Activities and partial purification of extracellular proteases of Bacteroides nodosus from virulent and benign footrot

In an attempt to differentiate virulent and benign strains of B. nodosus, the extracellular proteolytic activity of these cultures was assayed with elastin, casein and hide powder azure, and the stability to heating at 55 degrees C was determined. Broth cultures of both strains hydrolysed 125I-labelled elastin, indicating that this activity is not a unique marker of virulence. When cultures were grown in Trypticase-arginine-serine broth medium modified by omitting Na2CO3 and thioglycollic acid, the total proteolytic activity and its stability at 55 degrees C could be used to differentiate isolates causing virulent or benign footrot lesions. However, when other broth cultures were used, these parameters could no longer be used to make such a distinction. The proteases of a virulent and benign strain of B. nodosus were partially purified and characterized. Four to five closely related proteases were detected by polyacrylamide gel electrophoresis at pH 8.8 in both types of isolates. The proteases are serine-type enzymes requiring a divalent metal ion such as calcium for activity. The proteases of the benign strain were somewhat less stable to heat than the enzymes of the virulent strain. Differences in the relative mobilities of the proteases of virulent and benign strains of B. nodosus, on electrophoresis at pH 8.8, suggest that this property may be used to distinguish virulent and benign strains.     

Proteinase isoenzyme patterns of Bacteroides nodosus: distinction between ovine virulent isolates, ovine benign isolates and bovine isolates

Bacteroides nodosus isolates from ovine virulent footrot and ovine benign footrot and bovine isolates of low virulence for sheep were distinguishable from each other by their proteinase isoenzyme patterns after polyacrylamide gel electrophoresis. Variants of low virulence were not always distinguishable from their virulent parent strains. The molecular weights of the isoenzymes ranged from 70000 to 129000. The relationship of isoenzyme patterns to virulence is discussed.     

Aggregation of Actinomyces strains by extracellular vesicles produced by Bacteroides gingivalis

The aggregation of Actinomyces viscosus and Actinomyces naeslundii with extracellular vesicles of Bacteroides gingivalis was studied. Factors influencing the aggregation phenomenon were examined. L-Arginine was found to effectively inhibit aggregation as was an antibody preparation directed against a B. gingivalis surface hemagglutinin. Aggregation occurred over a wide pH range and did not seem to be affected by high salt concentrations or the presence of carbohydrates. Treatment of the vesicle preparation with proteases, sodium dodecyl sulphate, and high temperatures diminished or eliminated aggregation, while similar treatment of the Actinomyces had no effect on aggregation.     

Molecular analysis of one of multiple protease-encoding genes from the prototype virulent strain of Bacteroides nodosus

The aim of these studies was to examine the organization of the Bacteroides nodosus protease-encoding gene(s). The extracellular serine proteases (38 kDa) from the prototype virulent strain of B. nodosus were purified and used to raise a specific antiserum in rabbits. This antiserum was used in a colony immunoassay to screen a genomic DNA library constructed in Escherichia coli using BamHI-digested B. nodosus DNA and the plasmid pBR322. An E. coli clone expressing a 50-kDa immunoreactive polypeptide was identified. No protease activity was detected in the culture media, or in crude soluble and membrane fractions prepared from this clone. Restriction mapping and deletion analysis of the recombinant plasmid, pEKM2, was used to locate the coding region to a 1.4-kb EcoRI-BamHI fragment which was subsequently sequenced. A large open reading frame was found to extend through the BamHI site from a putative start codon just downstream from the EcoRI site, which indicated that the complete gene was not isolated. Southern blotting demonstrated that there were at least three B. nodosus BamHI fragments which were homologous to the 0.4-kb PstI-BamHI fragment of pEKM2. Based on these results the existence of multiple protease genes in B. nodosus was postulated.     

Molecular cloning of the fimbrial subunit gene from a benign type B isolate of Bacteroides nodosus

Abstract The fimbrial subunit gene from the benign type B Bacteroides nodosus isolate AC/6 was cloned into the Sph I site of the multicopy vector plasmid pUC19. Five Escherichia coli recombinants that were positive in a colony immunoassay were shown, by Western transfer analysis, to produce an immunologically cross-reacting protein of identical molecular size to fimbrial subunits prepared from B. nodosus AC/6. Restriction endonuclease analysis showed that 4 of the recombinant plasmids carried a 6.7 kb Sph I fragment. Recloning experiments showed that the fimbrial subunit gene was located within a 2.5 kb Eco RI- Sph I fragment and that there was a Pst I site located within the structural gene or its regulatory region. These recombinant clones will prove useful for the construction of a multivalent recombinant vaccine for the control of ovine footrot.     

Characterization of black-pigmented Bacteroides strains isolated from animals

The aims of the study were: the isolation of strains of black-pigmented Bacteroides from the gingival sulcus of different animals, their biochemical and immunological characterization and comparison of their properties for classification within the genus. A total of 104 strains, isolated from cats, dogs, racoons and a jaguar, were characterized on the basis of fermentation of carbohydrates, metabolic end products, haemagglutination studies, enzymatic activities, catalase production and indirect immunofluorescence. No differences were observed between the strains regardless of their animal origin. The strains did not ferment carbohydrates, produce phenylacetic acid, show an array of enzyme activities or agglutinate sheep red blood cells. They were catalase-positive and so differed from the human oral strains of Bact. gingivalis. Immunofluorescence microscopy revealed that the animal strains shared at least one major antigen with Bact. gingivalis but none with Bact. asaccharolyticus. Apart from their catalase activity, the animal strains isolated were similar to those of human Bact. gingivalis strains.     

Characterization of black-pigmented Bacteroides strains isolated from animals

The aims of the study were: the isolation of strains of black-pigmented Bacteroides from the gingival sulcus of different animals, their biochemical and immunological characterization and comparison of their properties for classification within the genus. A total of 104 strains, isolated from cats, dogs, racoons and a jaguar, were characterized on the basis of fermentation of carbohydrates, metabolic end products, haemagglutination studies, enzymatic activities, catalase production and indirect immunofluorescence. No differences were observed between the strains regardless of their animal origin. The strains did not ferment carbohydrates, produce phenylacetic acid, show an array of enzyme activities or agglutinate sheep red blood cells. They were catalase-positive and so differed from the human oral strains of Bact. gingivalis. Immunofluorescence microscopy revealed that the animal strains shared at least one major antigen with Bact. gingivalis but none with Bact. asaccharolyticus. Apart from their catalase activity, the animal strains isolated were similar to those of human Bact. gingivalis strains.     

Quantitative test for Bacteroides nodosus pilus protein by an agglutination-absorption test

A pili agglutination-absorption test (PAAT) was developed for the quantitative measurement of pilus protein in cultures of Bacteroides nodosus and to quantify pili yields during purifications. The test was calibrated by recording the amount of pilus protein required to absorb a measurable amount of anti-pili antibody from antiserum. The amount of anti-pilus antibody in absorbed and unabsorbed serum was specifically measured by a Bact. nodosus K-agglutination test. The PAAT could be calibrated using any combination of crude or pure pili preparations and specific anti-pili serum or non-specific anti- Bact. nodosus serum. This had advantages over the use of radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques for measurement of pili because they require the use of highly purified reagents for calibration. The minimum quantity of pilus protein measurable by PAAT was 0.1 μg per test which was similar in sensitivity to that reported for RIA and ELISA. The reagents used in PAAT were stable for at least six months. The amount of pilus protein per bacterium as measured by PAAT was directly proportional to the average number of pili per bacterium as measured by electron microscopy. The test was Bact. nodosus serotype specific.     

Quantitative test for Bacteroides nodosus pilus protein by an agglutination-absorption test

A pili agglutination-absorption test (PAAT) was developed for the quantitative measurement of pilus protein in cultures of Bacteroides nodosus and to quantify pili yields during purifications. The test was calibrated by recording the amount of pilus protein required to absorb a measurable amount of anti-pili antibody from antiserum. The amount of anti-pilus antibody in absorbed and unabsorbed serum was specifically measured by a Bact. nodosus K-agglutination test. The PAAT could be calibrated using any combination of crude or pure pili preparations and specific anti-pili serum or non-specific anti-Bact. nodosus serum. This had advantages over the use of radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques for measurement of pili because they require the use of highly purified reagents for calibration. The minimum quantity of pilus protein measurable by PAAT was 0.1 microgram per test which was similar in sensitivity to that reported for RIA and ELISA. The reagents used in PAAT were stable for at least six months. The amount of pilus protein per bacterium as measured by PAAT was directly proportional to the average number of pili per bacterium as measured by electron microscopy. The test was Bact. nodosus serotype specific.     

Ultrastructure of the Bacteroides nodosus cell envelope layers and surface.

The surface structure and cell envelope layers of various virulent Bacteroides nodosus strains were examined by light microscopy and by electron microscopy by using negative staining, thin-section, and freeze-fracture-etch techniques. Three surface structures were described: pili and a diffuse material, both of which emerged from one or both poles of the bacteria (depending on the stage of growth and division), and large rodlike structures (usually 30 to 40 nm in diameter) associated with a small proportion of the bacterial population. No capsule was detected. The cell envelope consisted of four layers: a plasma membrane, a peptidoglycan layer, an outer membrane, and an outermost additional layer. The additional layer was composed of subunits, generally hexagonally packed with center-to-center spacing of 6 to 7 nm. The outer membrane and plasma membrane freeze-fractured through their hydrophobic regions revealing four fracture faces with features similar to those of other gram-negative bacteria. However, some unusual features were seen on the fracture faces of the outer membrane: large raised ring structure (11 to 12 nm in diameter) on cw 3 at the poles of the bacteria; complementary pits or ring-shaped depressions on cw 2; and small raised ring structures (7 to 8 nm in diameter) all over cw 2.     

Morphogenetic expression of Bacteroides nodosus fimbriae in Pseudomonas aeruginosa.

Type 4 fimbriae are found in a range of pathogenic bacteria, including Bacteroides nodosus, Moraxella bovis, Neisseria gonorrhoeae, and Pseudomonas aeruginosa. The structural subunits of these fimbriae all contain a highly conserved hydrophobic amino-terminal sequence preceding a variable hydrophilic carboxy-terminal region. We show here that recombinant P. aeruginosa cells containing the B. nodosus fimbrial subunit gene under the control of a strong promoter (pL, from bacteriophage lambda) produced large amounts of fimbriae that were structurally and antigenically indistinguishable from those produced by B. nodosus. This was demonstrated by fimbrial isolation and purification, electrophoretic and Western transfer analyses, and immunogold labeling and electron microscopy. These results suggest that type 4 fimbriated bacteria use a common mechanism for fimbrial assembly and that the structural subunits are interchangeable, thereby providing a basis for the development of multivalent vaccines.     

Characterization of highly virulent Escherichia coli strains by ribosomal DNA restriction fragment length polymorphism

Patterns of ribosomal DNA polymorphism were examined to compare carboxylesterase B type B1 strains and B2 strains of Escherichia coli isolated from extra-intestinal infections. DNA from 14 type B2 strains showing the presence of alpha-haemolysin and mannose-resistant haemagglutinin and lethality to mice and 14 type B1 strains lacking these characteristics, was digested with HindIII, EcoRI, BamHI or BglII restriction enzymes and analysed by Southern blotting. The obtained ribotypes clearly differentiated the B2 strains from the B1 strains. These results indicate that genotypes of the highly virulent B2 strains are different from that of the less virulent B1 strains.     

Keratinolytic proteinase produced by Candida albicans

Candida albicans was cultivated in various media that contained human stratum corneum, human scalp hair or keratin powder (cow's hoof) as a nitrogen source. Production of a keratinolytic proteinase (KPase) was observed when C. albicans was incubated in the medium containing stratum corneum. However, there was no production of a KPase that could digest human stratum corneum in the medium containing hair or keratin powder. alpha-fibrous protein extracted from human stratum corneum was digested by the KPase. The pH optimum of the enzyme was 4.0 and enzyme activity was inhibited by pepstatin A and chymostatin. The KPase, a kind of carboxyl proteinase, may be important for C. albicans to enable it to play a pathogenic role in vivo.     

Compositional difference of the exopolysaccharides produced by the virulent and virulence-deficient strains of Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, is known to produce phytotoxic polysaccharides. The extracellular polysaccharide (EPS) was isolated from virulent (BXO1) and virulence-deficient gum G mutant (BXO1002) strains of X. oryzae pv. oryzae and characterized using fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR). Data from the FT-IR suggested that the aldehyde (R-CHO) group and C=O of acid anhydride are present in BXO1 but absent in BXO1002. The (1)H-NMR spectra showed the presence of an acetyl amine of hexose or pentose, free amines of glucose, an beta-anomeric carbon of hexose and pentose, hydrogen next to hydroxyl group, an acetyl amine of hexose and pentose in the polysaccharides of both BXO1 and BXO1002, and the absence of alpha-anomeric carbon of hexose or pentose and the glucuronic acid in the polysaccharides produced by BXO1002. The test for glucuronic acid also confirmed the absence of glucuronic acid in the polysaccharides of BXO1002 and the presence glucuronic acid (32 microg/mg) in the polysaccharides produced by BXO1.     

Characterization of intermediate phenotypes induced by chemically undefined laboratory media in virulent Bordetella bronchiseptica strains

The expression of many virulence factors of Bordetella bronchiseptica is regulated by the bvgAS locus and reduced in response to environmental signals called modulators. Virulent strains can alternate between virulent (Bvg(+)), intermediate (Bvg(i)), and modulated (Bvg(+)mod) phenotypes. Potential vaccine antigens can be expressed by Bvg1 strains grown only in the absence of modulators. In the present study we evaluated filamentous hemagglutinin (FHA) and outer membrane protein (OMP) expression in Bvg(+) B. bronchiseptica strains grown in chemically undefined media: nutrient agar (NA), tryptic soy agar (TSA), tryptose phosphate broth (TPB), and brain-heart infusion (BHI). Our results suggest that TSA and TPB usually induce semimodulation, since Bvg(+) strains cultured in these media retained the expression of FHA and virulence-associated OMPs in the 30 kDa region, but failed to express other virulence markers such as OMPs in the regions of 90 and 200 kDa, though they expressed flagellin (avirulence marker). On the other hand, NA and BHI usually induce modulation. Thus the assayed chemically undefined media should not be used in vaccine production. Semimodulation induced by TSA and TPB can be accurately detected by SDS-PAGE Sarkosyl-insoluble OMP-enriched profiles. The reduction or absence of OMPs in the regions of 90 and 200 kDa is the most sensitive marker, and in some cases the presence of flagellin in intermediate profiles is another trait of the Bvg(i) phenotypes. Therefore these markers could be useful for selecting media for vaccine production. We also characterized the phenotype of Bvg(+) strains grown in Stainer-Scholte broth, an expensive medium, with and without glutathione, and we have detected no differences; this is the first attempt to reduce the cost of a Bordetella growth medium for veterinary vaccine production.     

Butyrate: a cytotoxin for Vero cells produced by Bacteroides gingivalis and Bacteroides asaccharolyticus

Culture filtrates of B. gingivalis and B. asaccharolyticus are cytotoxic for Vero cells. It is shown that the cytotoxic effect is due to the butyrate concentrations present in the culture filtrates of these strains. This cytotoxic effect proved to be reversible. Strains of the B. melaninogenicus subspecies intermedius and melaninogenicus did not produce butyrate and did not show cytotoxic activity towards Vero Cells.The significance of the production of toxic concentrations of butyrate for the etiology of especially periodontal diseases is discussed.     

Characterization of enterotoxin produced by four Yersinia enterocolitica strains of pig origin

All four isolates of Yersinia enterocolitica and one isolate of Y. frederiksenii from pigs were found to be enterotoxigenic. Whole-cell preparations of Y. enterocolitica isolates did not induce any change in the rabbit ligated gut test after 6 and 18 h of inoculation, but Y. frederiksenii on the other hand showed a positive gut response at 18 h. Cell-free supernatant (CFS) of all five isolates induced dilatation in the rabbit gut up to 6 h, after which Y. enterocolitica became negative, while Y. frederiksenii continued to show a reaction up to 18 h. CFS of all five isolates were also found positive with the infant mouse test. Of the five isolates of Yersinia, three gave a positive reaction for the permeability factor on rabbit skin. Yersinia enterotoxin could be concentrated by methanol extraction. It was stable at 100°C for 20 min and at 120°C for 15 min. However, its activity was lost at low (2.0) and high pH (10.0). Enterotoxic preparations of Y. enterocolitica lost part of their enterotoxic activity upon dialysis.