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β-barrel membrane proteins are essential for nutrient import, signalling, motility and survival. In Gram-negative bacteria, the β-barrel assembly machinery (BAM) complex is responsible for the biogenesis of β-barrel membrane proteins, with homologous complexes found in mitochondria and chloroplasts. Here we describe the structure of BamA, the central and essential component of the BAM complex, from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane β-barrel domain. Three structural features shed light on the mechanism by which BamA catalyses β-barrel assembly. First, the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the β-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. And third, the β-barrel can undergo lateral opening, suggesting a route from the interior cavity in BamA into the outer membrane.

Polyserositis caused by Haemophilus parasuis is an important disease that affects mostly weaned pigs. Recent studies have shown that virulence can differ among strains recovered from distinct body sites and also that it may be related to the presence of certain outer membrane proteins (OMPs). The objective of this study was to compare the OMP and DNA profiles of H. parasuis strains isolated from systemic and respiratory sites from diseased and healthy pigs. Strains evaluated in this study were processed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and repetitive-PCR techniques. Two experiments were conducted in order to better define the relationship among genotype, phenotype, and site of isolation. Experiment 1 included 53 H. parasuis isolates recovered from healthy and diseased pigs from unrelated herds. Experiment 2 included 31 isolates of H. parasuis obtained from diseased pigs involved in an outbreak in a large, multifarm system. Results showed that strains recovered from systemic sites had more homogeneous OMP and DNA profiles than those isolated from respiratory sites. Evaluation of isolates involved in the multifarm outbreak showed that only two H. parasuis strains were causing disease. These strains had homogeneous OMP and DNA profiles. However, it was noted that these two parameters were unrelated, since strains classified in the same genotype group expressed different OMP profiles. The homogeneity of OMP and DNA profiles of strains isolated from systemic sites strongly suggests the existence of clonal relationships between virulent strains and also suggests that expression of certain OMP profiles may be related to virulence.

Many pathogens of the Pasteurellaceae and Neisseriaceae possess a surface receptor that binds transferrin (Tf) as an initial step in an iron acquisition process. This receptor is comprised of two proteins, transferrin binding protein A (TbpA) and transferrin binding protein B (TbpB). Since the ability to recognize the iron-loaded form of Tf preferentially would be a useful attribute of these receptors, we examined this property in a number of bacterial species. In solid-phase binding assays with isolated membranes, only the receptor from Moraxella catarrhalis was capable of preferentially binding iron-loaded Tf. In a competitive affinity isolation assay which enabled us to resolve TbpA and TbpB, TbpA from all tested species was shown to bind both apo and iron-loaded Tf. Under these assay conditions TbpB from M. catarrhalis, Haemophilus somnus and Pasteurella haemolytica discriminated between apo and holo Tf, whereas TbpB from Neisseria meningitidis showed no discrimination. The ability of TbpB from N. meningitidis to bind iron-saturated hTf preferentially became evident in a TbpA- background or by using recombinant TbpB. In binding assays with recombinant fusion proteins, both intact TbpB and the N-terminal half of TbpB from all the tested species preferentially bound Fe-loaded Tf, indicating that this may be a conserved mechanism by which these organisms optimize their ability to acquire iron., (Copyright 1998 Academic Press.)

A fourth lipoprotein gene from Pasteurella haemolytica A1 was cloned and characterized. The plpD gene encodes a 31-kDa lipoprotein (Plp4) which could be recognized in Western immunoblot by sera from calves immunized with the culture supernatant vaccine Presponse. This suggests that Plp4 is one of the immunogenic molecules in the P. haemolytica A1 culture supernatant. The lipoprotein nature of Plp4 was confirmed by labelling with [3H]palmitate and inhibition of leader peptide cleavage with globomycin. A homology search with databanks showed extensive homology between Plp4 and a 31-kDa antigen from Haemophilus somnus and a 19.2-kDa antigen from Neisseria meningitidis. Additional homology of the distal half of Plp4 was identified with a number of bacterial outer membrane proteins belonging to the OmpA family. Plp4 appears to be a novel type of outer membrane protein that contains motifs typical of OmpA but which is also lipid modified.

Bacterial pathogens have evolved various strategies to acquire iron from the iron-restricted environment found in mammalian hosts. Borrelia burgdorferi should be no different with regard to its requirement for ferric iron, and previous studies have suggested that transferrin (Tf) may be a source of iron in vivo. By probing blots with Tf conjugated to horseradish peroxidase, we have identified an outer membrane protein (28 kDa) from B. burgdorferi B31 that bound holo-Tf but not apo-Tf. The 28-kDa protein bound human, rat, or mouse Tf and was produced only by low-passage (less than passage 5), virulent isolates of strain B31. In addition, the Tf-binding protein (Tbp) from strain B31 retained the ability to bind Tf after treatment with 2% sodium dodecyl sulfate-1% beta-mercaptoethanol and heating to 100 degrees C for 5 min. These properties are remarkably similar to those of the Tbp of Staphylococcus aureus and Tbp2 from Neisseria meningitidis. B. burgdorferi Sh-2-82 produced an outer membrane protein different in size, i.e., 26 kDa, but with properties similar to those of to the protein from strain B31, suggesting variation in B. burgdorferi Tbps. The exact role of the 28-kDa protein in iron acquisition by B. burgdorferi remains to be determined.

The present study was initiated to identify the region(s) of ovotransferrin involved in binding to the bacterial transferrin receptors from Haemophilus paragallinarum and Haemophilus avium. Ovotransferrin was digested with either trypsin or thermolysin to obtain its N-lobe and C-lobe fragments. The individual fragments were then purified by a combination of gel exclusion and ion-exchange chromatography. Solid phase binding experiments with the individual fragments demonstrated that the C-lobe fragments blocked the binding of horse radish peroxidase-conjugated ovotransferrin to the transferrin receptors and that much higher concentrations of the N-lobe fragment were required for any detectable blocking. Affinity isolation of the bacterial transferrin receptor from the two Haemophilus species revealed that both native ovotransferrin and its C-lobe fragment were capable of isolating two iron repressible outer membrane proteins. These 95 and 60 kDa outer membrane proteins correspond to Tbp1 and Tpb2, respectively. In contrast, the N-lobe fragment was capable of isolating Tbp2 of H. paragallinarum but not that of H. avium. The inability of the N-lobe and C-lobe fragments from ovotransferrin and human transferrin to support the growth of iron-limited cultures of H. paragallinarum and Neisseria meningitidis, respectively, suggested that interaction with both lobes is necessary for efficient iron acquisition.

A polyclonal antibody against the 35 kDa major outer-membrane protein of Pasteurella multocida cross-reacted with the 40 kDa major outer-membrane protein of Actinobacillus pleuropneumoniae and the 42 kDa major outer-membrane protein of Haemophilus parasuis. The N-terminal amino-acid sequences of these proteins revealed a strong homology with the putative 35 kDa porin protein of Pasteurella multocida (66.7 and 76.2%, respectively). Significant homologies were also evident between the 40 kDa and the 42 kDa protein (76.2%), and with non-specific porins of gram-negative bacteria.

In immunoblot analysis, a murine monoclonal antibody (MAb), 27-1, which was produced to an outer membrane protein (OMP) of Haemophilus somnus, showed that a major OMP is heat modifiable, having a molecular mass of 28 kDa when the N-lauroylsarcosine-insoluble OMP preparation was solubilized at 60 degrees C and a mass of 37 kDa when the OMP preparation was solubilized at 100 degrees C. The heat-modifiable OMP reacted intensely with convalescent sera obtained from calves with experimental H. somnus pneumonia in immunoblot analysis. Immunoelectron microscopic and antibody absorption studies revealed that the MAb 27-1 epitope was not surface exposed on the intact bacterium. However, a decrease in antibody reactivity to the heat-modifiable OMP in immunoblot analysis after absorption of convalescent serum with intact bacterial cells of H. somnus suggests that a surface-exposed portion of the heat-modifiable OMP is expressed on the intact bacterium. MAb 27-1 reacted with 45 of 45 strains of H. somnus tested in immunoblot analysis. The apparent molecular mass of the antigen varied among strains, and five reactivity patterns demonstrated by MAb 27-1 were observed. MAb 27-1 also reacted with six species in the family Pasteurellaceae, Escherichia coli, and Salmonella dublin, but not with the other eight species of gram-negative bacteria. The heat-modifiable OMP of H. somnus showed immunological cross-reactivity with the OmpA protein of E. coli K-12 and significant N-terminal amino acid sequence homology with the OmpA proteins of gram-negative bacteria. We conclude that a major, 37-kDa heat-modifiable OMP of H. somnus, which elicits an antibody response in H. somnus-infected animals, is a common antigen among H. somnus strains tested and is structurally related to the OmpA protein of E. coli.

Haemophilus parainfluenzae synthesizes an outer membrane protein adhesin which mediates binding to oral streptococci, salivary pellicle, and neuraminidase-treated erythrocytes. An indirect gold labeling technique and immunoelectron microscopy verified the location of this outer membrane protein. Further, a clustering of gold particles was observed in irregular patches at the cell surface.