IFN-γ-/- Mice Resist Actinobacillus pleuropneumoniae Infection by Promoting Early Lung IL-18 Release and PMN-I Accumulation.

Porcine pleuropneumonia is a common infectious disease of pigs caused by Actinobacillus pleuropneumoniae Interferon gamma (IFN-γ) expression increases in the lung of pigs after A. pleuropneumoniae infection, but the role of IFN-γ during the infection is still obscure. In this study, an IFN-γ-/- mouse infection model was established, and bacterial load, levels of inflammatory cytokines, and types of neutrophils in the lungs were studied at different times post-A. pleuropneumoniae infection. We found that wild-type (WT) mice were more susceptible to A. pleuropneumoniae than IFN-γ-/- mice. At 6 h postinfection (hpi), the expression of interleukin 18 (IL-18) and IL-1ß in the lungs of IFN-γ-/- mice was significantly increased compared to WT mice. The bacterial load and levels of inflammatory cytokines (IL-1ß and IL-6) of IFN-γ-/- mice were significantly reduced at 12 hpi compared to WT mice. After an initial loss, the numbers of lung polymorphonuclear (PMN)-I cells dramatically increased in the lungs of IFN-γ-/- but not WT mice, whereas PMN-II cells continually decreased. Finally, in vivo administration of IL-18 significantly reduced clinical scores and bacterial load in the lungs of A. pleuropneumoniae-infected mice. This study identifies IFN-γ as a target for regulating the inflammatory response in the lung and provides a basis for understanding the course of clinical bacterial pneumonia and for the formulation of treatment protocols.

iTRAQ-based quantitative proteomic analysis of peripheral blood serum in piglets infected with Actinobacillus pleuropneumoniae.

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae (APP) is a swine respiratory disease with an important impact around the world either as a single infection or part of the porcine respiratory disease complex. The data of interaction between hosts and pathogens has becoming more crucial for exploration of the mechanism. However, up to now, comparatively little information is available on the systemic and dynamic changes that occur in pig serum in response to APP infection. This study used iTRAQ to identify differentially expressed proteins (DEPs) in pig serum in response to APP infection. Compared with the APP un-infected group (S0),there were 137 up-regulated and 68 down-regulated proteins at 24 h (S24), and 81 up-regulated and 107 down-regulated proteins at 120 h (S120). At 24 h, the immune response was not significantly enriched, but cell adhesion, cytosol, Golgi apparatus, GTP and ATP binding and regulation of cell cycle were extremely active, implying host preparation of immune response starting. Subsequently, innate immune response, negative regulation of apoptotic process, immunological synapse, adaptive immune response, the regulation of inflammatory response, positive regulation of T cell proliferation were more enhanced at 120 h then that of 24 h, representing innate immunity transferring to the adaptive, while endocytosis, cell adhesion and platelet aggregation showed obvious decline. The pathways of T cell receptor signaling pathway, cytokine-cytokine receptor interaction, complement and coagulation cascades, leukocyte transendothelial migration were active remarkably during all infection period, and more pathways could connect to form innate immune defense networks. Surprisingly, the pathways like amoebiasis, rheumatoid arthritis and malaria had been found up-regulated. As a conclusion, APP could delay host inflammatory response to the infection at early stage, and induced innate immunity to convert from adhesion, interaction into complement activation, proteasome digestion, bacterial invasion at later stage. This would increase our understanding of the porcine distinct response to APP infection.

Update on Actinobacillus pleuropneumoniae-knowledge, gaps and challenges.

Porcine pleuropneumonia, caused by the bacterial porcine respiratory tract pathogen Actinobacillus pleuropneumoniae, leads to high economic losses in affected swine herds in most countries of the world. Pigs affected by peracute and acute disease suffer from severe respiratory distress with high lethality. The agent was first described in 1957 and, since then, knowledge about the pathogen itself, and its interactions with the host, has increased continuously. This is, in part, due to the fact that experimental infections can be studied in the natural host. However, the fact that most commercial pigs are colonized by this pathogen has hampered the applicability of knowledge gained under experimental conditions. In addition, several factors are involved in development of disease, and these have often been studied individually. In a DISCONTOOLS initiative, members from science, industry and clinics exchanged their expertise and empirical observations and identified the major gaps in knowledge. This review sums up published results and expert opinions, within the fields of pathogenesis, epidemiology, transmission, immune response to infection, as well as the main means of prevention, detection and control. The gaps that still remain to be filled are highlighted, and present as well as future challenges in the control of this disease are addressed.

Apa2H1, the first head domain of Apa2 trimeric autotransporter adhesin, activates mouse bone marrow-derived dendritic cells and immunization with Apa2H1 protects against Actinobacillus pleuropneumoniae infection.

Actinobacillus pleuropneumoniae is the causative pathogen of porcine pleuropneumonia, which results in large economic losses in the pig industry worldwide. There are, however, no effective subunit vaccines are available in the market owing to the various serotypes and the absence of cross-protection against this pathogen. Therefore, the selection of protective components is of great significance for vaccine development. We previously showed that trimeric autotransporter adhesins are important virulence factors of A. pleuropneumoniae. To determine the potential role in vaccine development of the functional head domain (Apa2H1) of Apa2, a trimeric autotransporter adhesin found in A. pleuropneumoniae, we obtained nature-like trimeric Apa2H1 using a prokaryotic expression system and co-culture of Apa2H1 with bone marrow derived dendritic cells (BMDCs) in vitro resulted in maturation of BMDCs, characterised by the up-regulation of CD83, MHC-II, CCR7, ICAM-I and the increased expression of factors related to B lymphoid cells stimulation, such as proliferation-inducing ligand (APRIL), B lymphocyte stimulator (BLyS) and B cell activating factor (BAFF). The in vivo results showed that vaccination with Apa2H1 resulted in the robust production of antigen-specific antibodies, modestly induced mixed Th1 and Th2 immunity, impaired bacterial colonization and dissemination, and improved mouse survival rates. This study is the first to show that Apa2H1 is antigenic and can be used as a component of a subunit vaccine against A. pleuropneumoniae infection, providing valuable reference material for the development of an effective vaccine against A. pleuropneumoniae.

Surface Polysaccharide Mutants Reveal that Absence of O Antigen Reduces Biofilm Formation of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an O-antigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR, and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA.

Trimeric autotransporter adhesins contribute to Actinobacillus pleuropneumoniae pathogenicity in mice and regulate bacterial gene expression during interactions between bacteria and porcine primary alveolar macrophages.

Actinobacillus pleuropneumoniae is an important pathogen that causes respiratory disease in pigs. Trimeric autotransporter adhesin (TAA) is a recently discovered bacterial virulence factor that mediates bacterial adhesion and colonization. Two TAA coding genes have been found in the genome of A. pleuropneumoniae strain 5b L20, but whether they contribute to bacterial pathogenicity is unclear. In this study, we used homologous recombination to construct a double-gene deletion mutant, ΔTAA, in which both TAA coding genes were deleted and used it in in vivo and in vitro studies to confirm that TAAs participate in bacterial auto-aggregation, biofilm formation, cell adhesion and virulence in mice. A microarray analysis was used to determine whether TAAs can regulate other A. pleuropneumoniae genes during interactions with porcine primary alveolar macrophages. The results showed that deletion of both TAA coding genes up-regulated 36 genes, including ene1514, hofB and tbpB2, and simultaneously down-regulated 36 genes, including lgt, murF and ftsY. These data illustrate that TAAs help to maintain full bacterial virulence both directly, through their bioactivity, and indirectly by regulating the bacterial type II and IV secretion systems and regulating the synthesis or secretion of virulence factors. This study not only enhances our understanding of the role of TAAs but also has significance for those studying A. pleuropneumoniae pathogenesis.

The Adh adhesin domain is required for trimeric autotransporter Apa1-mediated Actinobacillus pleuropneumoniae adhesion, autoaggregation, biofilm formation and pathogenicity.

Actinobacillus pleuropneumoniae is a causative agent of porcine pleuropneumonia, which is a highly contagious endemic disease of pigs. Adhesion is a critical first step in the infection process. Trimeric autotransporter adhesions (TAAs) have been identified as novel virulence factors; however, little is known on their roles in A. pleuropneumoniae pathogenicity. Here, our data show that YadA-like head region (Adh) of Apa1 was the optimal adhesion functional domain via segment expression and adhesion assays in vitro. Additionally, Adh induced partial protection against A. pleuropneumoniae 5b L20 and serotypes 1, 3, and 5a in mice. The deletion of Adh gene significantly decreased autoaggregation, biofilm formation and adherence to host cells in vitro. Furthermore, with delaying of clinical symptoms, reducing production of pro-inflammatory cytokines and lessening the lung injury after infection, Adh deletion strain (5bϕAdh) significantly reduced the pathogenicity to piglets. To elucidate the mechanism of lung injury, the differentially expressed genes in the lung tissues of piglets infected with the 5b L20 or 5bϕAdh strains were investigated using microarray analysis and validated by qRT-PCR. Compared with the 5b L20 infected piglets, 495 genes were differentially expressed in 5bϕAdh infected lung tissue (221 upregulated and 274 downregulated). Especially, the antigen processing and presentation gene IFI30 was increased following infection with the 5bϕAdh strain. Thus, Adh may enhance pathogenicity by depressing host immune recognition. We conclude that the head domain of the A. pleuropneumoniae trimeric autotransporter Apa1 regulates autoagglutination, biofilm formation, adhesion to host cells and pathogenicity.

Differential gene expression profiling of Actinobacillus pleuropneumoniae during induction of primary alveolar macrophage apoptosis in piglets.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the causative agent of porcine pleuropneumonia, a disease that causes serious problems for the swine industry. Successful infection by this bacterium requires breaking the first line of defence in the lungs, the primary alveolar macrophages (PAMs). Therefore, exploring A. pleuropneumoniae-PAM interactions will provide vital groundwork for the scientific control of this infectious disease, which has been little studied up to now. In this work, PAMs were isolated from piglets and co-incubated with A. pleuropneumoniae serovar 5b strain L20 in vitro, and their interaction, PAM cell death, and differential gene expression of A. pleuropneumoniae in response to PAM cell death were observed and analysed using confocal microscopy, electron microscopy, RT-PCR, Western blot, flow cytometry and the use of a gene expression profile chip. A. pleuropneumoniae quickly adhered to and invaded PAMs, inducing apoptosis, which was confirmed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The highest percentage of apoptosis in cells was confirmed using flow cytometry when the cells were infected at a multiplicity of infection (MOI) of 10 and incubated for 5 h, with higher expression of activated caspase-3 as measured by Western blot. Using microarray gene chips with 2868 probes containing nearly all of the genomic sequence of A. pleuropneumoniae serotype 5b strain L20, a total of 185 bacterial genes were found to be differentially expressed (including 92 up-regulated and 93 down-regulated genes) and involved in the process of apoptosis, as compared with the expression of control bacteria cultured without PAMs in BHI medium (mean expression ratios >1.5-fold, p < 0.05). The up-regulated genes are involved in energy metabolism, gene transcription and translation, virulence related gene such as LPS, Trimeric Autotransporter Adhesin, RTX and similar genes. The down-regulated genes are involved in amino acid, cofactor, and vitamin metabolism, and also include ABC transporters. These data demonstrate that A. pleuropneumoniae induces apoptosis of PAMs and undergoes complex changes in gene transcription, including expression changes in known and potential virulence factors. Some potentially novel virulence targets have been identified, suggesting new strategies for the development of vaccines and medicines for both preventive and clinical use.

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.

PCR specific for Actinobacillus pleuropneumoniae serotype 3.

Serotypes 3 and 8 of Actinobacillus pleuropneumoniae, the aetiological agent of porcine pleuropneumonia, have been reported to predominate in the UK. Direct serotyping of isolates of the organism is typically determined by the immunological reactivity of rabbit serum to its surface polysaccharides, but the method has limitations, for example, cross-reactions between serotypes 3, 6 and 8. This study describes the development of a serotype 3-specific pcr, based on the capsule locus, which can be used in a multiplex format with the organism's specific gene apxIV. The pcr test was evaluated on 266 strains of A pleuropneumoniae and 121 strains of other organisms, including all the major respiratory bacterial pathogens of pigs. The test was highly specific and sensitive and should be useful for differentiating strains of serotypes 3, 6 and 8, and in seroprevalence and epidemiological surveys in regions where serotype 3 is prevalent, such as the UK.

Multiplex PCR that can distinguish between immunologically cross- reactive serovar 3, 6, and 8 Actinobacillus pleuropneumoniae strains.

We describe a highly sensitive and specific multiplex PCR, based on capsular loci and the species specific apxIV gene, that unequivocally differentiates serovar 3, 6, and 8 Actinobacillus pleuropneumoniae strains that are cross-reactive in conventional immunological tests.

Current research priorities in chronic fatigue syndrome/myalgic encephalomyelitis: disease mechanisms, a diagnostic test and specific treatments.

Chronic fatigue syndrome (CFS) is an illness characterised by disabling fatigue of at least 6 months duration, which is accompanied by various rheumatological, infectious and neuropsychiatric symptoms. A collaborative study group has been formed to deal with the current areas for development in CFS research--namely, to develop an understanding of the molecular pathogenesis of CFS, to develop a diagnostic test and to develop specific and curative treatments. Various groups have studied the gene expression in peripheral blood of patients with CFS, and from those studies that have been confirmed using polymerase chain reaction (PCR), clearly, the most predominant functional theme is that of immunity and defence. However, we do not yet know the precise gene signature and metabolic pathways involved. Currently, this is being dealt with using a microarray representing 47,000 human genes and variants, massive parallel signature sequencing and real-time PCR. It will be important to ensure that once a gene signature has been identified, it is specific to CFS and does not occur in other diseases and infections. A diagnostic test is being developed using surface-enhanced, laser-desorption and ionisation-time-of-flight mass spectrometry based on a pilot study in which putative biomarkers were identified. Finally, clinical trials are being planned; novel treatments that we believe are important to trial in patients with CFS are interferon-beta and one of the anti-tumour necrosis factor-alpha drugs.

High-throughput identification of conditionally essential genes in bacteria: from STM to TSM.

Signature-tagged mutagenesis (STM) provided the first widely applicable high-throughput method for detecting conditionally essential genes in bacteria by using negative selection to screen large pools of transposon (Tn) mutants. STM requires no prior knowledge of the bacterium's genome sequence, and has been used to study a large number of Gram-positive and Gram-negative species, greatly expanding the repertoires of known virulence factors for these organisms. Originally, hybridization of radiolabelled probes to colony or dot blots was used to detect differences in populations of tagged mutants before and after growth under a selective condition. Modifications of the tag detection method involving polymerase chain reaction (PCR) amplification and visualisation by gel electrophoresis have been developed and can be automated through the use of robotics. Genetic footprinting is another negative selection technique that uses PCR amplification to detect loss of mutants from a pool. Unlike PCR-STM, this technique allows direct amplification of Tn-flanking sequences. However, it requires the bacterium's whole genome sequence in order to design specific primers for every gene of interest. More recently, a number of techniques have been described that combine the negative-selection principle of STM and genetic footprinting with the genome-wide screening power of DNA microarrays. These techniques, although also requiring whole genome sequences, use either a form of linker-mediated or semi-random PCR to amplify and label Tn-flanking regions for hybridization to microarrays. The superior sensitivity microarray detection allows greater numbers of mutants to be screened per pool, as well as determination of the coverage/distribution of insertions in the library prior to screening, two significant advantages over STM.

Characterisation and genetic organisation of a 24-MDa plasmid from the Brazilian Purpuric Fever clone of Haemophilus influenzae biogroup aegyptius.

Strains of Haemophilus influenzae biogroup aegyptius causing septicaemia were identified in Brazil in the 1980s, causing the life-threatening illness of Brazilian Purpuric Fever (BPF). The strains were found to fall into a single clonal group, the BPF clone, characterised by their possession of the approximately 24MDa "3031" plasmid. In this work we report the characterisation and genetic organisation of this plasmid. Analysis of the gene content of what appears to be a typical broad host range conjugative plasmid, its presence in non-BPF strains as revealed by Southern hybridisation, and the recent discovery of plasmid-lacking BPF strains, has led us to conclude that it is unlikely to play a critical role in bacterial virulence. Establishing its entire sequence has nonetheless been an important step on the road to delineating, by comparison of BPF and non-BPF strains, chromosomal genetic loci that are involved in the special virulence of the BPF clone.

Actinobacillus pleuropneumoniae serotype 1 carrying the defined aroA mutation is fully avirulent in the pig.

The aroA gene from Actinobacillus pleuropneumoniae serotype 1 reference strain 4074 was isolated and sequenced. The gene complemented the aroA mutation in Escherichia coli AB2829. A kanamycin resistance cassette was inserted into the aroA gene and the mutant gene was reintroduced into A. pleuropneumoniae by allelic replacement. Intratracheal infection of susceptible pigs with A. pleuropneumoniae aroA caused no signs of respiratory disease or lung lesions in any of the animals at a dose 10(4) times the dose reliably known to induce acute pleuropneumonia; all animals infected with the unaltered control strain developed acute disease. The aroA mutant was rapidly eliminated from the lungs and tonsil of infected animals. The mutant may represent a safely attenuated strain for use in live bacterial vaccination or the delivery of antigen by the intranasal route. However, the residence time of the mutant in the respiratory tract of the pig may be too short for it to be useful in generating a protective immune response.

A novel heme protein, the Cu,Zn-superoxide dismutase from Haemophilus ducreyi.

Haemophilus ducreyi, the causative agent of the genital ulcerative disease known as chancroid, is unable to synthesize heme, which it acquires from humans, its only known host. Here we provide evidence that the periplasmic Cu,Zn-superoxide dismutase from this organism is a heme-binding protein, unlike all the other known Cu,Zn-superoxide dismutases from bacterial and eukaryotic species. When the H. ducreyi enzyme was expressed in Escherichia coli cells grown in standard LB medium, it contained only limited amounts of heme covalently bound to the polypeptide but was able efficiently to bind exogenously added hemin. Resonance Raman and electronic spectra at neutral pH indicate that H. ducreyi Cu,Zn-superoxide dismutase contains a 6-coordinated low spin heme, with two histidines as the most likely axial ligands. By site-directed mutagenesis and analysis of a structural model of the enzyme, we identified as a putative axial ligand a histidine residue (His-64) that is present only in the H. ducreyi enzyme and that was located at the bottom of the dimer interface. The introduction of a histidine residue in the corresponding position of the Cu,Zn-superoxide dismutase from Haemophilus parainfluenzae was not sufficient to confer the ability to bind heme, indicating that other residues neighboring His-64 are involved in the formation of the heme-binding pocket. Our results suggest that periplasmic Cu,Zn-superoxide dismutase plays a role in heme metabolism of H. ducreyi and provide further evidence for the structural flexibility of bacterial enzymes of this class.

A histidine-rich metal binding domain at the N terminus of Cu,Zn-superoxide dismutases from pathogenic bacteria: a novel strategy for metal chaperoning.

A group of Cu,Zn-superoxide dismutases from pathogenic bacteria is characterized by histidine-rich N-terminal extensions that are in a highly exposed and mobile conformation. This feature allows these proteins to be readily purified in a single step by immobilized metal affinity chromatography. The Cu,Zn-superoxide dismutases from both Haemophilus ducreyi and Haemophilus parainfluenzae display anomalous absorption spectra in the visible region due to copper binding at the N-terminal region. Reconstitution experiments of copper-free enzymes demonstrate that, under conditions of limited copper availability, this metal ion is initially bound at the N-terminal region and subsequently transferred to an active site. Evidence is provided for intermolecular pathways of copper transfer from the N-terminal domain of an enzyme subunit to an active site located on a distinct dimeric molecule. Incubation with EDTA rapidly removes copper bound at the N terminus but is much less effective on the copper ion bound at the active site. This indicates that metal binding by the N-terminal histidines is kinetically favored, but the catalytic site binds copper with higher affinity. We suggest that the histidine-rich N-terminal region constitutes a metal binding domain involved in metal uptake under conditions of metal starvation in vivo. Particular biological importance for this domain is inferred by the observation that its presence enhances the protection offered by periplasmic Cu,Zn-superoxide dismutase toward phagocytic killing.

A promoter probe plasmid based on green fluorescent protein : a strategy for studying meningococcal gene expression.

Many bacterial genes are regulated in an environment-responsive fashion, and from the perspective of a pathogen, the host represents just another environment. Many genes that contribute to virulence are differentially expressed in response to host environments that they encounter during colonization and invasion (1). Recognition of this has led to the development of selection or reporter systems that utilize the increased activity of promoters during growth in vivo to identify genes that are selectively expressed during infection, and, thus, may contribute to the infection process (2-5). One of these techniques, differential fluorescence induction (2,3), involves the use of a promoter-probe plasmid that utilizes a variant of green fluorescent protein (GFP) as its reporter. The technique has been used successfully to identify novel Salmonella typhimurium genes that are selectively expressed following exposure to acid environments (3) and during infection of macrophages (2). GFP reporter systems have also been used to evaluate in vivo gene expression in other organisms including Staphylococcus aureus (6), Listeria monocytogenes (7), and Mycobacterium marinum (8). This chapter describes the use of the GFP-promoter-probe plasmid, pJSK411, which is suitable for the evaluation of differential gene expression in Neisseria meningitidis (Fig.1). Fig. 1. Map of pJSK411 demonstrating restriction sites within the multiple cloning site. The binding site for the 401 US primer overlies the XhoI site and the 41 1DS primer binding site lies within the coding region of GFPmut3.