Gene cloning of Porphyromonas gingivalis specific antigens recognized by serum of adult periodontitis patient.

1. Porphyromonas gingivalis is believed an important pathogen of adult periodontitis. A gene library of P. gingivalis 381 was constructed in lambda phage vector L47.1. The library was probed with serum obtained from patients of severe adult periodontitis. Two clones, lambda MDBG101 and lambda MDBG103 which were expressed, 200 and 160 kDa respectively, were selected and further studied. 2. The expressed antigens in these two clones were also reacted with rabbit antiserum against whole cells, capsular fraction and cell surface fraction of P. gingivalis. 3. Genes coding protein antigens in lambda MDBG101 and lambda MDBG103 were subcloned into high-copy-number plasmid vector pACYC184 and subclones obtained were designated as MD101 and MD103. Recombinant plasmids, pMD101 and pMD103, differed in their restriction endonuclease digestion. 4. Immunodiffusion analysis showed that cloned proteins from MD101 and MD103 reacted with antiserum against P. gingivalis but did not react with antiserum against Prevotella intermedia, Prevotella loescheii and Prevotella asaccharolyticus. 5. These data suggest that P. gingivalis species-specific antigens has been successfully cloned and expressed in Escherichia coli. Since these cloned specific antigens were recognized by adult periodontitis patient sera, the recombinant antigen will be useful material for the development of serodiagnosis of P. gingivalis infection in adults periodontitis.     

Purification and characterization of a DnaK-like and a GroEL-like protein from Porphyromonas gingivalis

Heat-shock proteins of Porphyromonas gingivalis were demonstrated and two of them were purified and further characterized. The amplified de novo synthesis of two different proteins, with apparent molecular weights of 75 kDa and 68 kDa, was observed by autofluorography when a P. gingivalis culture incubated in a 14C-labeled amino acid mixture was shifted from 37 degrees C to 44 degrees C. Both proteins possessed ATP-binding abilities and were purified to almost homogeneity employing affinity chromatography on ATP-agarose followed by preparative SDS-PAGE. Purified 75 kDa and 68 kDa proteins had isoelectric points of 4.4 and 4.6, respectively. They were shown to be immunoreactive with commercial anti-DnaK and anti-GroEL polyclonal antibodies, respectively. Immunoblotting analysis of whole cells using antiserum raised against each purified protein from P. gingivalis, confirmed elevated synthesis of both proteins during thermal shock. A GroEL protein reacted strongly with antiserum against the 68 kDa protein. However, a DnaK protein reacted weakly with antiserum to the 75 kDa protein. Analysis of the N-terminal amino acid sequence of the DnaK-like protein (75 kDa) showed a high degree of homology with those of the HSP70 family including both prokaryotic and eukaryotic cells. The N-terminal amino acid analysis of the GroEL-like protein (68 kDa) indicated that it was identical to those of cloned GroEL homologues from P. gingivalis.     

Serological properties and immunobiological activities of lipopolysaccharides from black-pigmented and related oral Bacteroides species

Lipopolysaccharides (LPS) from five species of oral Bacteroides, B. gingivalis strains 381 and ATCC 33277, B. oralis ATCC 33269, B. loescheii ATCC 15930, B. intermedius ATCC 25611 and B. corporis ATCC 33547, were extracted from whole cells by the phenol/water procedure, and subsequently purified by treatment with nuclease and ultracentrifugation. The LPS were composed of hexoses, glucosamine, fatty acids and phosphorus. Heptose and 2-keto-3-deoxyoctonate were not detected. The LPS preparations from B. gingivalis strains 381 and ATCC 33277 presented very similar SDS-polyacrylamide gel electrophoresis patterns when stained with ammoniacal silver. They produced a fused precipitin band against an antiserum to B. gingivalis 381 LPS in immunodiffusion tests. Antisera raised against the LPS from B. loescheii and B. intermedius reacted with the LPS prepared from all the oral Bacteroides strains except those of B. gingivalis. All the LPS preparations were mitogenic for spleen cells of BALB/c (nu/nu) mice, but not for thymus cells from C3H/HeN mice. The LPS induced marked mitogenic responses and polyclonal B cell activation for spleen cells of not only C3H/HeN (LPS responder) mice, but also C3H/HeJ (LPS nonresponder) mice. The mitogenic responses were not suppressed significantly upon addition of polymyxin B to the reaction mixture. These LPS also enhanced interleukin-1 production by murine peritoneal macrophages and mouse cell line J744. 1 macrophages. Hydrolysis of B. gingivalis ATCC 33277 LPS in 1 m-HCl at 100 degrees C for 1 h yielded lipid and polysaccharide. The lipid portion was largely composed of fatty acids and glucosamine, and was mitogenic for spleen cells from C3H/HeJ as well as C3H/HeN mice, while the polysaccharide portion induced no significant mitogenic responses under similar experimental conditions.     

Iron-regulated outer membrane proteins in the periodontopathic bacterium, Bacteroides gingivalis

Hemin has been implicated in the pathogenesis of the oral pathogen, Bacteroides gingivalis. In order to elucidate the role of hemin (iron) in the growth and expression of outer membrane proteins, B. gingivalis strain W50 was grown with and without hemin to induce iron-limitation. Cells grew slower under iron stress and growth was completely inhibited in the absence of added hemin. The outer membrane protein profiles of B. gingivalis grown under iron-replete and iron-restricted conditions were studied by extrinsic radiolabelling with [125I] and polyacrylamide gel-electrophoresis. The induction of 10 surface proteins, with apparent molecular weights of 26, 29, 50, 56, 58, 60, 62, 71, 77, and 80 Kd, was observed in B. gingivalis grown under iron-restricted conditions. These proteins were repressed under iron-replete conditions. We postulate the involvement of the iron-regulated proteins in hemin uptake and virulence in B. gingivalis.     

Bacteroides gingivalis and Bacteroides intermedius recognize different sites on human fibrinogen.

Bacteroides (Porphyromonas) gingivalis and Bacteroides (Porphyromonas) intermedius have been implicated in the etiology of human periodontal diseases. These organisms are able to bind and degrade human fibrinogen, and these interactions may play a role in the pathogenesis of periodontal disease. In attempts to map the bacterial binding sites along the fibrinogen molecule, we have found that strains of B. gingivalis and B. intermedius, respectively, recognize spatially distant and distinct sites on the fibrinogen molecule. Isolated reduced and alkylated alpha-, beta-, and gamma-fibrinogen chains inhibited binding of 125I-fibrinogen to both Bacteroides species in a concentration-dependent manner. Plasmin fragments D and to some extent fragment E, however, produced a concentration-dependent inhibition of 125I-fibrinogen binding to B. intermedius strains but did not affect binding of 125I-fibrinogen to B. gingivalis strains. Radiolabeled fibrinogen chains and fragments were compared with 125I-fibrinogen with respect to specificity and reversibility of binding to bacteria. According to these criteria, gamma chain most closely resembled the native fibrinogen molecule in behavior toward B. gingivalis strains and fragments D most closely resembled fibrinogen in behavior toward B. intermedius strains. The ability of anti-human fibrinogen immunoglobulin G (IgG) to inhibit binding of 125I-fibrinogen to B. intermedius strains was greatly reduced by absorbing the IgG with fragments D. Absorbing the IgG with fragments D had no effect on the ability of the antibody to inhibit binding of 125I-fibrinogen to B. gingivalis strains. A purified staphylococcal fibrinogen-binding protein blocked binding of 125I-fibrinogen to B. intermedius strains but not to B. gingivalis strains.     

Immune-enhanced phagocytosis of Neisseria gonorrhoeae by macrophages: characterization of the major antigens to which opsonins are directed

antisera were prepared in rabbits against whole organisms of colony type 1 Neisseria gonorrhoeae strains F62 and B (fron gonococcal urethritis) and 7122 (a strain typical of those associated with disseminated gonococcal infection), and against purified outer membrane components from the same strains including pili and principal outer membrane protein. Antibody levels to pili, principal outer membrane protein and lipopolysaccharide were determined using a quantitative enzyme-linked immunosorbent assay. Each antiserum was heat-inactivated and tested for opsonic for its homologous strain, and this immune-enhanced phagocytosis was decreased by adsorption with homologous purified outer membrane components: pili greater than lipopolysaccharide greater than principal outer membrane protein. Opsonic activity was approximately equal for antiserum to purified pili and antiserum to the whole organisms for each of the three strains, and purified antibody to pili was highly opsonic. The F(ab')2 fragments of antibody to pili were not opsonic, indicating a role for the Fc receptor on the phagocyte membrane in immune-enhanced phagocytosis of gonococci.     

Analysis of outer membrane proteins which are associated with growth of Bacteroides thetaiotaomicron on chondroitin sulfate.

By analyzing outer membrane proteins of Bacteroides thetaiotaomicron on two-dimensional polyacrylamide gels, we were able to identify 10 protein spots that were associated with growth on chondroitin sulfate but not with growth on glucuronic acid or other monosaccharides. These proteins were distinct from the outer membrane polypeptides that were associated with growth on two other negatively charged polysaccharides, polygalacturonic acid and heparin. Of the 10 protein spots that were associated with growth on chondroitin sulfate, 4 could be detected on immunoblots with antiserum that had been raised against outer membranes from bacteria grown on chondroitin sulfate and then cross-adsorbed with membranes from bacteria grown on glucose. Synthesis of these four proteins appeared to be regulated coordinately with synthesis of the two enzymes that degrade chondroitin sulfate, chondroitin lyase I and II. Although one of the four proteins (Mr 110,000) was similar in molecular weight to the chondroitin lyases, the cross-adsorbed antiserum which detected this outer membrane protein did not cross-react with either of these two enzymes.     

Characterization of Bacteroides gingivalis by direct fluorescent antibody staining and cellular fatty acid profiles

Bacteroides gingivalis is a newly proposed species which includes strains isolated from the mouth. Thirteen strains of B. gingivalis isolated from three geographic locations in the United States and France were examined with direct fluorescent antibody staining and analysis of total cellular fatty acids and compared with 16 strains of B. asaccharolyticus of nonoral origin by the same methods. Bacteroides gingivalis strains reacted with the B. gingivalis conjugate (fluorescein isothiocyanate labeled antibody reagent) only, while the B, asaccharolyticus strains reacted with the B. asaccharolyticus conjugate only. The B. gingivalis strains showed negative fluorescence with fluorescein isothiocyanate conjugates for other black-pigmented Bacteroides species. The specificity of the B. gingivalis conjugate was demonstrated by its failure to stain 88 strains of aerobic and anaerobic bacteria other than B. gingivalis. The fatty acid profiles of B. gingivalis and B. asaccharolyticus were readily distinguishable. The B. gingivalis profile was also distinguishable from those of other pigmenting Bacteroides species on the basis of concentration ratios among the characteristic components. These results support the species separation of B. gingivalis and B. asaccharolyticus. Further, they indicate the usefulness of cellular fatty acid profiles as an adjunct to the use of specific fluorescent antibody conjugates for identification of Bacteroides species.     

Molecular cloning and expression of a major surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis in Escherichia coli

A major immunodominant surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis 381 has been purified and its amino-terminal amino acid sequence has been determined. Using oligonucleotide probes corresponding to the sequence, we identified a recombinant plasmid clone carrying a single 4.2-kb BamHI fragment from pUC19 libraries of P. gingivalis. The BamHI fragment transferred to the bacteriophage T7 RNA polymerase/promoter expression vector system produced a slightly larger (77-kDa) protein, a precursor form, immunoreactive to the antibody against the 75-kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 75-kDa protein. Genomic Southern analysis revealed a single copy of the 75-kDa protein gene per genome among all P. gingivalis strains tested, and that no homologous genes are present in other black-pigmented Bacteroides species. These observations suggest that the 75-kDa protein gene may be useful as a specific DNA probe to classify or to detect this organism.     

Localization of a Porphyromonas gingivalis 26-kilodalton heat-modifiable, hemin-regulated surface protein which translocates across the outer membrane.

We recently identified a 26-kDa hemin-repressible outer membrane protein (Omp26) expressed by the periodontal pathogen Porphyromonas gingivalis. We report the localization of Omp26, which may function as a component of a hemin transport system in P. gingivalis. Under hemin-deprived conditions, P. gingivalis expressed Omp26, which was then lost from the surface after a shift back into hemin-rich conditions. Experiments with 125I labeling of surface proteins to examine the kinetics of mobilization of Omp26 determined that it was rapidly (within less than 1 min) lost from the cell surface after transfer into a hemin-excess environment. When cells grown under conditions of hemin excess were treated with the iron chelator 2,2'-bipyridyl, Omp26 was detected on the cell surface after 60 min. One- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses using purified anti-Omp26 monospecific polyclonal immunoglobulin G antisera established that Omp26 was heat modifiable (39 kDa unheated) and consisted of a single protein species. Immunogold labeling of negatively stained and chemically fixed thin-section specimens indicated that Omp26 was associated with the cell surface and outer leaflet of the P. gingivalis outer membrane in hemin-deprived conditions but was buried in the deeper recesses of the outer membrane in hemin-excess conditions. Analysis of subcellular fractions of P. gingivalis grown either in hemin-excess or hemin-deprived conditions detected Omp26 only in the cell envelope fraction, not in the cytoplasmic fraction or culture supernatant. Limited proteolytic digestion of hemin-deprived P. gingivalis with trypsin and proteinase K verified the surface location of Omp26 as well as its susceptibility to proteolytic digestion. Heat shock treatment of hemin-excess-grown P. gingivalis also resulted in Omp26 translocation onto the outer membrane surface even in the presence of hemin. Furthermore, hemin repletion of heat-shocked, hemin-deprived P. gingivalis did not result in Omp26 translocation off the outer membrane surface, suggesting that thermal stress inactivates this transmembrane event. This newly described outer membrane protein appears to be associated primarily with the outer membrane, in which it is exported to the outer membrane surface for hemin binding and may be imported across the outer membrane for intracellular hemin transport.     

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.     

Chemical, immunobiological and antigenic characterizations of lipopolysaccharides from Bacteroides gingivalis strains.

Lipopolysaccharides (LPS) were extracted from whole cells of seven strains of Bacteroides gingivalis--381, ATCC 33277, BH18/10, OMZ314, OMZ406, 6/26 and HW24D-1--by the phenol/water procedure, and purified by treatment with nuclease and by repeated ultracentrifugation. These LPS were composed of hexoses, hexosamines, fatty acids, phosphorus and phosphorylated 2-keto-3-deoxyoctonate (KDO). The major components of the lipid portion of these LPS were hexadecanoic, 3-hydroxyhexadecanoic, branched 3-hydroxypentadecanoic and branched 3-hydroxyheptadecanoic acids. All the LPS preparations induced marked mitogenic and in vitro polyclonal B cell activation responses in spleen cells from both C3H/HeN and C3H/HeJ mice, exhibited no definitive preparatory activity in the local Shwartzman reaction in rabbits, but were active in the chromogenic Limulus amoebocyte lysate test. A monoclonal antibody (mAb) raised against the LPS from B. gingivalis strain 6/26 reacted with LPS from all other B. gingivalis strains tested. Other mAbs raised against LPS from B. gingivalis strains 381 and 6/26 reacted with the LPS from strains 381, ATCC 33277, BH18/10 and 6/26 (these strains were termed LPS serogroup I), as revealed by ELISA and immunodiffusion. The LPS from these strains except for 6/26 showed almost identical patterns in SDS-PAGE stained with ammoniacal silver. A mAb raised against the LPS from B. gingivalis HW24D-1 reacted with the LPS from strains OMZ314, HW24D-1 and OMZ409 (LPS serogroup II). These LPS, except OMZ409, exhibited very similar profiles in SDS-PAGE. These results indicate that there are at least two different antigenic groups present among LPS from B. gingivalis strains, as well as a common, species-specific antigen.     

副溶血弧菌SH112株OmpA蛋白的高效表达及免疫学特性

【目的】我们前期研究表明副溶血弧菌SH112株的OmpA蛋白在该菌的致病过程中发挥重要作用,是亚单位疫苗研制的潜在靶标抗原。本研究进一步对ompA(VPA1186)基因进行克隆表达,并研究其免疫学特性。【方法】扩增去除信号肽序列的成熟外膜蛋白OmpA的基因片段,定向克隆至表达载体,基因测序后对其编码蛋白质进行生物信息学分析。重组蛋白His-OmpA经纯化后,免疫ICR小鼠制备鼠多抗血清。Western blotting检测该蛋白的免疫原性及鼠多抗血清的特异性。动物实验验证其免疫保护率。【结果】成功表达分子量约为40.0 kDa的重组蛋白His-OmpA。制备的鼠多抗血清ELISA效价可达1∶50000以上。Westernblotting检测结果显示,该血清可与His-OmpA蛋白、总外膜蛋白和全菌蛋白发生特异性反应,说明所表达的目的蛋白保持原蛋白的免疫原性。此外,该高免血清可与其他主要血清型的副溶血弧菌发生特异性交叉反应,而与其他非副溶血弧菌菌株无交叉反应,表明该血清特异性较高,且提示OmpA蛋白可能是副溶血弧菌属的共同保护性抗原。小鼠免疫保护实验结果表明,该蛋白可提供约35%的免疫保护率。【结论】OmpA蛋白可作为诊断副溶血弧菌感染和亚单位疫苗研制的靶蛋白,为进一步开展该蛋白的功能研究提供了参考。     

Molecular size variation of the hemagglutinating adhesin HA-Ag2, a common antigen of Bacteroides gingivalis

The array of Bacteroides gingivalis W83 antigens revealed by crossed immunoelectrophoresis includes one antigen that is associated with an erythrocyte-binding capacity, termed the hemagglutinating adhesin HA-Ag2. This antigen was excised from crossed-immunoelectrophoresis plates to produce two polyclonal antisera, VL 011 and WL 303, whose restricted specificity for HA-Ag2 was assessed using crossed immunoelectrophoresis, crossed immunoelectrophoresis with an intermediate gel, and crossed immunoaffinoelectrophoresis. Both antisera, when used to probe blots of an EDTA cell surface extract of B. gingivalis W83, reacted with two bands, at 33 and 38 kDa, which were also detected by a monoclonal antibody (Naito et al. 1985. Infect. Immun. 50: 231-235), specific for a hemagglutinin of B. gingivalis. Antiserum WL 303 was used to examined by immunoblotting the distribution of HA-Ag2 among a variety of human and animal strains of B. gingivalis. All human strains tested showed two major bands at 33 and 38 kDa in the EDTA cell surface extract, and at 43 and 49 kDa in outer membrane preparations. Only one band, at 29 kDa, was detected in EDTA cell surface extracts from the animal strains, while the outer membrane preparation of a single strain showed a positive reaction. We concluded that HA-Ag2 is an antigen common to human and animal strains of B. gingivalis and that its subunits may show heterogeneity in apparent molecular mass.     

Localization of the Fusobacterium nucleatum T18 adhesin activity mediating coaggregation with Porphyromonas gingivalis T22.

Adherence of pathogenic bacteria is often an essential first step in the infectious process. The ability of bacteria to adhere to one another, or to coaggregate, may be an important factor in their ability to colonize and function as pathogens in the periodontal pocket. Previously, a strong and specific coaggregation was demonstrated between two putative periodontal pathogens, Fusobacterium nucleatum and Porphyromonas gingivalis. The interaction appeared to be mediated by a protein adhesin on the F. nucleatum cells and a carbohydrate receptor on the P. gingivalis cells. In this investigation, we have localized the adhesin activity of F. nucleatum T18 to the outer membrane on the basis of the ability of F. nucleatum T18 vesicles to coaggregate with whole cells of P. gingivalis T22 and the ability of the outer membrane fraction of F. nucleatum T18 to inhibit coaggregation between whole cells of F. nucleatum T18 and P. gingivalis T22. Proteolytic pretreatment of the F. nucleatum T18 outer membrane fraction resulted in a loss of coaggregation inhibition, confirming the proteinaceous nature of the adhesin. The F. nucleatum T18 outer membrane fraction was found to be enriched for several proteins, including a 42-kDa major outer membrane protein which appeared to be exposed on the bacterial cell surface. Fab fragments prepared from antiserum raised to the 42-kDa outer membrane protein were found to partially but specifically block coaggregation. These data support the conclusion that the 42-kDa major outer membrane protein of F. nucleatum T18 plays a role in mediating coaggregation with P. gingivalis T22.     

Non-heritable change of a spirochaete's phenotype by decoration of the cell surface with exogenous lipoproteins

Genetic transformation of Borrelia spp. is limited in development and has found application in only one species. For a non-genetic approach for manipulating the phenotype of these spirochaetes, we determined whether exogenous recombinant lipoproteins would incorporate in the cell's outer membrane. Using unlabelled or 125I-labelled Osp proteins, Osp-specific monoclonal antibodies, proteinase K and formaldehyde as reagents, we found that decoration of spirochaetes had the following characteristics. (i) Purified recombinant OspA or OspD lipoproteins associated with Borrelia burgdorferi and B. hermsii cells that lacked abundant lipoproteins of their own. (ii) This decoration of the cells with exogenous OspA did not affect cell's viability. (iii) The decoration was concentration and temperature dependent and stable for at least 24 h. (iv) Like native OspA, the recombinant OspA decorating the cells was accessible to antibodies and proteases and could be cross-linked to the integral outer membrane protein, P66. (v) Decoration of viable B. burgdorferi and B. hermsii with OspA rendered the cells susceptible to killing by OspA-specific antiserum. Such non-genetic alteration of the surface of a bacterium may be used to study functions and properties of lipoproteins in situ.     

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.     

Fimbria-mediated coaggregation between human oral anaerobes Treponema medium and Porphyromonas gingivalis.

Bacterial binding phenomena among different bacterial genera or species play an important role in bacterial colonization in a mixed microbiota such as in the human oral cavity. The coaggregation reaction between two gram-negative anaerobes, Treponema medium and Porphyromonas gingivalis, was characterized using fimbria-deficient mutants of P. gingivalis and specific antisera against purified fimbriae and bacterial whole cells. T. medium ATCC 700273 strongly coaggregated with fimbriate P. gingivalis strains ATCC 33277 and 381, but not with afimbriate strains including transposon-induced fimbria-deficient mutants and KDP98 as a fimA-disrupted mutant of P. gingivalis ATCC 33277. In the P. gingivalis-T. medium coaggregation assay, the presence of rabbit antiserum against the purified fimbriae or the whole cells of P. gingivalis ATCC 33277 produced different "aggregates" consisting predominantly of P. gingivalis cells with few spirochetes, but both preimmune serum and the antiserum against the afimbriate KDP98 cells did not inhibit the coaggregation reaction. Heated P. gingivalis cells lost their ability to bind both heated and unheated T. medium cells. This T. medium-P. gingivalis coaggregation reaction was inhibited by a cysteine proteinase inhibitor, leupeptin, and also by arginine and lysine, but not by EDTA or sugars including lactose. A binding assay on nitrocellulose membranes and immunoelectron microscopy demonstrated that a heat-stable 37 kDa surface protein on the T. medium cell attached to the P. gingivalis fimbriae.     

Studies on the function of cell surface glycoproteins. I. Use of antisera to surface membranes in the identification of membrane components relevant to cell-substrate adhesion

An antiserum prepared against purified surface membranes of transformed BHK21/C13 cells (C13/B4) reversibly rounded and detached hamster cells from plastic tissue culture plates but did not affect cells of other species. Antiserum treatment did not alter the growth rate of C13/B4 or BHK21/C13 cells; however, NIL-8 cells exposed to the antiserum detached from the substrate and stopped growing, but remained viable for up to 72 h in the presence of the antiserum. Rounding and detachment were not inhibited by DNP or cycloheximide. Antiserum-detached cells did not reattach in the presence of these inhibitors. F(ab)' fragments also induced rounding, thus ruling out the involvement of complement and ligand-induced rearrangement of surface antigens in rounding and detachment. Three different surface-reactive immunoglobulin preparations were used in indirect immunoprecipitation studies in an attempt to identify cell surface antigens involved in regulating adhesion and morphology. Antiserum against surface membranes (anti-M) and against material shed by the cells into serum-free medium (anti-SFM) caused rounding and detachment, but a third antiserum (anti-LIS) prepared against a partially purified glycoprotein did not. All three immunoglobulin preparations precipitated glycoproteins with an apparent mol wt of 120,000 daltons from a crude membrane preparation solubilized by Nonidet NP-40. The two immunoglobulin preparations that caused rounding precipitated an additional glycoprotein peak of 140,000 daltons. Extensive preabsorption of the extract with anti-LIS immunoglobulin enriched the anti-membrane and antiserum-free medium precipitates for the 140,000-dalton peak. Anti-M immunoglobulin eluted from intact cells and subsequently used to precipitate NP-40 solubilized membrane constituents also reacted with a group of glycoproteins of approximately 140,000 mol wt. Therefore, this group of glycoproteins was considered most likely to be the glycoproteins involved in substrate adhesion and maintenance of cellular morphology.     

Cloning, expression, and purification of Brucella suis outer membrane proteins

Brucella, an aerobic, nonsporeforming, nonmotile Gram-negative coccobacillus, is a NIH/CDC category B bioterror threat agent that causes incapacitating human illness. Medical defense against the bioterror threat posed by Brucella would be strengthened by development of a human vaccine and improved diagnostic tests. Central to advancement of these goals is discovery of bacterial constituents that are immunogenic or antigenic for humans. Outer membrane proteins (OMPs) are particularly attractive for this purpose. In this study, we cloned, expressed, and purified seven predicted OMPs of Brucella suis. The recombinant proteins were fused with 6-His and V5 epitope tags at their C termini to facilitate detection and purification. The B. suis surface genes were PCR synthesized based on their ORF sequences and directly cloned into an entry vector. The recombinant entry constructs were propagated in TOP 10 cells, recombined into a destination vector, pET-DEST42, then transformed into Escherichia coli BL21 cells for IPTG-induced protein expression. The expressed recombinant proteins were confirmed with Western blot analysis using anti-6-His antibody conjugated with alkaline phosphatase. These B. suis OMPs were captured and purified using a HisGrab plate. The purified recombinant proteins were examined for their binding activity with antiserum. Serum derived from a rabbit immunized intramuscularly with dialyzed cell lysate of Brucella rough mutant WRR51. The OMPs were screened using the rabbit antiserum and purified IgG. The results suggested that recombinant B. suis OMPs were successfully cloned, expressed and purified. Some of the expressed OMPs showed high binding activity with immunized rabbit antiserum.