Extraction, purification, and characterization of major outer membrane proteins from Wolinella recta ATCC 33238.

The outer membrane of Wolinella recta ATCC 33238 was isolated by French pressure cell disruption and differential centrifugation. Outer membrane proteins (OMPs) were solubilized by Zwittergent 3.14 extraction and separated by DEAE-Sephacel ion-exchange chromatography. The major OMPs that were found in W. recta ATCC 33238 and in several other Wolinella spp. consisted of proteins with apparent molecular masses of 51, 45, and 43 kDa. These three conserved proteins were purified to essential homogeneity by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and characterized chemically. Heating at between 75 and 100 degrees C revealed both the 43- and 51-kDa proteins to be heat modified from apparent molecular masses of 32 and 38 kDa, respectively. The 45-kDa protein was unmodified at all temperatures tested. Two-dimensional isoelectric focusing-SDS-PAGE revealed the 51-kDa protein to be composed of multiple pIs between a pH of 5.0 and greater than 8.0 while the 43- and 45-kDa proteins had a pI of approximately 6.0. N'-terminal amino acid sequence analysis of the first 30 to 40 amino acids and search of the Protein Identification Resource data base for similar proteins only revealed the 43-kDa protein to be similar to the P.69 OMP of Bordetella pertussis; however, the homology was weak (33%). Amino acid analysis revealed the 43-kDa protein to be noncharged and the 45- and 51-kDa proteins to be hydrophilic, containing between 38 to 42% polar residues but no cysteine. This study reports the purification and partial characterization of three conserved proteins in W. recta ATCC 33238.

Purification and immunological characterization of a GroEL-like protein from Bordetella pertussis.

A GroEL-like protein from Bordetella pertussis was purified. This protein was found to have the tetradecameric subunit structure typical of the GroEL family of proteins and to contain epitopes similar to those of other members of this family, including a human GroEL-like protein. Active immunization of neonatal mice with the B. pertussis GroEL-like protein provided little protection against an aerosol challenge with B. pertussis. Antibodies to this protein were elicited in mice by a standard diphtheria-tetanus-pertussis (DTP) vaccine but not by an experimental acellular pertussis vaccine. Since the Bordetella GroEL-like protein was found to contain epitopes similar to those on the mammalian analog, the potential exists that vaccination with standard DTP vaccines may induce antibodies which react with the mammalian GroEL analog.

Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis.

Comparison of lipopolysaccharides (LPS) from phase variants of different strains of Bordetella phase variants of different strains of Bordetella pertussis has shown a difference in their composition, antigenicity and reactogenicity. Phase I variants of B. pertussis, with the exception of strain 134, contain a preponderance of LPS I whereas the major component of LPS of phase IV variants is LPS II. Sera raised to LPSs of phase I strains, other than 134, cross-react with each other but not with phase IV LPSs; and similarly all sera raised to phase IV LPSs cross-react with each other and with LPS from 134 phase I. The LPSs of all phase I variants, including that of 134, are approximately ten-fold or more reactive in the limulus amoebocyte lysate assay (LAL) than phase IV LPSs. In the human mononuclear cell pyrogen assay phase IV LPSs also stimulated a lower response than phase I LPSs. The B. pertussis phase I LPSs are 10-times more reactive than Escherichia coli standard endotoxin in the LAL assay but 100-times less reactive than E. coli LPS in the monocyte test for pyrogen. The SDS-PAGE profiles of B. pertussis LPSs are quite different from those of B. parapertussis and B. bronchiseptica strains. B. pertussis LPSs produced a typical lipo-oligosaccharide (LOS) pattern. B. bronchiseptica LPS produced a similar pattern but was antigenically distinct from B. pertussis LPSs I and II. B. parapertussis in contrast produced a ladder pattern typical of smooth type LPS.

Serum sensitivity and lipopolysaccharide characteristics in Bordetella bronchiseptica, B. pertussis and B. parapertussis.

The viability of four strains of Bordetella bronchiseptica, two strains of B. pertussis and one strain of B. parapertussis exposed to hyperimmune and pre-colostrum porcine serum was examined. Viable cell numbers (cfu/ml) of the B. pertussis strains and a rough strain of B. bronchiseptica (CSU-P-1) decreased by 99% and 99.99%, respectively, after exposure for 1 h to porcine hyperimmune serum. In contrast, smooth B. bronchiseptica strains and the B. parapertussis strain showed no significant decrease in viable cell numbers after the same treatment. B. bronchiseptica strain CSU-P-1 also showed a 99% decrease in viable cell numbers after exposure to pre-colostrum porcine serum for 1 h whereas the other strains tested showed no decrease in viable numbers under the same conditions. Heating the hyperimmune and pre-colostrum serum at 56 degrees C for 30 min resulted in the loss of bactericidal activity suggesting the involvement of complement in both systems. Analysis of silver-stained SDS-PAGE profiles of lipopolysaccharide (LPS) extracted from the bacterial cells indicated that the smooth strains of B. bronchiseptica and the B. parapertussis strain possessed high mol. wt O-side chain-like material, whereas the B. pertussis strains and B. bronchiseptica strain CSU-P-1 did not. Gel filtration of acid-hydrolysed LPS samples indicated two distinct carbohydrate peaks for the strains with high mol. wt O-side chain-like material, whereas the other strains each yielded one distinct peak. Western-blot analysis indicated a positive reaction for anti-B. bronchiseptica antibodies to the high mol. wt O-side chain-like material of all serum-resistant strains used in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel process for preparing an acellular pertussis vaccine composed of non-pyrogenic toxoids of pertussis toxin and filamentous hemagglutinin.

A novel process for preparing non-pyrogenic toxoids of pertussis toxin (PT) and filamentous hemagglutinin (FHA) is described. The process consists of chromatographies on perlite then on hydroxylapatite. Purification yields for PT and FHA are 62 and 68%, respectively. The purification process takes advantage of the novel use of perlite (a filter aid) for the simultaneous purification of PT and FHA. The hydroxylapatite, in addition to removing the remaining contaminants, also concentrates the antigens. The resulting PT and FHA are approximately 95% pure, and are non-pyrogenic as judged by the rabbit pyrogen test. The purification process is simple, inexpensive, and does not use blood components or toxic substances. The mild conditions in which the PT and FHA are purified ensure the recovery of native protein. The purified PT and FHA are detoxified in the presence of glycerol using glutaraldehyde and formaldehyde, respectively, to produce antigenic components of an acellular pertussis vaccine. The final PT and FHA toxoids are immunogenic in guinea-pigs and have been shown to be protective in the mouse intracerebral challenge test.

[The phenotypic properties of freshly isolated strains of Bordetella]. / Izuchenie fenotipicheskikh svoistv svezheizolirovannykh shtammov bordetell.

As the result of our investigations, newly isolated B. pertussis and B. bronchiseptica strains were studied. The results of these investigations showed that B. pertussis strains isolated under the conditions of immunoprophylaxis were characterized by sufficient stability of the main phenotypical properties which determined their pathogenicity: B. pertussis toxin, fimbrial agglutinogens and filamentous hemagglutinin. At the same time B. bronchiseptica strains isolated from animals proved to be phenotypically variable both in vivo and in the process of in vitro passage.

Cloning of the recA gene of Bordetella pertussis and characterization of its product.

A recA gene of Bordetella pertussis was identified in a plasmid library by complementation of a recA mutation in E coli and subcloned as a 2.1-kb Sph I DNA fragment. Southern hybridization experiments showed no similarity to the E coli recA gene, but very strong similarity to other Bordetella species. E coli recA mutant cells containing the B pertussis recA gene at high gene dosage were resistant to DNA-damaging agents such as methyl methane sulfonate or 4-nitroquinoline-N-oxide, displayed induction of SOS functions, and were able to promote DNA recombination, but not induction of phage lambda. The latter phenotype distinguishes the B pertussis recA gene product from the corresponding proteins from most other Gram-negative organisms. Amino acid sequence comparisons revealed a high degree of structural conservation between prokaryotic RecA proteins.

Preliminary X-ray crystallographic analysis of holotoxin from Bordetella pertussis.

Pertussis (whooping cough) is a serious infectious disease caused by the bacterium Bordetella pertussis. One of the major virulence factors is a protein known as pertussis toxin, which is composed of six subunits, with a total molecular weight of 106,000. Enzymatic transfer of ADP-ribose from NAD to a family of GTP-binding proteins is effected by the largest subunit (S1 or the A monomer), while binding of host cells and entry of S1 to the interior is a function of the other subunits (the B oligomer). The holotoxin crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 98.4 A, b = 164.2 A and c = 195.2 A. The crystals are suitable for high-resolution X-ray diffraction analysis.

Simple, efficient purification of filamentous hemagglutinin and pertussis toxin from Bordetella pertussis by hydrophobic and affinity interaction.

Two major antigens of Bordetella pertussis, filamentous hemagglutinin (FHA) and pertussis toxin (PT), were efficiently purified from culture filtrate by exploiting their relative hydrophobicities and differences in affinity to sialic acid-containing protein. High yields of FHA (40 to 80 mg/liter) and PT (8 to 16 mg/liter) were first produced by growing the bacteria in the modified CL medium. The FHA and PT in the culture filtrate were adsorbed onto butyl-Sepharose by hydrophobic interaction at appropriately high ionic strength. Elution of the antigens was effected by decreasing their hydrophobicities with a buffer of low ionic strength. FHA was then separated from PT with an affinity column of fetuin-Sepharose. The fraction passing through the column contained purified FHA, and the fetuin-bound PT was eluted with buffered MgCl2. The FHA and PT purified by these steps were electrophoretically and serologically identical to the reference purified FHA and PT preparations. Approximately 16 to 32 mg of purified FHA and 4 to 8 mg of purified PT were obtained from 1 liter of culture filtrate. The described procedure for making FHA and PT antigens from B. pertussis for serologic and immunologic use is very simple, efficient, and reproducible.

Location of the three major agglutinogens of Bordetella pertussis by immuno-electronmicroscopy.

When the three serotypes of Bordetella pertussis (types 1,2,3; 1,2 and 1,3) were labelled with agglutinins and protein-A gold, agglutinogen 1 was found on fimbriae and on the cell surface of types 1,2,3 and 1,2 but on the cell surface only of non-fimbriate type 1,3 organisms. In contrast, agglutinogen 2 was located on fimbriae only. Agglutinogen 3 was not labelled. When protein-A gold was replaced by immunoglobulin-G gold, agglutinogen 3 was found on the cell surface only, even of fimbriate bacteria of type 1,2,3. The implications of these findings for acellular vaccines are discussed.

Quantitation of pertussis toxin in an enzyme linked immunosorbent assay with improved specificity.

The quantitation of pertussis toxin (PT) in two sandwich ELISAs was tested for specificity. The detection of the captured PT was obtained by using either polyspecific rabbit anti Bordetella pertussis serum (RaBp-ELISA) or a monoclonal anti-PT antibody (McaPT-ELISA). No major differences in the estimation of PT in highly purified preparations were noted using either ELISA variants. In contrast, the quantitation of PT in crude extracts of B. pertussis cultures by the RaBp-ELISA was found to be over-estimated and showed greater variability when compared to the McaPT-ELISA. Comparison of the distribution of PT in the eluate fractions following partial purification by hydroxylapatite chromatography revealed that the results of the McaPT-ELISA were more specific as judged by SDS-PAGE analysis.

Variations in the carbohydrate regions of Bordetella pertussis lipopolysaccharides: electrophoretic, serological, and structural features.

Structural and immunological differences between the two components that are usually present in unequal quantities in Bordetella pertussis endotoxin preparations and are visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been studied by using strains 1414, A100, and 134, all in phase I. According to analyses by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thin-layer chromatography, the minor (8%) component of the endotoxin of strain 1414 (endotoxin 1414) appeared to be the predominating component of endotoxins A100 and 134. The masses of the carbohydrate chains isolated from endotoxin A100 and from the major component of endotoxin 1414 were 1,649 and 2,311 atomic mass units, respectively, as determined by 252Cf plasma desorption mass spectrometry. Comparison of the 1H nuclear magnetic resonance spectra of these chains established that four N-acetyl groups, an N-methyl group, and a 6-deoxy function, which characterize the nonreducing, distal trisaccharide of the glycose chain of strain 1414, were absent from that of strain A100. The antigenicity of endotoxin 1414, as measured by enzyme-linked immunosorbent assay, was higher than that of endotoxin A100, but fell below it when the glycose chain of endotoxin 1414 was deprived of seven sugars by treatment with nitrous acid. This observation suggests that at least three (distal, proximal, and intermediate) regions of the glycose chain of endotoxin 1414 carry antigenic determinants. One of these, located in the distal trisaccharide, is absent from both endotoxins A100 and 134.

Biological activities and chemical composition of purified tracheal cytotoxin of Bordetella pertussis.

Specific destruction of ciliated epithelial cells lining the large airways is the primary respiratory tract cytopathology associated with human Bordetella pertussis infections. We have purified a single low-molecular-weight glycopeptide, tracheal cytotoxin (TCT), that appears to cause this pathology. By using a combination of solid-phase extraction and reversed-phase high-pressure liquid chromatography, about 700 nmol of biologically active peptide can be isolated from 1 liter of B. pertussis culture supernatant (approximately 60% yield). TCT at concentrations of 1 microM destroyed the ciliated cell population when incubated with respiratory epithelium in vitro. This concentration of TCT is similar to the concentrations found in the culture supernatant of growing B. pertussis. Purified TCT also inhibited DNA synthesis of hamster trachea epithelial cells in a quantitative, dose-dependent fashion. Endotoxin was not detected in the purified material, and neither B. pertussis nor Escherichia coli endotoxin could duplicate the biological activities of TCT. Amino acid and amino sugar analyses of purified TCT revealed the presence of glucosamine, muramic acid, alanine, glutamic acid, and diaminopimelic acid in molar ratios of 1:1:2:1:1. This suggests that TCT, the released ciliostatic principle of B. pertussis, is a disaccharide tetrapeptide subunit of peptidoglycan.

Unusual composition of peptidoglycan in Bordetella pertussis.

The composition of the peptidoglycan of Bordetella pertussis and the nature of its turnover products was determined by a new combination of analytical techniques: high performance liquid chromatography of an enzymatic peptidoglycan hydrolysate and fast atom bombardment mass spectrometry and fast atom bombardment collision-activated dissociation tandem mass spectrometry. Sixteen major components of the peptidoglycan were purified, and assignment of complete or partial chemical structures was achieved for nine and seven species, respectively. At this level of resolution, a previously unrecognized heterogeneity of monomeric (five new species; nine total) and dimeric species (five new species; five total) was detected. No species containing diaminopimelyl-diaminopimelic acid cross-links or lysyl-arginine substitutions were found. Previous estimates of total cross-linkage and average chain length were revised downward to 32% and 21 disaccharide residues, respectively. Detection of a chemically novel species, a disaccharide octapeptide monomer, in both the peptidoglycan hydrolysate and culture supernatant fluid, suggests that an N-acetyl-muramyl-L-alanine amidase acts on the intact peptidoglycan of Bordetella and participates in cell wall turnover. Five peptidoglycan turnover products were identified in the supernatant fluid of late logarithmic phase cultures, including the 1,6-anhydro monomeric species known as tracheal cytotoxin. Peptidoglycan turnover was detected at a low rate of approximately 10%/generation, a value sufficient to account for the generation of all tracheal cytotoxin found in culture supernatant fluids.

[Use of an immunoblotting method for identifying the individual proteins in the antigenic complexes of Bordetella pertussis]. / Ispol'zovanie metoda immunoblottinga dlia identifikatsii individual'nykh belkov v antigennykh kompleksakh kokliushnogo mikroba.

The composition of antigenic complexes isolated from the supernatant fluid of B. pertussis culture has been studied by means of immunoblotting techniques. In preparations obtained from B. pertussis strains 305 and 475 fragments of the molecule of fimbrial hemagglutinin, three subunits of B. pertussis toxin and agglutinogens 2 and 3 have been detected with the use of antisera to B. pertussis protective substances.

Primary structure of the peptidoglycan-derived tracheal cytotoxin of Bordetella pertussis.

The etiological agent of whooping cough, Bordetella pertussis, destroys the ciliated epithelial cells lining the large airways of infected individuals. This cytopathology can be reproduced in respiratory epithelium by tracheal cytotoxin (TCT), a small peptidoglycan-related molecule purified from the culture supernatant of growing B. pertussis organisms. Using fast atom bombardment mass spectrometry, we analyzed the positive- and negative-ion spectra of the purified, biologically active material and assigned a mass of 921 daltons to TCT. Analysis of fragment ions in these spectra as well as the spectra of the methyl ester and acetylated derivatives of TCT unambiguously defined the primary structure of TCT as N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramylalanyl-gamma- glutamyldiaminopimelylalanine. TCT is therefore identical with the ciliostatic anhydropeptidoglycan monomer released by Neisseria gonorrhoeae and with the neurologically active slow-wave sleep-promoting factor FSu. These and other structurally related glycopeptides containing muramic acid thus form a family of molecules with remarkably diverse biological activities.

Lipopolysaccharides in a traditional pertussis vaccine.

Analysis of the lipopolysaccharide (LPS, endotoxin) in cell sonicates of four Danish vaccine strains of Bordetella pertussis (3803, 3825, 3843 and 3860) and of purified strain 3803 LPS in sodium dodecyl sulphate-polyacrylamide gel electrophoresis by silver staining, showed identical profiles. The LPS profile revealed a dominant, brownish LPS II band and a minor, faster-migrating, black-stained LPS I band. However, the ratio of LPS I to LPS II in the preparation of purified LPS differed slightly from the cell sonicates. Using marker LPS, the molecular weights of LPS I and LPS II were estimated at 5.4 and 6.0 kD, respectively. Seven different lots of whole cell pertussis vaccine were assayed for LPS in the Limulus Amoebocyte Lysate test and were found to contain 0.9-2.8 micrograms LPS/ml. No significant difference in the content of LPS in similar dilutions of the individual strains was observed. In addition, the distribution of free and cell-bound LPS in four pertussis vaccines was investigated. Most of the LPS was found to exist as free LPS. During several months, the course of both LPS and pertussis toxin (Pt) release in freshly killed B. pertussis preparations was followed. In the first few weeks, 35-50% of the LPS was released and after 5-6 months of storage 60-80% had been released. In contrast, less than 10% of the biologically active pertussis toxin was released during the experimental period. The possibility of producing a safer whole cell pertussis vaccine by reducing the amount of free LPS without reducing the protective value correspondingly is discussed.

Surface proteins of Bordetella pertussis.

Bordetella pertussis cells express multiple virulence-associated surface proteins, including adenylate cyclase, agglutinogens 2 and 3, filamentous hemagglutinin, pertussis toxin, and outer-membrane protein (Omp) 30/32 and Omp91. Surface proteins that are not virulence-associated include three peptidoglycan-associated Omps of apparent molecular weights 40,000, 25,000, and 18,000. Omp40 is an anion-selective porin and is the most abundant surface protein of virulent and avirulent cells. Three independent approaches--immunomicroscopy, surface radioiodination, and isolation of Triton X-100-insoluble envelope proteins--suggest that the Triton-insoluble fraction of the B. pertussis cell envelope is the outer membrane. Agglutinogens 2 and 3 and filamentous hemagglutinin lie outside the outer membrane, the first two as fimbriae and the last as a microcapsule. Adenylate cyclase and pertussis toxin are present in the outer membrane but may be present transiently or present in small amounts.

The purification and characterization of an acellular pertussis vaccine.

An acellular pertussis vaccine manufactured by Biken was investigated for purity, potency and toxicity. The vaccine was composed of almost equal proportions of pertussis toxin (PT) and filamentous hemagglutinin (FHA). The purity of the vaccine was 97-99%. The protective effects of component vaccines containing various ratios of PT and FHA were tested and it was found that the ratio of 1:1 provided the most effective vaccine.

Immuno-electronmicroscopy of fimbriae-like structures on Bordetella pertussis serotype 1.3.

Fimbriae-like filaments were demonstrated on the surface of Bordetella pertussis, serotype 1.3, by negative staining and electronmicroscopy. Immunoelectronmicroscopy with a monoclonal antibody specific for strains possessing agglutinogen 3, and colloidal gold, gave strong labelling of these structures. However, incubation with adsorbed polyclonal anti-agglutinogen 3 serum gave only weak labelling of the distal parts of the filaments and of the bacterial surface. The different binding patterns of the two antisera suggested that the epitopes involved were dissimilar. Thus, agglutinogen 3, as defined by conventional adsorbed sera, appeared to be associated with the fimbriae-like structures but was not necessarily identical to the fimbrial subunit protein. The monoclonal antibody, however was more likely directed against the subunits of the fimbriae-like structures on serotype 1.3 bacteria.