtuB, with the modelled HgbA barrel having approximately the same elliptical cross-section and height as that of BtuB. Extracellular loop regions of HgbA are predicted to be more extended than those of the E. coli outer membrane receptors, potentially facilitating a protein-protein interface with hemoglobin. Fold recognition modelling of the HgbA loop regions showed that 10 out of 11 predicted loops are highly homologous to known structures of protein loops that contribute to heme/iron or protein-protein interactions. Strikingly, HgbA loop 2 has structural homology to a loop in bovine endothelial nitric acid oxidase that is proximal to a heme-binding site; and HgbA loop 7 contains a histidine residue conserved in a motif that is involved in heme/hemoglobin interactions. These findings implicate HgbA loops 2 and 7 in recognition and binding of hemoglobin or the heme ligand.

Surface polysaccharides and iron-uptake systems of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the etiologic agent of porcine pleuropneumonia. Infection by A. pleuropneumoniae is a multifactorial process governed by many virulence factors acting alone or, more often, in concert to establish the pathogen in the porcine host. The aim of this short review is to present recent data concerning important surface molecules of A. pleuropneumoniae; namely, lipopolysaccharides, capsular polysaccharides, and a subset of outer membrane proteins involved in iron uptake.

Cloning and characterisation of type 4 fimbrial genes from Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumoniae. Little is known about the mechanisms by which A. pleuropneumoniae colonises the respiratory tract. Fimbriae are common mediators of bacterial adherence to mucosal epithelia and have been observed on the surface of A. pleuropneumoniae cells. Here we report the identification and characterisation of the type 4 fimbrial structural gene (apfA) from A. pleuropneumoniae. In addition a number of open reading frames were identified in A. pleuropneumoniae that have significant homology to type 4 fimbrial biogenesis genes from other species, including a putative leader specific peptidase (apfD). A. pleuropneumoniae apfA codes for a predicted polypeptide of approximately 16kDa, removal of the leader sequence at the predicted cleavage site would yield a 14.5kDa polypeptide. The first 30 residues of the mature polypeptide are well conserved with other members of the group A type 4 fimbriae family. The signal sequence of ApfA is 13 amino acids in length and, unusually, the residue that precedes the cleavage site is alanine rather than glycine which is found in most other type 4 fimbriae. The C-terminus of ApfA possesses cysteine residues that are conserved in type 4 fimbriae of many species. In other type 4 fimbriae the distal C-terminal cysteines form a disulphide bond that produces a loop, which is important for the function of fimbriae and also comprises a major antigenic determinant. A motif within the predicted loop in ApfA was found to be highly conserved in type 4 fimbriae of other HAP organisms (Haemophilus, Actinobacillus, Pasteurella). The A. pleuropneumoniae type 4 fimbrial biogenesis genes showed the strongest homology to putative type 4 fimbrial genes of Haemophilus ducreyi. A. pleuropneumoniae apfA gene was shown to be present and highly conserved in different serotypes of A. pleuropneumoniae. Recombinant ApfA was produced and used to raise anti-ApfA antisera.

Characterization of the in vitro adhesion of Actinobacillus pleuropneumoniae to swine alveolar epithelial cells.

Actinobacillus pleuropneumoniae biovar 1 serotypes 2, 5a, 9 and 10 strains were tested for their ability to adhere to alveolar epithelial cells in culture. For the serotypes 5a, 9 and 10 strains, optimal adherence was observed after growth of bacterial cells in a NAD-restricted medium (0.001% NAD). This condition was also associated with the expression of a 55 kDa outer membrane protein (OMP) and of fimbriae. For the serotype 2 strain, adherence and expression of fimbriae and a 55 kDa OMP was less influenced by the growth conditions. The N-terminal amino acid sequence of the 55 kDa OMP had no homology with any known sequence, suggesting that it is an as yet unknown protein. Adherence capabilities were significantly reduced following treatment of the bacteria with proteolytic enzymes or heat. These findings suggest that proteins are involved in adhesion. The hydrophobic bond-breaking agent tetramethylurea was unable to inhibit the adherence of A. pleuropneumoniae to alveolar epithelial cells. Treatment of the bacteria with sodium metaperiodate resulted in lower adhesion scores for the serotypes 2 and 9 strains but the inhibition of adhesion was clearly lower than after treatment with proteolytic enzymes. This indicates that, besides proteins, carbohydrates might also be involved in adhesion of A. pleuropneumoniae to alveolar epithelial cells. The finding that inhibition of adhesion was very high when bacteria were treated with a combination of sodium metaperiodate and pronase also suggests that more than one adhesin is involved.

Association of the RTX proteins of Actinobacillus pleuropneumoniae with hemolytic, CAMP, and neutrophil-cytotoxic activities.

The immunoglobulin G from a monospecific rabbit antiserum to the 110-kDa RTX hemolysin of Actinobacillus pleuropneumoniae serotype 1 was used to determine that the related RTX proteins in isolates from serotypes 2 to 12 were also responsible for the hemolytic, CAMP, and neutrophil-cytotoxic activities produced by this bacterium. These proteins share common neutralizing epitopes.