Immunochemical characterization of the outer membrane complex of Serratia marcescens and identification of the antigens accessible to antibodies on the cell surface

Serratia marcescens New CDC O14:H12 contains major outer membrane proteins of 43.5 kDal, 42 kDal (the porins) and 38 kDal (the OmpA protein) which can be separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Immunoblotting of whole cell or outer membrane preparations using antiserum raised against the whole cells revealed similar complex patterns of antigens. The OmpA protein was the major immunogen, although six other outer membrane proteins were also detected; the porins reacted only weakly with antibodies in this system. Immunoabsorption of antisera with whole cells showed that only the O antigenic chains of lipopolysaccharide and the H (flagella) antigens were accessible to antibody on the cell surface. Failure to detect the OmpA protein and other envelope antigens in this way suggests that their antigenic sites are not able to react with antibodies and are possibly masked by the O antigen.     

Treponema pallidum in gel microdroplets: a novel strategy for investigation of treponemal molecular architecture

Controversy exists regarding the constituents and antigenic properties of the Treponema pallidum outer membrane; a major point of contention concerns the cellular location(s) of the spirochaete's lipoprotein immunogens. To address these issues and circumvent problems associated with prior efforts to localize treponemal surface antigens, we developed a novel strategy for investigating T. pallidum molecular architecture. Virulent treponemes were encapsulated in porous agarose beads (gel microdroplets) and then probed in the presence or absence of Triton X-100. Intact., encapsulated treponemes were not labelled by monospecific antisera directed against four major T. pallidum lipoproteins or a candidate T. pailidum outer membrane protein (TpN50) with C-terminal sequence homology to Escherichia coli OmpA or by human or rabbit syphilitic serum. Each of these immunologic reagents, however, labelled encapsulated treponemes co-incubated with detergent. In contrast, antibodies generated against isolated T, pal-lidum outer membranes labelled intact organisms and the pattern of fluorescence was consistent with the distribution of rare outer membrane proteins visualized by freeze-fracture electron microscopy. In addition to providing strong evidence that the protein portions of treponemal lipoproteins are located within the periplasmic space, these studies have extended our understanding of the topographical relationships among T. pallidum cell envelope constituents. They also demonstrate the feasibility of generating antibodies against rare outer membrane proteins and detecting them on the surfaces of virulent treponemes.     

Characteristics of antisera against periodate-resistant membrane antigens from Neisseria gonorrhoeae

Crude outer membrane (OM) was prepared by extraction of bacteria of the Neisseria gonorrhoeae strains 8551. V, and VII, with an EDTA-containing buffer. The preparations contained the lipopolysaccharide (LPS) and at least 10 proteins as shown by SDS-polyacrylamide gel electrophoresis. Immunization of rabbits with untreated OM resulted in production of antibodies against several antigens, including LPS. Antisera raised against periodate-treated OM did not contain antibodies against LPS. These latter antisera agglutinated heat-treated (100 degrees C, 60 min) gonoccal cells by means of antibodies to one or more common agglutinogens and against a strain-specific agglutinogen that was susceptible to digestion with proteolytic enzymes. Both side agglutination and a plate agglutination test could be used to detect antibodies against these agglutinogens.     

Protein import into yeast mitochondria is inhibited by antibodies raised against 45-kd proteins of the outer membrane.

Import of several precursor proteins into isolated yeast mitochondria is inhibited by rabbit antiserum raised against the total mitochondrial outer membrane or against electrophoretically purified 45-kd outer membrane proteins. Antisera against other outer membrane proteins are only marginally active or inactive. Inhibition by the antiserum against 45-kd proteins is only weak with untreated mitochondria, but reaches 80-90% with mitochondria that had been pretreated with 0.1 mg/ml trypsin. This trypsin pretreatment by itself inhibits precursor import only slightly (30-50%). Selective inhibition of import does not correlate with binding of the various IgGs to the mitochondrial surface and is also observed with the corresponding Fab fragments. Inhibition by antibodies against 45-kd outer membrane proteins strongly suggests the existence of a mitochondrial surface protein mediating protein import and offers a means of isolating this protein.     

Raising antibodies against OprD, an outer membrane protein of Pseudomonas aeruginosa using translational fusions to MalE.

OprD is an outer membrane porin of Pseudomonas aeruginosa that mediates uptake of basic amino acids, peptides as well as carbapenem antibiotics. Polyclonal antibodies were raised against the OprD porin by creating protein fusions between the Escherichia coli maltose binding protein and four OprD fragments. These were expressed in E. coli and shown to be exported to the periplasm. The fusion proteins were purified by amylose affinity chromatography and used to immunize rabbits intramuscularly. We established that MalE fusions to OprD fragments retain maltose and amylose binding activities in vivo and in vitro, confirming proper folding of the MalE domain of hybrid proteins. Furthermore, we demonstrate that this strategy can be used to obtain specific antibodies against bacterial outer membrane proteins (OMPs).     

Antigenic cross-reactivity of major outer membrane proteins in enterobacteriaceae species.

The protein constituents in the outer membrane (OM) of several serotypes of Escherichia coli and some other Enterobacteriaceae cross-reacted antigenically. Solubilized OM preparations of these bacteria were applied in interfacial precipitin tests to antisera elicited in rabbits against whole bacterial cells, absorbed with their appropriate lipopolysaccharide before testing. The resulting immunecomplexes were analysed on polyacrylamide gels. Protein profiles of the immunoprecipitates showed a considerable antigenic cross-reactivity of outer membrane proteins between most E. coli serotypes. Cross-reactivity, though substantially lower, was also found with OM from three other Enterobacteriaceae species, but was not detectable with Pseudomonas aeruginosa OM. When OM preparations were solubilized at room temperature, the peptidoglycan-bound proteins in the molecular weight range 37,000 to 41,000 predominated in the protein profiles of the immunecomplexes. In profiles of immunecomplexes obtained with boiled OM preparations, a heat-modifiable protein (mol. wt 33,000) predominated. The major OM proteins of the Gram-negative bacterium may therefore play a role as common surface antigens of the family of Enterobacteriaceae.     

Isolation and Characterization of Three Porin-Like Proteins from Vibrio vulnificus: Effect of Different Growth Media on Their Production

The effect of the culture media on the composition of the outer membrane protein of Vibrio vulnificus strain 393 from human blood was examined. Only one major outer membrane protein, with an apparent molecular weight of 37,000 (37K protein) and 34,000 (34K protein), was formed in the cells grown in 3% NaCl-BHI broth and chemically defined medium, respectively. The production of one major outer membrane protein was also observed in other isolates from humans and asari clam when they were grown in 3% NaCl-BHI broth. On the other hand, three major outer membrane proteins, with apparent molecular weights of 48,000 (48K protein), 37,000 (37K protein), and 34,000 (34K protein), were produced in the cells grown in 3% NaCl-nutrient broth. Three proteins, 48K, 37K, and 34K from strain 393, were purified and the amino acid compositions were determined. Although there was a little difference in the composition of amino acid among three proteins, the amino acid compositions of the three porin-like proteins showed characteristic properties of the porins of Escherichia coli and Salmonella typhimurium. Immunoblot analysis of the outer membrane proteins from four vibrios, E. coli, and S. typhimurium using monospecific antisera against these three porin-like proteins showed that only the antiserum against 37K protein cross-reacted with the outer membrane proteins from all the strains tested.     

Outer membrane protein H for protective immunity against Pasteurella multocida

Pasteurella multocida, a Gram-negative facultative anaerobic bacterium, is a causative animal pathogen in porcine atrophic rhinitis and avian fowl cholera. For the development of recombinant subunit vaccine against P. multocida, we cloned and analyzed the gene for outer membrane protein H (ompH) from a native strain of Pasteurella multocida in Korea. The OmpH had significant similarity in both primary and secondary structure with those of other serotypes. The full-length, and three short fragments of ompH were expressed in E. coli and the recombinant OmpH proteins were purified, respectively. The recombinant OmpH proteins were antigenic and detectable with antisera produced by either immunization of commercial vaccine for respiratory disease or formalin-killed cell. Antibodies raised against the full-length OmpH provided strong protection against P. multocida, however, three short fragments of recombinant OmpHs, respectively, showed slightly lower protection in mice challenge. The recombinant OmpH might be a useful vaccine candidate antigen for P. multocida.     

The capsular polysaccharide of Haemophilus influenzae type B prevents killing by complement and antibodies against outer membrane protein a

Abstract The ability of antibodies, raised in rabbits against purified outer membrane protein a ( M _r 47 000) of Haemophilus influenzae type b, to promote complement-dependent killing of these encapsulated organisms was investigated. Killing of encapsulated strains was not induced by these antibodies in conjunction with either human, mouse, rabbit or guinea-pig complement. Acapsular mutants were effectively killed by complement in the presence of antibodies against protein a . Killing was dependent on the presence of the 47-kDa protein a and was not influenced by the outer membrane protein subtype or lipopolysaccharide serotype of the strain. The killing-promoting activity could be absorbed from the sera with cells of strains with the same protein a , purified protein a , but not by purified lipopolysaccharide and capsular polysaccharide. Binding experiments showed that the encapsulated strain and its acapsular mutant bound antibodies against protein a with the same rate and to the same extent, indicating that the capsule probably interferes with complement activation or insertion of the membrane attack complex into the bacterial cell.     

Identification of the outer membrane proteins of Campylobacter pyloridis and antigenic cross-reactivity between C. pyloridis and C. jejuni

The outer membrane and surface exposed proteins of four strains of the gastric Campylobacter-like organism Campylobacter pyloridis were identified by SDS-PAGE of Sarkosyl-insoluble membranous material and 125I-surface-labelled whole bacteria. Although constant outer membrane proteins (molecular mass 61, 54 and 31 kDa) were observed in these strains, several variable 125I-labelled surface proteins were detected. C. pyloridis does not appear to express a single surface-exposed major outer membrane protein like that of C. jejuni and C. coli. Putative flagella proteins were identified from isolated flagella and acid-extractable surface material and by immunoblotting with anti-flagella antibodies. Several major protein antigens were observed by immunoblotting with anti-C. pyloridis antisera. At least two of these antigens cross-reacted with anti-C. jejuni antiserum. This cross-reaction appears to be caused primarily by flagellar antigens. However, one major protein antigen (61 kDa) was not cross-reactive with C. jejuni and may, therefore, be useful in serological tests for the specific diagnosis of C. pyloridis infections.     

Surface antigen analysis of group B Neisseria meningitidis outer membrane by monoclonal antibodies: Identification of bactericidal antibodies to class 5 protein

Twenty-four monoclonal antibodies (mAbs) against group B Neisseria meningitidis surface antigens were analyzed by immunoenzymatic assays and by a bactericidal test. Two mAbs were specific to polysaccharide B and one to lipopolysaccharide. The others were directed against outer membrane proteins ranging in molecular mass from 25 to 200 kDa. The outer membrane protein epitopes recognized by the mAbs were not conformational and were located on the outer surface of the microorganism. Linear epitopes on the class 5 protein, exposed on the surface of the membrane, were able to induce bactericidal antibodies to the homologous strain. The susceptibility of Neisseria meningitidis to these antibodies was unchanged when this organism was cultivated under conditions of iron depletion. These results demonstrate that peptides derived from class 5 proteins are potentially important in synthetic peptide or in recombinant protein vaccines containing linear bactericidal epitopes.     

Identification of Epstein-Barr virus terminal protein 1 (TP1) in extracts of four lymphoid cell lines, expression in insect cells, and detection of antibodies in human sera.

The terminal proteins TP1 and TP2 are putative products of Epstein-Barr virus (EBV) genes expressed during the latent cycle of the virus. They are predicted to code for 53- and 40-kilodalton integral membrane proteins. We used the baculovirus Autographa californica nuclear polyhedrosis virus as an expression vector to produce TP1 in large amounts in insect cells. The DNA sequences used to express TP1 originated from a TP1 cDNA derived from an M-ABA/CBL1 cDNA library. Rabbit antisera raised against procaryotic TP1 fusion proteins recognized a monomer and a dimer of the recombinant TP1 protein in the infected insect cells. Immunofluorescence studies of living insect cells showed that the recombinant protein is located in the plasma membrane. The insect cells infected with the recombinant baculovirus producing TP1 provided a test system to screen human antisera for TP1 antibodies. A total of 168 human EBV-positive and EBV-negative antisera were studied. TP1 antibodies were detected only in sera from nasopharyngeal carcinoma patients (16 out of 42). Rabbit antiserum raised against the recombinant TP1 protein expressed in the baculovirus system specifically recognized a protein of about 54 kilodaltons in the lymphoblastoid cell lines M-ABA and M-ABA/CBL1 and in the Burkitt's lymphoma cell lines BL18 and BL72. This protein could be located in the total membrane fraction of M-ABA cells and is upregulated by treating the cells with 12-O-tetradecanoylphorbol-13-acetate.     

Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes.

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface. In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.     

Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface.In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.     

Combinations of polyclonal or monoclonal antibodies to proteins of the outer membranes of the two infectious forms of vaccinia virus protect mice against a lethal respiratory challenge

Previous studies demonstrated that antibodies to live vaccinia virus infection are needed for optimal protection against orthopoxvirus infection. The present report is the first to compare the protective abilities of individual and combinations of specific polyclonal and monoclonal antibodies that target proteins of the intracellular (IMV) and extracellular (EV) forms of vaccinia virus. The antibodies were directed to one IMV membrane protein, L1, and to two outer EV membrane proteins, A33 and B5. In vitro studies showed that the antibodies to L1 neutralized IMV and that the antibodies to A33 and B5 prevented the spread of EV in liquid medium. Prophylactic administration of individual antibodies to BALB/c mice partially protected them against disease following intranasal challenge with lethal doses of vaccinia virus. Combinations of antibodies, particularly anti-L1 and -A33 or -L1 and -B5, provided enhanced protection when administered 1 day before or 2 days after challenge. Furthermore, the protection was superior to that achieved with pooled immune gamma globulin from human volunteers inoculated with live vaccinia virus. In addition, single injections of anti-L1 plus anti-A33 antibodies greatly delayed the deaths of severe combined immunodeficiency mice challenged with vaccinia virus. These studies suggest that antibodies to two or three viral membrane proteins optimally derived from the outer membranes of IMV and EV, may be beneficial for prophylaxis or therapy of orthopoxvirus infections.     

Fibronectin is a component of the sodium dodecyl sulfate-insoluble transglutaminase substrate

Liver plasma membranes contain a morphologically distinct protein complex which serves as a substrate for the plasma membrane-associated transglutaminase. The complex, which appears as a two-dimensional sheet, is insoluble in sodium dodecyl sulfate and reducing agents and has been named SITS for sodium dodecyl sulfate-insoluble transglutaminase substrate (Tyrrell, D. J., Sale, W. S., and Slife, C. W. (1988) J. Biol. Chem. 263, 1946-1951). Polyclonal antibodies raised against SITS were used to probe for soluble constituents of the matrix. Immunoblots showed that proteins of 230, 35, and 32 kDa reacted with the anti-SITS antiserum when the soluble fraction from a liver homogenate was examined. The 230-kDa protein was identified as fibronectin after observing cross-reactivity of anti-SITS antiserum with authentic fibronectin and cross-reactivity of anti-fibronectin antiserum with the 230-kDa cytosolic protein and purified SITS. Preincubating anti-SITS antiserum with purified fibronectin decreased immunostaining of the 230-kDa cytosolic protein and authentic fibronectin. Immunoblots of the plasma membrane fraction using anti-SITS and anti-fibronectin antisera showed that both antisera reacted with proteins at the top of the stacking gel (SITS) and of 230 kDa. In addition, the anti-SITS antiserum reacted with proteins of 85, 35, and 32 kDa. Immunofluorescence microscopy revealed that the anti-SITS and anti-fibronectin antisera both react with isolated SITS and with the same filamentous structures associated with intact plasma membranes. These studies show that fibronectin is a component of the plasma membrane matrix, SITS. This finding is consistent with the proposed role of this matrix which is to mediate cell-cell adhesion between hepatocytes in the tissue.     

Antigenic variability of the outer membrane antigens of Legionella pneumophila serogroups 1 to 8

The outer membrane proteins of Legionella pneumophila serogroups 1 to 8 were prepared from broken cells by selective solubilization using sodium lauryl sarcosinate. The isolated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets. Rabbit antisera against each of the eight serogroups of L. pneumophila were obtained by immunizing each animal with live bacteria. The transferred proteins were revealed using these antisera and peroxidase-labeled swine anti-rabbit immunoglobulins. Antigenic determinants common to all eight serogroups were found in at least three outer membrane antigens (19, 29, and 45 kilodaltons (kDa)). However, cross-absorption experiments revealed that these three antigens were immunologically related, but not identical among serogroups. The antigenic relationships observed with two of these three antigens correlated well with cross-reactions observed in immunofluorescence. When a monoclonal antibody directed against L. pneumophila serogroup 1 lipopolysaccharide was used to reveal a blot of serogroup 1 outer membrane antigens, the 29- and 45-kDa bands appeared. This demonstrates a strong association between lipopolysaccharide and outer membrane proteins.     

The identification of outer membrane proteins and flagella of Campylobacter jejuni

The outer membrane proteins of five clinical isolates of Campylobacter jejuni were identified by 125I-surface labelling and SDS-PAGE of outer membrane preparations. All isolates expressed a major outer membrane protein of variable molecular weight (43 000-46 000: 43K-46K). Several constant surface proteins were also identified including a 27K protein which was surface-exposed and acid-extractable but was not present in the outer membrane preparations. Isolated flagella comprised a major 62K protein and a minor 87K protein. Both proteins were absent in an aflagellate variant. The 62K protein was immunoblotted and immunoprecipitated by rabbit anti-flagella antisera.     

Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the Asd+ vector pYA292, and the construct was introduced into the avirulent delta cya delta crp delta asd S. typhimurium chi 3987 for oral immunization of birds. The gene encoding the 21-kDa protein was expressed equivalently in B. avium 197, delta asd E. coli chi 6097, and S. typhimurium chi 3987 and was localized primarily in the cytoplasmic membrane and outer membrane. In preliminary studies on oral inoculation of turkey poults with S. typhimurium chi 3987 expressing the gene encoding the B. avium 21-kDa protein, it was determined that a single dose of the recombinant Salmonella vaccine failed to elicit serum antibodies against the 21-kDa protein and challenge with wild-type B. avium 197 resulted in colonization of the trachea and thymus with B. avium 197.     

Locus-specific detection of HLA-DQ and -DR antigens by antibodies against synthetic N-terminal octapeptides of the beta chain

Antibodies against synthetic peptides representing the class-II antigen HLA-DR and -DQ beta chain N-terminal sequences were prepared in rabbits. The two octapeptides only share two amino acids and enzyme-linked immuno-assays showed the antisera only to bind to its own antigen. Both peptide antisera detected a 29 kDa component in immunoblots of Raji and AL-34 cell plasma membrane proteins separated by SDS gel electrophoresis. The binding of either N-terminal peptide antiserum was selectively inhibited only by the peptide used as antigen. Indirect immunofluorescence analysis by flow cytofluorometry showed specific surface immunofluorescence in 1:100-1:1000 dilutions in lymphoblastoid and blood mononucleated cells. In the latter the binding was primarily confined to monocytes and a subpopulation of lymphocytes. It is concluded that locus-specific immunological reagents to distinguish between beta chains of HLA-DR and -DQ have been prepared by the preparation by the production of antibodies against the N-terminal sequences of each polypeptide.