Characterization of Bacteroides asaccharolyticus and B. melaninogenicus oral isolates

Experiments were designed to characterize a number of oral "pigmented" Bacteroides isolates with regard to their pathogenicity in an experimental model system and a number of other properties. T these include fatty acid determination, hemagglutination studies, collagenase and protease activities, and vitamin K dependency. Oral B, asaccharolyticus and B, melaninogenicus isolates differed from one another in phenylacetic acid production, hemagglutination, collagenase activity, and pathogenicity. All B. asaccharolyticus were found to be pathogenic in the vivo mixed infection model and this property could be correlated with biochemical enzymatic activities.     

Investigation of antigenic expression of Bacteroides fragilis by immunogold labelling and immunoblotting with a monoclonal antibody

Abstract Electron microscopy and immunogold labelling with monoclonal antibody (McAb) Bfl identified an antigen expressed on some in vitro and in vivo grown Bacteroides fragilis NCTC9343 cells.
Immunoprecipitation with this McAb was used to enrich for B. fragilis NCTC9343 cells expressing the Bfl antigen. The McAb Bfl bound to an epitope close to the surface of the outer membrane, but the fibrous capsular network radiating from the bacterial surface was not labelled. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting identified 3 high- M _r bands which resisted heating and protease digestion but were partially sensitive to sodium periodate treatment.     

Molecular relationships between U snRNP proteins as investigated by rabbit antisera and peptide mapping.

Each of the major U snRNP polypeptides from human cells was purified by electroelution from SDS-polyacrylamide gels. Rabbit antisera could be obtained against the individual proteins 70K, A, B', B and D, although rabbits failed to elicit antibodies against E, F and G. A strong structural homology was found between proteins B' and B, against which patients with connective tissue diseases produce predominantly anti-Sm autoantibodies. Thus, rabbit antisera against B' strongly crossreact with B and vice versa. Peptide patterns of the proteins B' and B obtained with chymotrypsin are identical with the exception of one fragment in each case. Polypeptide D, the third major Sm-antigenic protein, is structurally distinct from B' and B, as evidenced by the failure of anti-D antisera to crossreact with B' or B and vice versa, as well as by the different peptide patterns observed for proteins D and B' or B. The U1 specific polypeptide A and the U2 specific polypeptide B" share homologous regions, as indicated by the crossreactivity of anti-A antisera with protein B", and the occurrence of common fragments in the peptide patterns of the two proteins. Further homologies between other snRNP protein pairs were not detected.     

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.     

Localization of sequential antigenic determinants on bovine beta-casein with synthetic peptides and antisera from mouse, rabbit, and goat.

The antigenic determinants of bovine beta-casein (beta-CN) were localized by using twenty overlapping peptides encompassing the entire sequence of beta-CN and anti-beta-CN antisera from outbred mouse, rabbit and goat. The profile of the reactions was characteristic to the species, the dominant antigenic regions being 80-95, 143-158 and 195-209 in mouse, 1-16 in rabbit and 100-115 in goat. Regions 1-16, 100-115, 121-136 and 143-158 were antigenic in all three species. The number of antigenic regions recognized by goat was much fewer than that by mouse and rabbit, possibly because of the homology between bovine and goat beta-CN. A mixture of the twenty peptides could absorb about 50-60% of beta-CN specific antibodies from each species. Furthermore, the mouse and rabbit anti-beta-CN antibodies were also specific to the phosphorylated regions. We therefore conclude that the major antigenic determinants on beta-CN would be largely sequential and include the phosphorylated sites.     

Taxonomic relationships among strains of the anaerobic bacterium Bacteroides ruminicola determined by DNA and extracellular polysaccharide analysis.

DNA and extracellular polysaccharide (EPS) analyses were performed on 14 strains of Bacteroides ruminicola. The guanine-plus-cytosine (G+C) base contents, determined from the buoyant densities of chromosomal DNAs, showed a broad range of values, from 37.6 to 50.9 mol%. DNA hybridization showed generally low DNA relatedness among the strains. Seven strains formed two groups of closely related bacteria consisting of five (group 1) and two (group 2) strains, and another strain, E42g, showed moderate relatedness to group 1 strains. However, the remaining six strains were not related to any of the other strains. DNA reassociation indicates that the strains constitute a genetically diverse group representing as many as nine separate species. EPS analysis showed that the strains produced EPS with rather uniform sugar compositions, which did not correlate with strain relationships determined by DNA analysis. Four strains had EPS with acidic sugars or unknown compounds. The EPS of strain 20-63 contained the unusual acidic sugar 4-O-(1-carboxyethyl)-rhamnose. This monosaccharide has been shown to occur in nature in only one other bacterial species.     

Taxonomic relationships among strains of the anaerobic bacterium Bacteroides ruminicola determined by DNA and extracellular polysaccharide analysis.

DNA and extracellular polysaccharide (EPS) analyses were performed on 14 strains of Bacteroides ruminicola. The guanine-plus-cytosine (G+C) base contents, determined from the buoyant densities of chromosomal DNAs, showed a broad range of values, from 37.6 to 50.9 mol%. DNA hybridization showed generally low DNA relatedness among the strains. Seven strains formed two groups of closely related bacteria consisting of five (group 1) and two (group 2) strains, and another strain, E42g, showed moderate relatedness to group 1 strains. However, the remaining six strains were not related to any of the other strains. DNA reassociation indicates that the strains constitute a genetically diverse group representing as many as nine separate species. EPS analysis showed that the strains produced EPS with rather uniform sugar compositions, which did not correlate with strain relationships determined by DNA analysis. Four strains had EPS with acidic sugars or unknown compounds. The EPS of strain 20-63 contained the unusual acidic sugar 4-O-(1-carboxyethyl)-rhamnose. This monosaccharide has been shown to occur in nature in only one other bacterial species.     

Host range,RFLP, and antigenic relationships betweenRhizobium fredii strains andRhizobium sp. NGR234

Rhizobium fredii is a nitrogen-fixing symbiont from China that combines broad host range for nodulation of legume species with cultivar specificity for nodulation of soybean. We have compared 10R. fredii strains withRhizobium sp. NGR234, a well known broad host range strain from Papua New Guinea. NGR234 nodulated 16 of 18 tested lugume species, and nodules on 14 of the 16 fixed nitrogen. TheR. fredii strains were not distinguishable from one another. They nodulated 13 of the legumes, and in only nine cases were nodules effective. All legumes nodulated byR. fredii were included within the host range of NGR234. Restriction fragment length polymorphisms (RFLPs) were detected with four DNA hybridization probes: the regulatory and commonnod genes,nodDABC; the soybean cultivar specificity gene,nolC; the nitrogenase structural genes, nifKDH; and RFRS1, a repetitive sequence fromR. fredii USDA257. A fifth locus, corresponding to a second set of soybean cultivar specificity genes,nolBTUVWX, was monomorphic. Using antisera against whole cells of threeR. fredii strains and NGR234, we separated the 11 strains into four serogroups. The anti-NGR234 sera reacted with a singleR. fredii strain, USDA191. Only one serogroup, which included USDA192, USDA201, USDA217, and USDA257, lacked cross reactivity with any of the others. Although genetic and phenotypic differences amongR. fredii strains were as great as those between NGR234 andR. fredii, our results confirm that NGR234 has a distinctly wider host range thanR. fredii.     

Human placental steryl-sulfatase. Enzyme purification, production of antisera, and immunoblotting reactions with normal and sulfatase-deficient placentas

The steryl-sulfatase of normal human placental microsomes was solubilized and enriched about 350-fold. Chromatography on Sepharose 6B of the purified enzyme preparation revealed a single protein peak which eluted according to an apparent molecular mass of 270 +/- 30 kDa; when electrophorized on sodium dodecyl sulfate polyacrylamide gel the sulfatase migrated according to a molecular mass of 64 +/- 4 kDa. Estrogensulfatase activity was co-purified with the steryl-sulfatase activity; obviously, both activities belong to the same enzyme species. The purified sulfatase was injected into three rabbits. Antisera produced by the rabbits yielded a single sharp immunoprecipitation line in Ouchterlony double diffusion experiments when tested with the isolated sulfatase or with a solubilized microsomal fraction of normal placentas. The activity of sulfatase preparations incubated with antiserum was precipitated by addition of polyethylene glycol followed by centrifugation; none of the antibodies reacting with the sulfatase therefore appeared to interfere with its enzymatic activity. Using these antisera, steryl-sulfatase protein could be detected by immunoblotting analysis in solubilized microsomal fractions of normal placentas but not in solubilized microsomal fractions of three steryl-sulfatase activity-deficient placentas. This finding argues in favour of human placental steryl-sulfatase deficiency being due to extremely diminished or absent enzyme protein in the placenta.     

Study of the antigenic relationship between animal and human immunoglobulins using antisera to human immunoglobulins]

Cross-reactivity of monospecific antisera to human immunoglobulins with animal sera of 10 species was studied by immunoelectrophoresis and radial immunodiffusion. Antisera to IgG were shown to reveal IgG of all the species studied, antisera to IgM and especially to IgA cross reacted less extensively. The greatest number of cross reactions were given by the antisera obtained as a result of hyperimmunization. Hyperimmune monospecific antisera to human IgG, IgA, and IgM can be used for the identification of animal immunoglobulins during their isolation from the sera and for their quantitation by radial immunodiffusion.     

Structural features of methyl-accepting taxis proteins conserved between archaebacteria and eubacteria revealed by antigenic cross-reaction.

A number of eubacterial species contain methyl-accepting taxis proteins that are antigenically and thus structurally related to the well-characterized methyl-accepting chemotaxis proteins of Escherichia coli. Recent studies of the archaebacterium Halobacterium halobium have characterized methyl-accepting taxis proteins that in some ways resemble and in other ways differ from the analogous eubacterial proteins. We used immunoblotting with antisera raised to E. coli transducers to probe shared structural features of methyl-accepting proteins from archaebacteria and eubacteria and found substantial antigenic relationships. This implies that the genes for the contemporary methyl-accepting proteins are related through an ancestral gene that existed before the divergence of arachaebacteria and eubacteria. Analysis by immunoblot of mutants of H. halobium defective in taxis revealed that some strains were deficient in covalent modification of methyl-accepting proteins although the proteins themselves were present, while other strains appeared to be missing specific methyl-accepting proteins.     

Role of nucleosides, 5-phosphoribosyl-1-pyrophosphate, and ribose 1-phosphate in the biosynthesis of phosphosphingolipids in Bacteroides melaninogenicus.

Using a deenergized spheroplast system from Bacterioides melaninogenicus, the sphingolipid precursor 3-ketodihydrosphingosine is not incorporated into the complete sphingolipids, ceramide phosphorylethanolamine, or ceramide phosphorylglycerol unless supplied with glutamine, ATP, ADP, or AMP. Adenosine, inosine, and certain other nucleosides were as effective as ATP. Purine bases and ribose, however, were inactive in this system. 5-Phosphoribosyl-1-pyrophosphate and ribose 1-phosphate also stimulated conversion of 3-ketodihydrosphingosine into ceramide phosphorylethanolamine and ceramide phosphorylglycerol, whereas ribose 5-phosphate showed only slight activity. Hypoxanthine was the main product formed from inosine and adenosine but there was no evidence for nucleotide formation. Adenosine stimulated³²P_i incorporation into cell phospholipids indicating that ribose 1-phosphate, formed via purine nucleoside phosphorylase, could be the compound stimulating sphingolipid synthesis in this system.     

Immunological studies on a yeast peptido-galactomannan. Nature of antigenic determinants reacting with rabbit antisera and those involved delayed hypersensitivity in guinea pigs.

The antigenicity of the peptido-phosphogalactomannan (PPGM) of Cladosporium werneckii and the carbohydrate and peptide moieties isolated from it were studied in rabbits and guinea pigs. In rabbits, the antisera produced against whole C. werneckii cells reacted only with the carbohydrate portions of the antigen. The major portion of the antibody was directed towards the long phosphogalactomannan carbohydrate chains. O-Acetyl substituents were shown to play important roles in the determinants on these chains since selective removal of these groups eliminated their reactivity with antibody. A relatively small amount of the antibody response was directed towards the more numerous, short, mannose-containing chains. Concanavalin A, on the other hand, precipitated the polymer by virture of its reactivity with these units and not with the phosphogalactomannan chains.Delayed type (cell-mediated) immunity to C. werneckii was studied in guinea pigs. PPGM was able to elicit strong delayed skin-tests as was a peptide-rich fraction derived from PPGM by mild acid hydrolysis. Pure carbohydrate chains of PPGM were completely inactive. In contrast, two of the modified peptides derived from PPGM were able to elicit a response although they were less active than PPGM. It was concluded that the determinants responsible for delayed hypersensitivity reside in the peptide portion of the antigen but that the technique used to isolate the peptides (alkaline β-elimination) partially destroyed the determinants.     

A comparison of the antigenic characteristics of rat and human Pneumocystis carinii by immunoblotting

The antigenic characteristics of rat Pneumocystis carinii obtained from infected lungs and grown in tissue culture were compared with the properties of human P. carinii obtained from the lungs of AIDS and non-AIDS patients by the immunoblotting technique, using different sources of antibody. Major immunoreactive bands of 45, 50, and 116 kd were found in both lung and tissue culture-derived rat P. carinii, suggesting the organism retains its antigenic characteristics in short-term culture. The principal immunoreactive bands in human P. carinii included a band of 40 kd, and to a lesser extent, a band of 66 kd; these antigens were found in the lungs of six and seven AIDS patients but in only one of eight non-AIDS patients with pneumocystosis. The rat and human P. carinii antigens reacted with sera from immunized rabbits, from rats with pneumocystosis and prolonged environmental exposure to the organism, from AIDS and non-AIDS P. carinii patients, and from healthy blood donors. Reactivity of these antigens could be removed by adsorption of antisera with P. carinii-infected lungs but not with normal lungs or lungs infected with bacteria and fungi. We conclude that rat and human P. carinii have shared, as well as species-specific, antigenic determinants, which should be useful for a variety of studies with this organism.