High Sensitivity Crosslink Detection Coupled With Integrative Structure Modeling in the Mass Spec Studio.

The Mass Spec Studio package was designed to support the extraction of hydrogen-deuterium exchange and covalent labeling data for a range of mass spectrometry (MS)-based workflows, to integrate with restraint-driven protein modeling activities. In this report, we present an extension of the underlying Studio framework and provide a plug-in for crosslink (XL) detection. To accommodate flexibility in XL methods and applications, while maintaining efficient data processing, the plug-in employs a peptide library reduction strategy via a presearch of the tandem-MS data. We demonstrate that prescoring linear unmodified peptide tags using a probabilistic approach substantially reduces search space by requiring both crosslinked peptides to generate sparse data attributable to their linear forms. The method demonstrates highly sensitive crosslink peptide identification with a low false positive rate. Integration with a Haddock plug-in provides a resource that can combine multiple sources of data for protein modeling activities. We generated a structural model of porcine transferrin bound to TbpB, a membrane-bound receptor essential for iron acquisition in Actinobacillus pleuropneumoniae Using mutational data and crosslinking restraints, we confirm the mechanism by which TbpB recognizes the iron-loaded form of transferrin, and note the requirement for disparate sources of restraint data for accurate model construction. The software plugin is freely available at www.msstudio.ca.

Truncation of the lipopolysaccharide outer core affects susceptibility to antimicrobial peptides and virulence of Actinobacillus pleuropneumoniae serotype 1.

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.

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.

fhuA of Actinobacillus pleuropneumoniae encodes a ferrichrome receptor but is not regulated by iron.

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.

A PCR assay used to study aerosol transmission of Actinobacillus pleuropneumoniae from samples of live pigs under experimental conditions.

The study describes a polymerase chain reaction (PCR) assay for the detection of Actinobacillus pleuropneumoniae. The test is based on the amplification of the omlA gene coding for an outer membrane protein of A. pleuropneumoniae. To test the specificity of the reaction, 19 other bacterial species related to A. pleuropneumoniae or isolated from pigs were assayed. They were all found negative in the PCR assay. The detection threshold of the test was 10(2) A. pleuropneumoniae CFU/assay. The test was then applied to the detection of A. pleuropneumoniae from tonsillar biopsies and tracheobronchial lavage fluids of pigs without a culture step. The detection of A. pleuropneumoniae in these samples was performed by PCR, by conventional culture and by bacteriology with immunomagnetic beads. The number of samples that were found positive by PCR was almost three times higher than the number of samples from which A. pleuropneumoniae was isolated by both bacteriological techniques. The detection of A. pleuropneumoniae in these samples allowed us to demonstrate its aerosol transmission to pigs under experimental conditions. The trial involved 18 specific pathogen free pigs. Six pigs, infected with A. pleuropneumoniae, were located in a unit A, together with four non-infected animals (contact pigs). Eight non-infected pigs (reporter pigs) were located in a unit B, adjacent to A. We detected A. pleuropneumoniae in samples from infected animals but also from 'contact' (unit A) and 'reporter' (unit B) pigs. The results of this study show that the simple preparation of the samples followed by the PCR assay may be a useful tool for epidemiological studies.

Characterization of a large transferrin-binding protein from Actinobacillus pleuropneumoniae serotype 7.

The binding of transferrin at the surface of Actinobacillus pleuropneumoniae (A. pp.) is mediated by two proteins of approximately 60 and 100 kDa. The 60 kDa protein has been shown to be highly divergent among different serotypes and to induce a serotype-specific protective immune response. In this study we have characterized the 100 kDa transferrin-binding protein of A. pp. serotype 7 and designated it as TfbB. The tfbB gene was found to be located immediately downstream of the tfbA gene. It was cloned and sequenced, and antibodies raised against the isolated recombinant protein detected, with a constant intensity, a 100 kDa protein in A. pp. serotypes 2, 4, 6, 7, 8, 9, 10 and 11, and a polypeptide of approximately 103 kDa in serotypes 1, 3, 5A and 12. In addition, comparative analysis of the deduced amino acid sequence showed more than 40% identity with the large transferrin-binding proteins of Neisseria meningitidis and Haemophilus influenzae. The TfbB protein was expressed in E. coli outer membranes in a conformation eliciting porcine transferrin-specific binding activity. Sera of pigs immunized with these TfbB-containing E. coli membranes recognized functional membrane-associated TfbB protein whereas no such reaction was observed upon immunization with isolated recombinant TfbB protein. A preliminary animal experiment showed that TfbB-containing outer membrane preparations from recombinant E. coli can reduce significantly the mortality of an A.pp. infection with the homologous strain.

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

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

Phenotypic variation in the outer membrane protein composition of Actinobacillus (Haemophilus) pleuropneumoniae: non-specific effect of exogenous pyridine nucleotide supply.

Actinobacillus pleuropneumoniae, grown in batch culture, was provided with pyridine nucleotides at concentrations that limited the final growth yield (pyridine nucleotide-deficient cultures) or did not determine the final extent of growth (pyridine nucleotide-sufficient cultures). Sarkosyl-extracted outer membranes from stationary phase, pyridine nucleotide-sufficient organisms contained 23,000 Mr and 43,000 Mr polypeptides that were absent (23,000 Mr) or barely detectable (43,000 Mr) in outer membranes from stationary phase, pyridine nucleotide-deficient organisms or exponential phase organisms. When growth ceased due to exhaustion of pyridine nucleotide, the ratio of the major outer membrane polypeptides (31,000, 38,000 and 69,000 Mr) was altered, becoming more like the ratio found with exponential phase organisms. Similar results were obtained when growth ceased due to glucose exhaustion at low biomass concentrations demonstrating that diverse nutrient deprivations can induce similar changes in outer membrane protein profile. All of these polypeptides were recognized by porcine immune sera indicating their production by A. pleuropneumoniae growing in vivo.