Identification of Genes Involved in Biosynthesis of Actinobacillus Pleuropneumoniae Serotype 1 O-antigen and Biological Properties of Rough Mutants

Journal of Endotoxin Research. 2002  |  Pubmed ID: 11981443

Actinobacillus pleuropneumoniae is an important pathogen of swine. Lipopolysaccharide (LPS) has been identified as the major adhesin of A. pleuropneumoniae and it is involved in adherence to porcine respiratory tract cells. We previously generated seven rough LPS mutants of A. pleuropneumoniae serotype 1 by using a mini-Tn10 transposon mutagenesis system [Rioux S, Galarneau C, Harel J et al. Isolation and characterization of mini-Tn10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1. Can J Microbiol 1999; 45: 1017-1026]. The purpose of the present study was to characterize these mutants in order to learn more about LPS O-antigen biosynthesis genes and their organization in A. pleuropneumoniae, and to determine the surface properties and virulence in pigs of these isogenic mutants. By mini-Tn10 insertions in rough mutants, four putative genes (ORF12, ORF16, ORF17, and ORF18) involved in O-antigen biosynthesis in A. pleuropneumoniae serotype 1 were found within a region of 18 ORFs. This region is homologous to the gene cluster of serotype-specific O-polysaccharide biosynthesis from A. actinomycetemcomitans strain Y4 (serotype b). Two mutants showed homology to a protein with identity to glycosyltransferases (ORF12); two others had the mini-Tn10 insertion localized in genes encoding for two distinct proteins with identity to rhamnosyltransferases (ORF16 and ORF17) and three showed homology to a protein which is known to initiate polysaccharide synthesis (ORF18). These four ORFs were also present in A. pleuropneumoniae serotypes 9 and 11 that express an O-antigen that serologically cross-reacts with serotype 1. Evaluation of some biological properties of rough mutants seems to indicate that the absence of O-chains does not appear to have an influence on the virulence of the bacteria in pigs and on the overall surface hydrophobicity, charge and hemoglobin-binding activity, or on LAL activation. An acapsular mutant was included in the present study in order to compare the influence of O-chains and capsule polysaccharides on different cell surface properties. Our data suggest that capsular polysaccharides and not O-chains polysaccharides have a major influence on surface properties of A. pleuropneumoniae serotype 1 and its virulence in pigs.

Molecular Cloning and Characterization of the Ferric Hydroxamate Uptake (fhu) Operon in Actinobacillus Pleuropneumoniae

Microbiology (Reading, England). Sep, 2002  |  Pubmed ID: 12213932

The bacterium Actinobacillus pleuropneumoniae, a swine pathogen, utilizes ferrichrome as an iron source. This study details the molecular cloning and sequencing of the genes involved in the uptake of this hydroxamate siderophore. Four ferric hydroxamate uptake (fhu) genes, fhuC, fhuD, fhuB and fhuA, were identified in a single operon, and these were found to encode proteins homologous to proteins of the fhu systems of several bacteria, including Escherichia coli. The fhuA gene encodes the 77 kDa outer-membrane protein (OMP) FhuA, the receptor for ferrichrome. FhuD is the 35.6 kDa periplasmic protein responsible for the translocation of ferric hydroxamate from the outer to the inner membrane. FhuC (28.5 kDa) and FhuB (69.4 kDa) are cytoplasmic-membrane-associated proteins that are components of an ABC transporter which internalizes the ferric hydroxamate. Reference strains of A. pleuropneumoniae that represented serotypes 1 to 12 of this organism all tested positive for the four fhu genes. When A. pleuropneumoniae FhuA was affinity-tagged with hexahistidine at its amino terminus and expressed in an E. coli host, the recombinant protein reacted with an mAb against E. coli FhuA, as well as with a polyclonal pig serum raised against an A. pleuropneumoniae infection. Hence, the authors conclude that fhuA is expressed in vivo by A. pleuropneumoniae. Three-dimensional modelling of the OMP FhuA was achieved by threading it to the X-ray crystallographic structure of the homologous protein in E. coli. FhuA from A. pleuropneumoniae was found to have the same overall fold as its E. coli homologue, i.e. it possesses an N-terminal cork domain followed by a C-terminal beta-barrel domain and displays 11 extracellular loops and 10 periplasmic turns.

FhuA of Actinobacillus Pleuropneumoniae Encodes a Ferrichrome Receptor but is Not Regulated by Iron

Infection and Immunity. May, 2003  |  Pubmed ID: 12704168

The swine pathogen Actinobacillus pleuropneumoniae possesses a 75-kDa outer membrane protein (OMP), FhuA, the receptor for ferrichrome, a hydroxamate-type siderophore. Polyclonal serum to FhuA reacted with OMP preparations from 12 serotypes of A. pleuropneumoniae under conditions of iron repletion and restriction. Reverse transcription-PCR confirmed that A. pleuropneumoniae fhuA expression is not upregulated in response to low iron levels. An A. pleuropneumoniae fhuA deletion mutant was generated and showed abolishment of ferrichrome uptake.

Identification, Cloning and Characterization of RfaE of Actinobacillus Pleuropneumoniae Serotype 1, a Gene Involved in Lipopolysaccharide Inner-core Biosynthesis

FEMS Microbiology Letters. Jun, 2003  |  Pubmed ID: 12798993

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and its lipopolysaccharides (LPS) have been identified as important adhesins involved in adherence to host cells. To better understand the role of LPS core in the virulence of this organism, the aim of the present study was to identify and clone genes involved in LPS core biosynthesis by complementation with Salmonella enterica serovar Typhimurium mutants (rfaC, rfaD, rfaE and rfaF). Complementation with an A. pleuropneumoniae 4074 genomic library was successful with Salmonella mutant SL1102. This Salmonella deep-rough LPS mutant is defective for the rfaE gene, which is an ADP-heptose synthase. Novobiocin was used to select transformants that had the smooth-LPS type, since Salmonella strains with wild-type smooth-LPS are less permeable, therefore more resistant to hydrophobic antibiotics like novobiocin. We obtained a clone that was able to restore the wild-type smooth-LPS Salmonella phenotype after complementation. The wild-type phenotype was confirmed using phage (Felix-O, P22c.2 and Ffm) susceptibility and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). One of the open reading frames contained in the 3.3-kb insert in the plasmid encoded a 475-amino-acid protein with 71% identity and 85% similarity to the RfaE protein of S. enterica. We then attempted to generate an A. pleuropneumoniae rfaE mutant by gene replacement. The rfaE gene seems essential in A. pleuropneumoniae viability as we were unable to isolate a heptose-less knockout mutant.

Characterization of the Novel Factor Paa Involved in the Early Steps of the Adhesion Mechanism of Attaching and Effacing Escherichia Coli

Infection and Immunity. Aug, 2003  |  Pubmed ID: 12874331

Nonenterotoxigenic porcine Escherichia coli strains belonging to the serogroup O45 have been associated with postweaning diarrhea in swine and adhere to intestinal epithelial cells in a characteristic attaching and effacing (A/E) pattern. O45 porcine enteropathogenic E. coli (PEPEC) strain 86-1390 induces typical A/E lesions in a pig ileal explant model. Using TnphoA transposon insertion mutagenesis on strain 86-1390, we found a mutant that did not induce A/E lesions. The insertion was identified in a gene designated paa (porcine A/E-associated gene). Sequence analysis of paa revealed an open reading frame of 753 bp encoding a 27.6-kDa protein which displayed 100, 51.8, and 49% homology with Paa of enterohemorrhagic E. coli O157:H7 strains (EDL933 and Sakai), PEB3 of Campylobacter jejuni, and AcfC of Vibrio cholerae, respectively. Chromosomal localization studies indicated that the region containing paa was inserted between the yciD and yciE genes at about 28.3 min of the E. coli K-12 chromosome. The presence of paa and eae sequences in the porcine O45 strains is highly correlated with the A/E phenotype. However, the observation that three eae-positive but paa-negative PEPEC O45 strains were A/E negative provides further evidence for the importance of the paa gene in the A/E activity of O45 strains. As well, the complementation of the paa mutant restored the A/E activity of the 86-1390 strain, showing the involvement of Paa in PEPEC pathogenicity. These observations suggest that Paa contributes to the early stages of A/E E. coli virulence.

Binding of Actinobacillus Pleuropneumoniae to Phosphatidylethanolamine

Infection and Immunity. Aug, 2003  |  Pubmed ID: 12874346

The gram-negative bacterium Actinobacillus pleuropneumoniae is the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia, a disease that causes important economic losses to the swine industry worldwide. In general, the initial step of bacterial colonization is attachment to host cells. The purpose of the present study was to evaluate the binding of A. pleuropneumoniae serotype 1 to phospholipids, which are the major constituents of biological membranes. Phospholipids serve as receptors for several bacteria, including respiratory pathogens. To study this effect, we used thin-layer chromatography overlay binding assays to test commercial phospholipids such as phosphatidic acid, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidylethanolamine (PE). Our results indicate that A. pleuropneumoniae serotype 1 binds to PE but not to the other phospholipids tested. Serotypes 5b and 7, which, along with serotype 1, are the most prevalent serotypes of A. pleuropneumoniae in North America, share the ability to bind PE. Inhibition of binding with a monoclonal antibody against A. pleuropneumoniae serotype 1 O antigen and the use of isogenic lipopolysaccharide (LPS) mutants of A. pleuropneumoniae serotype 1 showed that the O antigen seems to be implicated in the binding to PE, at least for A. pleuropneumoniae serotype 1. A. pleuropneumoniae was also shown to bind to a phospholipid extracted from swine lungs by using the method of Folch. Chemical staining with molybdenum blue and ninhydrin, migration with neutral, acidic, and basic solvent systems, and mass spectrometry analysis all indicated that this lipid is PE. This study is, to the best of our knowledge, the first description of A. pleuropneumoniae binding to phospholipids. Our data also suggest that LPS O antigens could be involved in binding to PE.

Identification and Preliminary Characterization of a 75-kDa Hemin- and Hemoglobin-binding Outer Membrane Protein of Actinobacillus Pleuropneumoniae Serotype 1

Canadian Journal of Veterinary Research = Revue Canadienne De Recherche Vétérinaire. Oct, 2003  |  Pubmed ID: 14620863

The reference strains representing serotypes 1 to 12 of Actinobacillus pleuropneumoniae biotype 1 were examined for their ability to utilize porcine hemoglobin (Hb) or porcine hemin (Hm) as iron sources for growth. In a growth promotion assay, all of the reference strains were able to use porcine Hb, and all strains except 2 were able to use porcine Hm. Using a preliminary characterization procedure with Hm- or Hb-agarose, Hm- and Hb-binding outer membrane proteins (OMPs) of approximately 75 kDa were isolated from A. pleuropneumoniae serotype 1 strain 4074 grown under iron-restricted conditions. Matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) analysis revealed a number of common tryptic peptides between the Hb-agarose- and Hm-agarose-purified 75 kDa OMPs, strongly suggesting that these peptides originate from the same protein. A database search of these peptide sequences revealed identities with proteins from various Gram-negative bacteria, including iron-regulated OMPs, transporter proteins, as well as TonB-dependent receptors. Taken together, our data suggest that A. pleuropneumoniae synthesizes potential Hm- and Hb-binding proteins that could be implicated in the iron uptake from porcine Hb and Hm.

Molecular Cloning of Haemoglobin-binding Protein HgbA in the Outer Membrane of Actinobacillus Pleuropneumoniae

Microbiology (Reading, England). Jun, 2004  |  Pubmed ID: 15184559

From the porcine pathogen Actinobacillus pleuropneumoniae cultivated in iron-deficient or haem-deficient media, haemoglobin (Hb)-agarose affinity purification was exploited to isolate an outer-membrane protein of approximately 105 kDa, designated HgbA. Internal peptide sequences of purified HgbA were used to design oligonucleotide primers for PCR amplification, yielding amplicons that showed partial sequences with homology to hgbA of Pasteurella multocida. Upon screening two genomic libraries of A. pleuropneumoniae serotype 1 strain 4074, positive clones were assembled into an ORF of 2838 bp. HgbA (946 aa) includes a signal peptide of 23 aa and the deduced HgbA sequence (104 890 Da) also demonstrated a possible Ton box. The promoter region of hgbA from A. pleuropneumoniae serotype 1 showed consensus for -35 and -10 sequences and a putative Fur-binding site. RT-PCR confirmed that hgbA of A. pleuropneumoniae is upregulated in response to diminished levels of iron in the culture medium. While an internally deleted hgbA mutant was unable to use pig Hb as sole source of iron for growth, flow cytometry confirmed its Hb binding; the internally deleted sequences may not be required for Hb binding, but appear necessary for the iron supply from Hb. HgbA is required for growth of A. pleuropneumoniae in the presence of Hb as sole iron source.

Surface Polysaccharides and Iron-uptake Systems of Actinobacillus Pleuropneumoniae

Canadian Journal of Veterinary Research = Revue Canadienne De Recherche Vétérinaire. Apr, 2004  |  Pubmed ID: 15188950

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.

Structural Analysis of the Lipopolysaccharide Derived Core Oligosaccharides of Actinobacillus Pleuropneumoniae Serotypes 1, 2, 5a and the Genome Strain 5b

Carbohydrate Research. Aug, 2004  |  Pubmed ID: 15261591

The structures of the core oligosaccharides of the lipopolysaccharides (LPS) from Actinobacillus pleuropneumoniae serotypes 1, 2, 5a and 5b were elucidated. The LPS's were subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structures for the core oligosaccharides were determined on the basis of the combined data from these experiments. [carbohydrate formula see text] For serotype 1: R is (1S)-GalaNAc-(1-->4,6)-alpha-Gal II-(1-->3)-beta-Gal I-(1-->, and R' is H For serotype 2: R is beta-Glc III-(1-->, and R' is D-alpha-D-Hep V-(1--> For serotypes 5a and 5b: R is H and R' is D-alpha-D-Hep V-(1--> All oligosaccharides elaborated a conserved inner core structure, as illustrated. All sugars were in the pyranose ring form apart from the open-chain N-acetylgalactosamine, the identification of which in the serotype 1 LPS was of interest.

Isolation of an Atypical Strain of Actinobacillus Pleuropneumoniae Serotype 1 with a Truncated Lipopolysaccharide Outer Core and No O-antigen

Journal of Clinical Microbiology. Jul, 2005  |  Pubmed ID: 16000496

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.

Truncation of the Lipopolysaccharide Outer Core Affects Susceptibility to Antimicrobial Peptides and Virulence of Actinobacillus Pleuropneumoniae Serotype 1

The Journal of Biological Chemistry. Nov, 2005  |  Pubmed ID: 16188878

We reported previously that the core oligosaccharide region of the lipopolysaccharide (LPS) is essential for optimal adhesion of Actinobacillus pleuropneumoniae, an important swine pathogen, to respiratory tract cells. Rough LPS and core LPS mutants of A. pleuropneumoniae serotype 1 were generated by using a mini-Tn10 transposon mutagenesis system. Here we performed a structural analysis of the oligosaccharide region of three core LPS mutants that still produce the same O-antigen by using methylation analyses and mass spectrometry. We also performed a kinetic study of proinflammatory cytokines production such as interleukin (IL)-6, tumor necrosis factor-alpha, IL1-beta, MCP-1, and IL8 by LPS-stimulated porcine alveolar macrophages, which showed that purified LPS of the parent strain, the rough LPS and core LPS mutants, had the same ability to stimulate the production of cytokines. Most interestingly, an in vitro susceptibility test of these LPS mutants to antimicrobial peptides showed that the three core LPS mutants were more susceptible to cationic peptides than both the rough LPS mutant and the wild type parent strain. Furthermore, experimental pig infections with these mutants revealed that the galactose (Gal I) and d,d-heptose (Hep IV) residues present in the outer core of A. pleuropneumoniae serotype 1 LPS are important for adhesion and overall virulence in the natural host, whereas deletion of the terminal GalNAc-Gal II disaccharide had no effect. Our data suggest that an intact core-lipid A region is required for optimal protection of A. pleuropneumoniae against cationic peptides and that deletion of specific residues in the outer LPS core results in the attenuation of the virulence of A. pleuropneumoniae serotype 1.

Identification of a Surface Protein of Streptococcus Suis and Evaluation of Its Immunogenic and Protective Capacity in Pigs

Infection and Immunity. Jan, 2006  |  Pubmed ID: 16368985

A Streptococcus suis surface protein reacting with convalescent-phase sera from pigs clinically infected by S. suis type 2 was identified. The apparent 110-kDa protein, designated Sao, exhibits typical features of membrane-anchored surface proteins of gram-positive bacteria, such as a signal sequence and an LPVTG membrane anchor motif. In spite of high identity with the partially sequenced genomes of S. suis Canadian strain 89/1591 and European strain P1/7, Sao does not share significant homology with other known sequences. However, a conserved avirulence domain that is often found in plant pathogens has been detected. Electron microscopy using an Sao-specific antiserum has confirmed the surface location of the Sao protein on S. suis. The Sao-specific antibody reacts with cell lysates of 28 of 33 S. suis serotypes and 25 of 26 serotype 2 isolates in immunoblots, suggesting its high conservation in S. suis species. The immunization of piglets with recombinant Sao elicits a significant humoral antibody response. However, the antibody response is not reflected in protection of pigs that are intratracheally challenged with a virulent strain in our conventional vaccination model.

Haemophilus Parasuis Invades Porcine Brain Microvascular Endothelial Cells

Microbiology (Reading, England). Jan, 2006  |  Pubmed ID: 16385123

Haemophilus parasuis, an important swine pathogen, is the aetiological agent of Glässer's disease. It is responsible for cases of polyserositis, meningitis and pneumonia in young pigs. To date, 15 serotypes have been described, although several non-typable isolates are frequently recovered from diseased animals. The pathogenesis of H. parasuis infection is poorly understood. To cause meningitis, H. parasuis would have to cross the blood-brain barrier (BBB), composed of brain microvascular endothelial cells (BMEC). The objective of this study was to investigate the ability of H. parasuis to interact with porcine brain microvascular endothelial cells (PBMEC). It was demonstrated that the serotype 5 reference strain of H. parasuis, Nagasaki (originally recovered from a case of meningitis), was able to adhere at very high levels to and, most importantly, invade PBMEC. These capacities were confirmed by electron microscopy. Actinobacillus pleuropnemoniae serotype 7 (strain WF 83), used as negative control, was not able to adhere to or invade PBMEC. Comparisons of the levels of adhesion and invasion by several H. parasuis field strains from different serotypes isolated from cases of either meningitis or pneumonia showed that isolates of serotypes 4 and 5 had a higher invasion capacity than isolates belonging to other serotypes. Inhibition studies demonstrated that PBMEC invasion by H. parasuis required rearrangement of actin microfilaments and microtubular cytoskeletal elements but not active bacterial DNA, RNA or protein synthesis. Characterization studies demonstrated that proteinaceous invasin(s) does not seem to play a major role in entry of H. parasuis into PBMEC. Intracellular viable H. parasuis were found in PBMEC up to 6 h after antibiotic treatment. Even at high bacterial doses, H. parasuis was not toxic to PBMEC. In swine, the invasion of endothelial cells of the BBB may play an important role in the pathogenesis of meningitis caused by H. parasuis.

Porcine Mannan-binding Lectin A Binds to Actinobacillus Suis and Haemophilus Parasuis

Developmental and Comparative Immunology. 2006  |  Pubmed ID: 16480769

Various collagenous lectins involved in innate immunity bind to surface oligosaccharides of bacteria and other microorganisms. We have been characterizing porcine plasma lectins that bind in a carbohydrate-dependent manner to surfaces of important bacterial pig pathogens including Actinobacillus suis (AS), A. pleuropneumoniae (APP), and Haemophilus parasuis (HP). A plasma protein with 32kDa subunits (pI 5.4 and 5.75) bound most isolates of HP, AS, and some APP. Partial amino acid sequences of this protein were similar to mammalian mannan-binding lectins (MBLs). The corresponding MBL-A cDNA sequences obtained by RT-PCR on liver tissue from pigs and cattle were homologous to the MBL1 gene of mice, rats and the MBL1P1 pseudogene of humans and chimpanzees. While human MBL-C, the product of the MBL2 gene, is known to bind various microorganisms, our studies in pigs provide the first direct evidence that MBL-A has bacteria-binding properties, and suggest it may have antibacterial functions in pigs.

Fhua and HgbA, Outer Membrane Proteins of Actinobacillus Pleuropneumoniae: Their Role As Virulence Determinants

Canadian Journal of Microbiology. Apr, 2006  |  Pubmed ID: 16699590

For the recently described serotype 15 of biotype I and serotypes 13 and 14 of biotype II of Actinobacillus pleuropneumoniae, fhuA and hgbA were detected by polymerase chain reaction and DNA sequencing. To determine the substrate specificity of the iron receptors FhuA and HgbA and to study their role in the virulence of A. pleuropneumoniae, we used two isogenic A. pleuropneumoniae serotype 1 deletion mutants of fhuA and hgbA. Different sources of iron and siderophores were tested in growth promotion assays. FhuA and HgbA are specific for their ligands ferrichrome and hemoglobin, respectively. The virulence of the two deletion mutant strains was evaluated in experimental infections using specific pathogen-free piglets. While the fhuA mutant (DG02) was as highly virulent as the parental strain S4074, the virulence of the hgbA mutant (DeltahgbA) was reduced. Our data indicate that both FhuA and HgbA are conserved among all serotypes and biotypes of A. pleuropneumoniae and that HgbA, the receptor for porcine hemoglobin, may play a role in virulence.

Transcriptional Modulation of Some Staphylococcus Aureus Iron-regulated Genes During Growth in Vitro and in a Tissue Cage Model in Vivo

Microbes and Infection / Institut Pasteur. Jun, 2006  |  Pubmed ID: 16969913

Staphylococcus aureus can proliferate in iron-limited environments such as the mammalian host. The transcriptional profiles of 460 genes (iron-regulated, putative Fur-regulated, membrane transport, pathogenesis) obtained for S. aureus grown in iron-restricted environments in vitro and in vivo were compared in order to identify new iron-regulated genes and to evaluate their potential as possible therapeutic targets in vivo. Iron deprivation was created in vitro by 2,2-dipyridyl, and in vivo, S. aureus was grown in tissue cages implanted in mice. Bacterial RNA was obtained from each growth condition and cDNA probes were co-hybridized on DNA arrays. Thirty-six upregulated and 11 downregulated genes were commonly modulated in animals and in the low-iron medium. Real-time PCR confirmed the iron-dependent modulation of four novel genes (SACOL0161, 2170, 2369, 2431) with a Fur box motif. Some genes expressed in the dipyridyl medium were not expressed in vivo (e.g., copA, frpA, SACOL1045). Downregulated genes included an iron-storage protein gene and genes of the succinate dehydrogenase complex, reminiscent of a small RNA-dependent regulation thus far only demonstrated in Gram-negative bacteria. The expression of iron-regulated genes in distinct low-iron environments provided insight into their relative importance in vitro and in vivo and their usefulness for vaccine and drug development.

Transcriptional Profiling of Actinobacillus Pleuropneumoniae Under Iron-restricted Conditions

BMC Genomics. 2007  |  Pubmed ID: 17355629

To better understand effects of iron restriction on Actinobacillus pleuropneumoniae and to identify new potential vaccine targets, we conducted transcript profiling studies using a DNA microarray containing all 2025 ORFs of the genome of A. pleuropneumoniae serotype 5b strain L20. This is the first study involving the use of microarray technology to monitor the transcriptome of A. pleuropneumoniae grown under iron restriction.

Outer Membrane Proteome of Actinobacillus Pleuropneumoniae: LC-MS/MS Analyses Validate in Silico Predictions

Proteomics. Jun, 2007  |  Pubmed ID: 17476711

The Gram-negative bacterial pathogen Actinobacillus pleuropneumoniae causes porcine pneumonia, a highly infectious respiratory disease that contributes to major economic losses in the swine industry. Outer membrane (OM) proteins play key roles in infection and may be targets for drug and vaccine research. Exploiting the genome sequence of A. pleuropneumoniae serotype 5b, we scanned in silico for proteins predicted to be localized at the cell surface. Five genome scanning programs (Proteome Analyst, PSORT-b, BOMP, Lipo, and LipoP) were run to construct a consensus prediction list of 93 OM proteins in A. pleuropneumoniae. An inventory of predicted OM proteins was complemented by proteomic analyses utilizing gel- and solution-based methods, both coupled to LC-MS/MS. Different protocols were explored to enrich for OM proteins; the most rewarding required sucrose gradient centrifugation followed by membrane washes with sodium bromide and sodium carbonate. This protocol facilitated our identification of 47 OM proteins that represent 50% of the predicted OM proteome, most of which have not been characterized. Our study establishes the first OM proteome of A. pleuropneumoniae.

Actinobacillus Pleuropneumoniae Vaccines: from Bacterins to New Insights into Vaccination Strategies

Animal Health Research Reviews / Conference of Research Workers in Animal Diseases. Jun, 2008  |  Pubmed ID: 18346296

With the growing emergence of antibiotic resistance and rising consumer demands concerning food safety, vaccination to prevent bacterial infections is of increasing relevance. Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease leading to severe economic losses in the swine industry. Despite all the research and trials that were performed with A. pleuropneumoniae vaccination in the past, a safe vaccine that offers complete protection against all serotypes has yet not reached the market. However, recent advances made in the identification of new potential vaccine candidates and in the targeting of specific immune responses, give encouraging vaccination perspectives. Here, we review past and current knowledge on A. pleuropneumoniae vaccines as well as the newly available genomic tools and vaccination strategies that could be useful in the design of an efficient vaccine against A. pleuropneumoniae infection.

Interactions of Haemophilus Parasuis and Its LOS with Porcine Brain Microvascular Endothelial Cells

Veterinary Research. Sep-Oct, 2008  |  Pubmed ID: 18387279

Haemophilus parasuis is a swine pathogen that causes Glässer's disease, which is characterized by polyserositis and meningitis. The pathogenesis of the H. parasuis infection is poorly understood. To cause meningitis, H. parasuis has to cross the blood-brain barrier (BBB) to gain access to the central nervous system (CNS). We recently showed that H. parasuis adheres to and invades porcine brain microvascular endothelial cells (PBMEC). The aim of this study was to evaluate the role of H. parasuis lipooligosaccharide (LOS) in the adhesion to PBMEC and to determine if H. parasuis (and/or its LOS) is able to induce apoptosis and activation of PBMEC. Results showed that adhesion of H. parasuis to PBMEC was partially mediated by LOS. Moreover, H. parasuis induces caspase-3-mediated apoptosis of PBMEC in a time--and dose--dependent manner, but its LOS did not seem to be involved in such a process. Furthermore, H. parasuis and, to a lesser extent, its LOS, was able to induce the release of IL-8 and IL-6 by PBMEC. Field strains of H. parasuis serotypes 4 and 5 induced similar levels of these inflammatory mediators. Our data suggest that H. parasuis uses cellular adhesion, induction of apoptosis and up-regulation of inflammatory mediators as mechanisms to invade the CNS via the BBB, and that LOS would play a certain but limited role in such pathological process.

Mutation in the LPS Outer Core Biosynthesis Gene, GalU, Affects LPS Interaction with the RTX Toxins ApxI and ApxII and Cytolytic Activity of Actinobacillus Pleuropneumoniae Serotype 1

Molecular Microbiology. Oct, 2008  |  Pubmed ID: 18713318

Lipopolysaccharides (LPS) and Apx toxins are major virulence factors of Actinobacillus pleuropneumoniae, a pathogen of the respiratory tract of pigs. Here, we evaluated the effect of LPS core truncation in haemolytic and cytotoxic activities of this microorganism. We previously generated a highly attenuated galU mutant of A. pleuropneumoniae serotype 1 that has an LPS molecule lacking the GalNAc-Gal II-Gal I outer core residues. Our results demonstrate that this mutant exhibits wild-type haemolytic activity but is significantly less cytotoxic to porcine alveolar macrophages. However, no differences were found in gene expression and secretion of the haemolytic and cytotoxic toxins ApxI and ApxII, both secreted by A. pleuropneumoniae serotype 1. This suggests that the outer core truncation mediated by the galU mutation affects the toxins in their cytotoxic activities. Using both ELISA and surface plasmon resonance binding assays, we demonstrate a novel interaction between LPS and the ApxI and ApxII toxins via the core oligosaccharide. Our results indicate that the GalNAc-Gal II-Gal I trisaccharide of the outer core is fundamental to mediating LPS/Apx interactions. The present study suggests that a lack of binding between LPS and ApxI/II affects the cytotoxicity and virulence of A. pleuropneumoniae.

Studies on the Interactions of Haemophilus Parasuis with Porcine Epithelial Tracheal Cells: Limited Role of LOS in Apoptosis and Pro-inflammatory Cytokine Release

Microbial Pathogenesis. Feb, 2009  |  Pubmed ID: 19013513

Haemophilus parasuis colonizes the upper respiratory tract of swine and causes Glässer's disease. We recently demonstrated that H. parasuis can adhere to newborn pig tracheal (NPTr) cells. However, the molecular mechanisms involved in upper respiratory tract colonization by H. parasuis are unknown. The aim of this work was to investigate the role of H. parasuis lipooligosaccharide (LOS) in bacterial adhesion to NPTr cells, the ability of the bacteria and its LOS to induce NPTr cells apoptosis, and their stimulating effect on cytokine release. Our results showed that LOS is partially involved in adhesion to NPTr cells. H. parasuis induced NPTr cells apoptosis in a caspase-3 dependent fashion, but LOS did not seem to be involved in such a process. H. parasuis and, to a lesser extent, its LOS stimulated IL-8 and IL-6 release by NPTr cells. In addition, H. parasuis serotype 4 field isolates induced higher levels of these mediators than did serotype 5 isolates. These results suggest that bacterial adhesion, induction of apoptosis and cytokine release are important events for H. parasuis colonization, but LOS appears to have a limited role in these processes.

Host-pathogen Interactions of Actinobacillus Pleuropneumoniae with Porcine Lung and Tracheal Epithelial Cells

Infection and Immunity. Apr, 2009  |  Pubmed ID: 19139196

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.

Microarray-based Comparative Genomic Profiling of Reference Strains and Selected Canadian Field Isolates of Actinobacillus Pleuropneumoniae

BMC Genomics. 2009  |  Pubmed ID: 19239696

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is a highly contagious respiratory pathogen that causes severe losses to the swine industry worldwide. Current commercially-available vaccines are of limited value because they do not induce cross-serovar immunity and do not prevent development of the carrier state. Microarray-based comparative genomic hybridizations (M-CGH) were used to estimate whole genomic diversity of representative Actinobacillus pleuropneumoniae strains. Our goal was to identify conserved genes, especially those predicted to encode outer membrane proteins and lipoproteins because of their potential for the development of more effective vaccines.

Modulation of Gene Expression in Actinobacillus Pleuropneumoniae Exposed to Bronchoalveolar Fluid

PloS One. 2009  |  Pubmed ID: 19578537

Actinobacillus pleuropneumoniae, the causative agent of porcine contagious pleuropneumonia, is an important pathogen of swine throughout the world. It must rapidly overcome the innate pulmonary immune defenses of the pig to cause disease. To better understand this process, the objective of this study was to identify genes that are differentially expressed in a medium that mimics the lung environment early in the infection process.

MalT Knockout Mutation Invokes a Stringent Type Gene-expression Profile in Actinobacillus Pleuropneumoniae in Bronchoalveolar Fluid

BMC Microbiology. 2009  |  Pubmed ID: 19751522

Actinobacillus pleuropneumoniae causes contagious pleuropneumonia, an economically important disease of commercially reared pigs throughout the world. To cause this disease, A. pleuropneumoniae must rapidly overcome porcine pulmonary innate immune defenses. Since bronchoalveolar fluid (BALF) contains many of the innate immune and other components found in the lungs, we examined the gene expression of a virulent serovar 1 strain of A. pleuropneumoniae after exposure to concentrated BALF for 30 min.

Transcriptional Profiling of Actinobacillus Pleuropneumoniae During the Acute Phase of a Natural Infection in Pigs

BMC Genomics. 2010  |  Pubmed ID: 20141640

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease which causes great economic losses worldwide. Many virulence factors are involved in the pathogenesis, namely capsular polysaccharides, RTX toxins, LPS and many iron acquisition systems. In order to identify genes that are expressed in vivo during a natural infection, we undertook transcript profiling experiments with an A. pleuropneumoniae DNA microarray, after recovery of bacterial mRNAs from serotype 5b-infected porcine lungs. AppChip2 contains 2033 PCR amplicons based on the genomic sequence of App serotype 5b strain L20, representing more than 95% of ORFs greater than 160 bp in length.

Investigation of the Species Origin of the St. Jude Porcine Lung Epithelial Cell Line (SJPL) Made Available to Researchers

Journal of Virology. May, 2010  |  Pubmed ID: 20200241

Evaluation of Some Staphylococcus Aureus Iron-regulated Proteins As Vaccine Targets

Veterinary Immunology and Immunopathology. Aug, 2010  |  Pubmed ID: 20416953

Staphylococcus aureus is an important pathogen that is responsible for a wide range of infections, including bovine mastitis. Previously, 54 genes from S. aureus that were up-regulated in an iron-restricted medium and in mice were identified. Seven of those genes were selected from five iron-acquisition systems (isd, feo, sir, sst, and fhu), and the proteins were evaluated as potential vaccine targets to prevent bovine mastitis. The antigenicity of the recombinant proteins obtained with each studied gene was evaluated in rabbits and/or cattle. Immune sera were used to test the bacterial accessibility of the native proteins. All the proteins were immunogenic in rabbits or cattle. IsdH, IsdB, FeoB and SstD were expressed on the bacterial surface, with IsdB and IsdH more expressed in an iron-restricted environment. The capacity of antibodies to prevent infection was measured in a mouse mastitis model. Preincubation of S. aureus with serum against IsdH or with the pool of sera against IsdB, SstD and FeoB led to decreased colonization of the mouse mammary glands. Lastly, cattle immunization with IsdH induced a strong and long-lasting immune response with IgG2 production. The protein IsdH appears to be a good vaccine candidate to prevent S. aureus bovine mastitis.

Biofilm Formation in Bacterial Pathogens of Veterinary Importance

Animal Health Research Reviews / Conference of Research Workers in Animal Diseases. Dec, 2010  |  Pubmed ID: 20969814

Bacterial biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that is attached to a surface. Biofilms protect and allow bacteria to survive and thrive in hostile environments. Bacteria within biofilms can withstand host immune responses, and are much less susceptible to antibiotics and disinfectants when compared with their planktonic counterparts. The ability to form biofilms is now considered a universal attribute of micro-organisms. Diseases associated with biofilms require novel methods for their prevention, diagnosis and treatment; this is largely due to the properties of biofilms. Surprisingly, biofilm formation by bacterial pathogens of veterinary importance has received relatively little attention. Here, we review the current knowledge of bacterial biofilms as well as studies performed on animal pathogens.

Effects of Growth Conditions on Biofilm Formation by Actinobacillus Pleuropneumoniae

Veterinary Research. Jan-Feb, 2010  |  Pubmed ID: 19737507

Biofilm formation is an important virulence trait of many bacterial pathogens. It has been reported in the literature that only two of the reference strains of the swine pathogen Actinobacillus pleuropneumoniae, representing serotypes 5b and 11, were able to form biofilm in vitro. In this study, we compared biofilm formation by the serotype 1 reference strain S4074 of A. pleuropneumoniae grown in five different culture media. We observed that strain S4074 of A. pleuropneumoniae is able to form biofilms after growth in one of the culture conditions tested brain heart infusion (BHI medium, supplier B). Confocal laser scanning microscopy using a fluorescent probe specific to the poly-N-acetylglucosamine (PGA) polysaccharide further confirmed biofilm formation. In accordance, biofilm formation was susceptible to dispersin B, a PGA hydrolase. Transcriptional profiles of A. pleuropneumoniae S4074 following growth in BHI-B, which allowed a robust biofilm formation, and in BHI-A, in which only a slight biofilm formation was observed, were compared. Genes such as tadC, tadD, genes with homology to autotransporter adhesins as well as genes pgaABC involved in PGA biosynthesis and genes involved in zinc transport were up-regulated after growth in BHI-B. Interestingly, biofilm formation was inhibited by zinc, which was found to be more present in BHI-A (no or slight biofilm) than in BHI-B. We also observed biofilm formation in reference strains representing serotypes 3, 4, 5a, 12 and 14 as well as in 20 of the 37 fresh field isolates tested. Our data indicate that A. pleuropneumoniae has the ability to form biofilms under appropriate growth conditions and transition from a biofilm-positive to a biofilm-negative phenotype was reversible.

Novel Genes Associated with Biofilm Formation of Actinobacillus Pleuropneumoniae

Veterinary Microbiology. Nov, 2011  |  Pubmed ID: 21530112

Actinobacillus pleuropneumoniae is a gram-negative bacterium and is the causative agent of swine pleuropneumonia, a highly contagious respiratory disease. Biofilm formation is an important ability possessed by numerous bacterial pathogens. The purpose of this study was to identify and characterize biofilm mutants of A. pleuropneumoniae serotype 1 strain S4074 created using a mini Tn-10 transposon. The transposon library was screened to identify mutants with a modified ability to form biofilms in polystyrene microtiter plates. A total of 1200 mutants were screened and the analysis identified 24 mutants that exhibited abnormal biofilm formation, at least 16 unique genes were identified. Most genes identified in the enhanced-biofilm mutants encoded proteins with unknown functions, whereas most genes identified in the biofilm-reduced mutants encoded proteins related to transport, protein synthesis and nucleic acid synthesis. Approximately 50% of genes, including hns, potD2, ptsI, tig and rpmF, identified in our screen have been previously associated with biofilm formation in A. pleuropneumoniae and other bacterial species, and thus validated the screening method. The rest of genes identified, such as APL_0049, APL_0637 and APL_1572, have not been previously associated with biofilm formation. Interestingly, gene APL_0049 was previously seen among the genes differentially expressed during a natural infection of pig lungs. Preliminary characterization of the mutants was also initiated by assessing their hydrophobicity, their biofilm matrix composition and their ability to adhere to a polystyrene surface or NPTr cells. Based on the preliminary characterization, some of the mutants identified appear to have deficiencies during the initial attachment or growth of the biofilm. In conclusion, transposon mutagenesis analysis allowed the identification of new genes associated with biofilm formation in A. pleuropneumoniae.

Antimicrobial Susceptibilities and Resistance Genes of Canadian Isolates of Actinobacillus Pleuropneumoniae

Microbial Drug Resistance (Larchmont, N.Y.). Dec, 2011  |  Pubmed ID: 22204596

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a severe and highly contagious respiratory disease responsible for economic losses in the swine industry worldwide. Although antimicrobial resistance in A. pleuropneumoniae has been recently reported in different countries, the current situation in Canada is unknown. The aim of the current study was to determine the antimicrobial susceptibilities of 43 strains of A. pleuropneumoniae isolated in Canada. In addition, antimicrobial resistance genes were detected with an oligonucleotide microarray. The impact of biofilm formation on susceptibility to antimicrobials was also evaluated. All isolates were susceptible to ceftiofur, florfenicol, enrofloxacin, erythromycin, clindamycin, trimethoprim/sulfamethoxazole, and tilmicosin. A low level of resistance was observed toward tiamulin, penicillin, and ampicillin as well as danofloxacin. We observed a high level of resistance to chlortetracycline (88.4%) and oxytetracycline (90.7%). The strains showing resistance to tetracycline antimicrobials contained at least one of the following tet genes: tetB, tetO, tetH, or tetC. Five isolates showed multiresistance to penicillins (bla(ROB-1)), streptomycin [aph3'' (strA)], sulfonamides (sulII), and tetracyclines (tetO) antimicrobials whereas three others showed multiresistance to streptomycin [aph3'' (strA)], sulfonamides (sulII), and tetracyclines (tetB, tetO, or tetB/tetH) antimicrobials. To the best of our knowledge, this is the first description of tetC gene in Pasteurellaceae. Finally, cells of A. pleuropneumoniae in a biofilm were 100 to 30,000 times more resistant to antimicrobials than their planktonic counterparts.