Characterization of the common antigenic lipopolysaccharide O-chains produced by Bordetella bronchiseptica and Bordetella parapertussis

Porins of Salmonella minnesota, R595, were purified by anion exchange chromatography and subsequently isolated in their monomeric form by chromatofocusing. Two forms of porin could be isolated, both with an apparent molecular mass of 37,000, but of differing isoelectric points (pI 4.6 versus pI of 4.9). Porins with pI 4.9 did not contain any detectable LPS, but porins with pI 4.6 were found to contain trace amounts of LPS (1.3 x 10(-4) micrograms LPS/1 microgram porin) as measured using a highly sensitive limulus assay. Unlike the LPS-associated porins the monomeric porins were biologically inert with regard to pore formation, but they were still able to bind C1q, a subcomponent of the first component of complement.     

Helical structure of Bordetella pertussis fimbriae.

The helical structures of Bordetella pertussis fimbriae of serotypes 2 and 6 were determined by optical diffraction analysis of electron micrographs of negatively stained paracrystalline bundles of purified fimbriae. The fimbrial structure is based on an axial repeat of 13 nm that contains five repeating units in two complete turns of a single-start helix. This structure was confirmed by direct measurements of mass per unit length for individual fimbriae performed by dark-field scanning transmission electron microscopy of unstained specimens. These data further established that the helically repeating unit is a monomer of fimbrial protein (Mr congruent to 22,000 for type 2 and Mr congruent to 21,500 for type 6). Radial density profiles calculated from the scanning transmission electron micrographs showed that the fimbria has peak density at its center, i.e., no axial channel, consistent with the results of conventional negative-staining electron microscopy. The radial profile gives an outermost diameter of approximately 7.5 nm, although the peripheral density is, on average, diffuse, allowing sufficient intercalation between adjacent fimbriae to give a center-to-center spacing of approximately 5.5 nm in the paracrystals. Despite serological and biochemical differences between type 2 and type 6 fimbriae, the packing arrangements of their fimbrial subunits are identical. From this observation, we infer that the respective subunits may have in common conserved regions whose packing dictates the helical geometry of the fimbria. It is plausible that a similar mechanism may underlie the phenomenon of phase variations in other systems of bacterial fimbriae.     

The structure of the carbohydrate backbone of the lipopolysaccharides from Bordetella hinzii and Bordetella bronchiseptica.

The structure of the core-lipid A region of the lipopolysaccharides from Bordetella hinzii and Bordetella bronchiseptica has been analyzed. Lipopolysaccharides were deacylated using strong alkaline hydrolysis, the products were separated by high performance anion-exchange chromatography and analyzed by NMR and mass spectrometry. The following structure of the products can be deduced from the experimental results: where for the product from Bordetella hinzii N = H, R = H, beta-FucN4N- or partially N-acetylated Sug-(1-3)-beta-FucN4N and for the product from Bordetella bronchiseptica N = alpha-Hep, R = H, beta-FucN4N, beta-FucN4NMe or partially N-acetylated Sug-(1-3)-beta-FucN4N or Sug-(1-3)-beta-FucN4NMe; Sug = 2,3-diamino-2,3, 4-trideoxy-hex-4-enuronopyranosyl.     

Immunochemical study of the antigenic structure of bacteria of genus Bordetella. IV. Fractionation of B. pertussis extracts and study of the immunochemical and biological properties of the isolated fractions]

Fractionation of an extract of pertussis microbes was carried out with the aid of gel-filtration on Sephadex G-100, ion-exchange chromatography on DEAE-cellulose, and preparative electrophoresis. Fractions differing in serological, immunogenic activity and the content of antigenic components were isolated. In using the method of gel-filtration of sefadex G-100 the greatest serological, immunogenic and histamine-sensitizing activity was possessed by the high-molecular fraction containing 8 of 11 antigenic components detected in the initial extract. The antigenic components were distributed into 5 fractions by the ion exchange chromatography on DEAE-cellulose. The greatest serological activity was possessed by fractions exiting from the column at the 0.01--0.04M interval of the phosphate buffer concentration. A method of preparative electrophoresis from the pertussis microbes extract was applied and two fractions were isolated from the anode and the cathode zones, each containing 4 antigenic components only, but possessing serological and immunogenic activity and having no histamine-sensitizing properties.     

Immunoelectrophoretic analysis of the antigenic structure of Sh. sonnei]

The authors studied the antigenic composition of 105 Sh. sonnei strains freshly isolated from patients suffering from acute dysentery and carriers. Immunophoregrams of pure S-and R-forms species were obtained. Up to 13 antigens differing by electrophoretic and diffusion mobility and immunological specificity were revealed among soluble Sh. sonnei antigens The position of common and specific antigens was determined on the immunophoregram. Along with the thermostable somatic O-antigen detected at the I phase of the S-forms, and two thermolabile O-antigen components at the II phase, and the R-forms, there was revealed a surface, relatively thermolabile, K-antigen of A-type capable of agglutinating live bacteria in the O-antiserum; position of the latter on the immunophoregram was also determined.     

The antigenic structure of poliovirus

We have solved the structure of the Mahoney strain of type 1 and the Sabin (attenuated vaccine) strain of type 3 poliovirus by X-ray crystallographic methods. By providing a three-dimensional framework for the interpretation of a wealth of experimental data, the structures have yielded insight into the architecture and assembly of the virus particle, have provided information regarding the entry of virus into susceptible cells, and defined the sites on the virus particle that are recognized by neutralizing monoclonal antibodies. Thus locating mutations in variants selected for resistance to neutralizing monoclonal antibodies has defined three antigenic sites of the surface of the virion, and provided clues as to the mechanisms by which viruses escape neutralization. Finally, comparison of the structures of the two strains, together with analysis of sequences of many poliovirus strains, have begun to define the structural changes associated with serotypic differences between polioviruses.     

Two trans-acting regulatory genes (vir and mod) control antigenic modulation in Bordetella pertussis.

Expression of virulence factors by Bordetella pertussis is altered by environmental signals (antigenic modulation) and is dependent on an activator encoded by a gene called vir. We have used TnphoA (Tn5 IS50L::phoA) gene fusions to define two sets of genes whose expression is either activated (vag loci) or repressed (vrg loci) by modulation signals. Both groups of genes appear to be regulated by the vir gene product in that, in the absence of modulators, null mutations in vir lead to the repression of vag gene fusions and derepression of vrg gene fusions. Mutants of B. pertussis were isolated that constitutively express virulence factors in the presence of the modulator MgSO4, nicotinic acid, or low incubation temperature. We designate the gene that carries such mutations mod (modulation) and have characterized one (mod-1) of these mod constitutive mutations. A method was developed for the insertional inactivation of the vir gene by using the integration of a suicide replicon. Inactivation of the vir gene in the mod-1 mutant, followed by transcomplementation with the cloned wild-type vir gene, gives the Mod-1 constitutive phenotype, showing that the mod-1 mutation defines a gene distinct from vir. The gene carrying the mod-1 mutation is linked to vir and was cloned on a recombinant cosmid (pLAF-C1) which transcomplements the vir-1::Tn5 mutation in B. pertussis 347. Introduction of pLAF-C1 into vir mutant and vir+ B. pertussis strains also gives the Mod-1 constitutive phenotype, indicating that mod-1 is a dominant allele. These data suggest that the mod gene product could have sensory functions for the environmental signals that affect the expression of vir-regulated genes of B. pertussis. The mod constitutive strains and plasmids described here also have applications in pertussis vaccine development.     

The antigenic structure of bovine serum albumin. Evidence for multiple, different, domain-specific antigenic determinants.

Using antiserum to native bovine albumin and antigenically active fragments of the protein, we have isolated antibodies directed to each of the three domains and to several subdomains of the albumin molecule. Using albumin and these fragments as inhibitors of the reaction between 125I-albumin and any given antibody population, we have demonstrated that: (a) each domain of albumin is antigenically distinct from each of the other domains; (b) each domain possesses a minimum of two different antigenic determinants; and (c) the entire albumin molecule possesses a minimum of six different, nonrepeating, antigenic determinants.     

The precise and entire antigenic structure of native lysozyme.

The exact boundary, residue, conformational and directional definitions of the three antigenic sites of native hen's egg-white lysozyme are described. The results clearly reveal that the three antigenic sites account quantitatively for the total antigenic reactivity of the protein. Thus the entire antigenic structure of lysozyme has now been precisely determined and is briefly discussed here, together with the power of the surface-stimulation synthetic concept.     

Revision of the antigenic structure of genus Listeria

O-antigenic structure of genus Listeria was studied, using antisera (obtained from rabbits) against different O-antigens of reference strains of each serovar. The titres of sera were determined by agglutination using antigens of the same reference strains as well. Some differences from the actual scheme were found: serum antifactor-IX gave a lower titre than expected against antigens 4ab and 6b, while the titre observed against antigen 4b was higher than the expected in this case. Serum antifactor-VIII presented a higher titre than could be expected against antigen 6b. The strains of serovars 4d and 4e used in this experience were impossible to distinguish, and could have been classified in the same serovar. We could not obtain serum antifactor-XI from serovar 6b after several trials. From these differences we propose some modifications of the current antigenic scheme of genus Listeria.     

The antigenic structure ofFrancisella tularensis

The ether antigen ofFrancisella tularensis was fractionated using DEAE-cellulose and CM-cellulose chromatography, and Sephadex G-200 gel filtration. The CM-cellulose chromatography did not appear to be a suitable method for separation of individual components of the complex ether antigen. Most of the polysaccharide material and substances with a high phosphorus content were eluted already in the first peak of the elution curve. This method could be used only to separate a component, yielding one immunoelectrophoretic precipitin line, localized towards the cathode. On the other hand, the DEAE-cellulose chromatography, and particularly the Sephadex G-200 gel filtration (especially when recycling was introduced), yielded a clear separation of relatively clean components of the ether antigen. The present work provides a comparison between the immunochemical (immunoelectrophoretic) properties of fractions obtained by these methods and components isolated by salting out with ammonium sulphate, ethanol or trichloracetic acid precipitation.