Yersinia enterocolitica immunodominant 60 kDa antigen, common to a broad range of bacteria, is a heat-shock protein

Monoclonal antibodies (mAbs) against the Yersinia enterocolitica immunodominant 60 kDa antigen, termed cross-reacting protein antigen (CRPA), were obtained by fusion of spleen cells from mice immunized with CRPA with murine myeloma cells. The reactivities of the mAbs were examined by Western blotting against extracts of Y. enterocolitica and 23 other species of Gram-positive and Gram-negative bacteria. Cross-reactions were recognized with a wide range of bacteria, but not with Gram-positive cocci. The reactivities were different for each mAb, suggesting that both species-specific and multiple cross-reactive epitopes were present on the CRPA molecule. CRPA was produced under heat-shock conditions in Y. enterocolitica and was shown to correspond immunologically to the GroEL protein in Escherichia coli, a protein involved in the morphogenesis of coliphage. In addition to CRPA, at least nine other major heat-shock proteins were detected by two-dimensional gel electrophoresis of extracts of heat-shocked Y. enterocolitica.     

A heat-modifiable outer membrane protein carries an antigen specific for the species Yersinia enterocolitica and Yersinia pseudotuberculosis

The outer membranes of gram-negative bacteria are considered to be of importance in host-bacteria interaction, in protective immunity, and occasionally in subclassification within a species. In this study, the outer membranes of several strains of Yersinia enterocolitica and Y. pseudotuberculosis were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the appearance of the major proteins depended on the temperature at which they were solubilized in SDS. A protein was identified with the use of two-dimensional gels and preparative SDS-PAGE, which was equivalent to the "heat-modifiable protein" (protein II) of other Enterobacteriaceae species. A monoclonal antibody, 4G1, was generated against an isolated preparation of this Y. enterocolitica protein. This antibody was tested with whole cell bacterial antigens of 46 individual bacterial strains. The reactive strains included only Y. enterocolitica and Y. pseudotuberculosis. In addition, the reactivity of the 4G1 monoclonal antibody preparation could be absorbed only with Y. enterocolitica and Y. pseudotuberculosis, and not with other strains of bacteria. The reactivity of this 4G1 monoclonal antibody was also tested by the Western Blot technique. Six individual strains were tested: a Y. enterocolitica serotype 0:3, a Y. enterocolitica serotype 0:9, an Escherichia coli, a Salmonella typhimurium, a Shigella flexneri, and a Klebsiella pneumoniae. The 4G1 antibody reacted with only the proteins of the two Y. enterocolitica strains. In conclusion, the equivalent of the heat-modifiable protein was present in Y. enterocolitica and Y. pseudotuberculosis. Moreover, this protein also carried a species-specific antigenic determinant.     

Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.     

Localization of IpaB protein in Escherichia coli K-12 MC1061 strain carrying Shigella sonnei form I plasmid pSS120

We partially purified antigens which reacted with shigellosis convalescent-monkey antisera. Hybridomas, which were constructed from mice immunized by the antigens, produced monoclonal antibodies recognizing IpaB protein. Using the monoclonal antibody against IpaB, evidence indicating that IpaB proteins were localized on the cell surface of invasive bacteria (Escherichia coli K-12 MC1061 harboring pSS120 plasmid) was obtained by immunoelectron microscopy.     

Effect of dyes on the quantitative recovery of Yersinia enterocolitica.

A total of 69 dyes were incorporated separately at different concentrations into an agar medium for evaluation of their effects on the quantitative recovery of five serotypes of Yersinia enterocolitica. One strain of Pseudomonas aeruginosa and one strain of Bacillus cereus were included for comparative purposes. Certain dyes were evaluated further for their selective properties with five additional serotypes of Y. enterocolitica, three strains of P. aeruginosa, and two of Engerobacter spp. Metanil yellow was the only dye which was tolerated bettr by Y. enterocolitica than by P. aeruginosa. Dye sensitivity was variable amond strains of the same serotype of Y. enterocolitica. In general, Y. enterocolitica showed a tolerance to dyes greater than that of gram-positive bacteria and similar to that of other gram-negative bacteria.     

Effect of dyes on the quantitative recovery of Yersinia enterocolitica.

A total of 69 dyes were incorporated separately at different concentrations into an agar medium for evaluation of their effects on the quantitative recovery of five serotypes of Yersinia enterocolitica. One strain of Pseudomonas aeruginosa and one strain of Bacillus cereus were included for comparative purposes. Certain dyes were evaluated further for their selective properties with five additional serotypes of Y. enterocolitica, three strains of P. aeruginosa, and two of Engerobacter spp. Metanil yellow was the only dye which was tolerated bettr by Y. enterocolitica than by P. aeruginosa. Dye sensitivity was variable amond strains of the same serotype of Y. enterocolitica. In general, Y. enterocolitica showed a tolerance to dyes greater than that of gram-positive bacteria and similar to that of other gram-negative bacteria.     

Analysis of a common-antigen lipopolysaccharide from Pseudomonas aeruginosa.

Lipopolysaccharide isolated from Pseudomonas aeruginosa PAO1 (O5 serotype) was separated into two antigenically distinct fractions. A minor fraction, containing shorter polysaccharide chains, reacted with a monoclonal antibody to a P. aeruginosa common antigen but did not react with antibodies specific to O5-serotype lipopolysaccharide. In contrast, fractions containing long polysaccharide chains reacted only with the O5-specific monoclonal antibodies. The shorter, common-antigen fraction lacked phosphate and contained stoichiometric amounts of sulfate, and the fatty acid composition of this fraction was similar to that of the O-antigen-specific fraction. The lipid A derived from the serotype-specific lipopolysaccharide cross-reacted with monoclonal antibodies against lipid A from Escherichia coli, while the lipid A derived from the common antigen did not react. We propose that many serotypes of P. aeruginosa produce two chemically and antigenically distinct lipopolysaccharide molecules, one of which is a common antigen with a short polysaccharide and a unique core-lipid A structure.     

Demonstration of peptidoglycan-associated Brucella outer-membrane proteins by use of monoclonal antibodies.

A monoclonal antibody (3D6) was produced which reacted only with Brucella sonicated cell extracts that had been lysozyme-treated after sonication. The monoclonal antibody (mAb) reacted with the three major outer-membrane proteins (OMPs) of B. melitensis B115 in Western blots. A large number of reactive bands ranging from 12 to 43 kDa were present in lysozyme-treated Escherichia coli and Yersinia enterocolitica sonicated cell extracts. In a latex agglutination inhibition immunoassay, mAb 3D6 showed better reactivity with purified peptidoglycan (PG) of B. melitensis B115 than with that of Escherichia coli. This mAb was also used in immunogold electron microscopy with whole Brucella cells and sections. No binding was observed on whole cells and immunogold labelling in sections was observed close to the outer membrane, in the periplasmic space and in the cytoplasm. These findings indicate that mAb 3D6 is specific for PG subunits. Immunoblot analysis of B. melitensis B115 rough sonicated cell extracts after SDS-PAGE, with or without lysozyme treatment, was performed using mAbs specific for Brucella OMPs of molecular masses of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 kDa, for PG and for rough lipopolysaccharide (R-LPS) and smooth lipopolysaccharide (S-LPS). mAbs specific for the 25-27, 31-34 and 36-38 kDa OMPs reacted with three to six bands. All of them except the band of lowest molecular mass reacted with the PG-specific mAb and not with R-LPS- and S-LPS-specific mAbs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies directed against plasmid-encoded released proteins of enteropathogenic Yersinia

Abstract Yersinia enterocolitica of serotypes O:3, O:8, O:9 and O:5,27 and Yersinia pseudotuberculosis of serotypes I and III release plasmid-encoded proteins into calcium-deficient medium. Mouse monoclonal antibodies were elicited against plasmid-encoded released proteins of Y. enterocolitica of serotype O:9. As shown by immunoblot analysis the monoclonal antibody Mab9–200 recognized the 46-kDa protein of Y. enterocolitica of serotypes O:3, O:9 and O:5,27, the 58-kDa protein of Y. enterocolitica of serotype O:8 and the 67-kDa protein of Y. pseudotuberculosis of serotypes I and III. Mab9–15 reacted with the 36-kDa protein of Y. enterocolitica of serotypes O:9, O:3 and O:8, and the 34-kd protein of Y. enterocolitica of serotype O:5,27 and Y. pseudotuberculosis of serotypes I and III. The 25-kDa proteins of Y. enterocolitica of serotypes O:3, O:9, O:8 and O:5,27, but not those of Y. pseudotuberculosis were recognized by the monoclonal antibody Mab-128. This species-specific recognition of epitopes could not be achieved by mouse polyclonal antibodies.     

Screening of murine monoclonal antibodies against living schistosomula of Schistosoma mansoni by radioimmunoassay

A radioimmunoassay was developed to screen supernatants of murine monoclonal antibodies against surface antigens of living schistosomula of Schistosoma mansoni. Of 196 clones screened, 10% bound schistosomula. Of these, 74% bound only schistosomula. The remaining molecules also reacted with soluble adult worm antigens and soluble egg antigens as determined by enzyme-linked immunosorbent assay. Immunoblot analysis demonstrated that monoclonal antibody 204-3E4 reacted with a 68 kDa protein, a glycoprotein that induces substantial resistance against S. mansoni infection. Recognition of an 18 kDa antigen by 204-3F1 antibody was stage-specific with the antigen being expressed in cercariae, 3- and 24-h-old parasites but not 4-day, lung stage or adult worms. Monoclonal antibody 204-4E3 reacted with purified S. mansoni paramyosin. These data indicate that radioimmunoassay using living schistosomula is a rapid alternative method to identify murine hybridomas that secrete antibodies which react with surface antigens of S. mansoni.     

A murine IgG1 monoclonal antibody thatbinds specifically to outer surface protein A of Lyme disease spirochete Borrelia burgdorferi

Abstract A murine monoclonal antibody, designated MA-2G9, directed against outer surface protein A (OspA) of the Lyme disease spirochete, Borrelia burgdorferi , has been produced. Antibody MA-2G9, IgG1 subclass, was purified by affinity chromatography on protein G Sepharose column and used for purification of OspA antigen from Borrelia burgdorferi cell lysate. Epitope specificity was studied by Western immunoblotting, using several strains of B. burgdorferi and non-Lyme disease bacteria such as Treponema pallidum and B. hermsii . The MA-2G9 monoclonal antibody reacted specifically with recombinant OspA aas well as with native OspA in sonicated B. burgdorferi strains. No reaction was observed with T. pallidum, Escherichia coli, Staphylococcus aureus and B. hermsii lysates. The MA-2G9 antibody also recognized the denatured form of OspA indicating that it is directed against sequential epitope and not conformational epitope.     

A method for studies of an El Tor-associated antigen of Vibrio cholerae O1

A method for studying the biotype El Tor associated mannose-sensitive haemagglutinin (MSHA) of V. cholerae O1 has been developed. By using crude MSHA adsorbed to chicken erythrocytes as solid phase antigen in an enzyme-linked immunosorbent assay (ELISA), antisera against V. cholerae of the El Tor biotype reacted in high titre with the MSHA-coated cells, whereas antisera against vibrios of the classical biotype did not bind significantly, i.e. in higher titre than pre-immune sera. The binding of anti-MSHA serum, or a monoclonal antibody against MSHA, to the MSHA-coated erythrocytes could be efficiently inhibited by crude MSHA as well as by El Tor vibrios whereas neither V. cholerae lipopolysaccharide nor different strains of classical vibrios had any inhibitory effect. These results support the existence of an El Tor-associated immunogen. They also suggest a possibility of determining antibodies against different haemagglutinins in ELISA without having access to purified antigens.     

Characterisation of a monoclonal antibody against the fimbrial F8 antigen of Escherichia coli

Abstract A monoclonal IgG1 antibody against F8 fimbriae was obtained with the hybridoma technique using spleen cells from C3H/f mice immunised with a fimbrial preparation of Escherichia coli 2980 (O18ac:K5:H⁻:F1C, F8) and Sp 2/0 Ag8 myeloma cells. The hybrid cells were cloned twice by limiting dilution and grown in tissue culture. The monoclonal antibody was purified from culture supernatants on Protein A Sepharose. It reacted with F8 fimbriae in colony blot, enzyme-linked immunosorbent assay (ELISA) and immunoblot after electrotransfer from sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of fimbrial preparations. The antibody bound to and agglutinated F8-fimbriated bacteria.     

Antibody inhibition of HeLa cell invasion by Yersinia enterocolitica

Antisera were prepared in rabbits against formalized and heat-killed bacteria of Yersinia enterocolitica serotype O:5,27 and against formalized bacteria of serotype O:8. Both strains used for immunization demonstrated adhesion to and invasion of HeLa cells. Coating of the bacteria with antibody did not greatly alter adhesion (i.e., extracellular attachment) to HeLa cells; however, antibody against formalized bacteria of both serotypes inhibited HeLa cell invasion by the homologous and heterologous strains. The Fab fragments from purified immunoglobulins also demonstrated cross-reacting inhibition of HeLa cell invasion. Antibody against heat-killed bacteria of serotype O:5,27 had no inhibitory activity. Adsorption of the antiserum against formalized bacteria of serotype O:5,27 with lipopolysaccharide from the homologous strain removed anti-lipopolysaccharide antibody but did not remove the inhibitory activity. The antiserum against formalized bacteria of serotype O:8 showed no antibody against lipopolysaccharide from serotype O:5,27 and no agglutinins against heat-killed bacteria of this strain. From these results, it is tentatively suggested that protein structures are important in mediating epithelial cell invasion by Y. enterocolitica.     

Application of released proteins (RPs) of Yersinia enterocolitica for serological diagnosis of yersiniosis. II. Gene recombination for production of the YopD protein

The aim of this study was to evaluate the usefulness of gene recombination technique using the pET-30 Ek/LIC expression vector for production a 36 kDa released protein called YopD and evaluate of this purified protein as antigen in serodiagnosis of yersiniosis. Protein YopD of Y. enterocolitica was expressing in Escherichia coli BL21 (DE3) using the pET-30 Ek/LIC expression vector. Purification of the expressed enzyme from suspensions of E. coli cells treated with Bug Buster Protein/Extraction Reagent was accomplished by immobilised metal (Ni2+) affinity column chromatography (His-trap). The IgM, IgG and IgA class antibodies to YopD were measured in 100 serum samples collected from patients suspected for yersiniosis and 100 blood donors. The obtained results were compared to the results of ELISA with released proteins isolated from the culture of Y. enterocolitica supernatant under calcium deficient conditions and commercial ELISA with recombinant released proteins. A very high (94.0-100.0%) specificity and good sensitivity (55.2-80.4%) were displayed by the ELISA with YopD in relation to other two ELISA. The results of our study showed that recombinant YopD protein purified by chromatography of bio-affinity may be used in serodiagnosis of yersiniosis as a high specific antigen free of Yersinia lipopolysaccharides.     

Monoclonal antibodies for detection of the H7 antigen of Escherichia coli.

Two murine monoclonal antibodies (MAbs) (2B7 and 46E9-9) reactive with the H7 flagellar antigen of Escherichia coli were produced and characterized. A total of 217 E. coli strains (48 O157:H7, 4 O157:NM, 23 O157:non-H7, 22 H7:non-O157, and 120 non-O157:nonH7), 17 Salmonella serovars, and 29 other gram-negative bacteria were used to evaluate the reactivities of the two MAbs by indirect enzyme-linked immunosorbent assay (ELISA). Both MAbs reacted strongly with all E. coli strains possessing the H7 antigen and with H23- and H24-positive E. coli strains. Indirect ELISA MAb specificity was confirmed by inhibition ELISA and by Western blotting (immunoblotting), using partially purified flagellins from E. coli O157:H7 and other E. coli strains. On a Western blot, MAb 46E9-9 was more reactive against H7 flagellin of E. coli O157:H7 than against H7 flagellin of E. coli O1:K1:H7. Competition ELISA suggested that MAbs 2B7 and 46E9-9 reacted with closely related H7 epitopes. When the ELISA reactivities of the MAbs and two commercially available polyclonal anti-H7 antisera were compared, both polyclonal antisera and MAbs reacted strongly with E. coli H7 bacteria. However, the polyclonal antisera cross-reacted strongly both with non-H7 E. coli and with many non-E. coli bacteria. The polyclonal antisera also reacted strongly with H23 and H24 E. coli isolates. The data suggest the need to define serotype-specific epitopes among H7, H23, and H24 E. coli flagella. The anti-H7 MAbs described in this report have the potential to serve as high-quality diagnostic reagents, used either alone or in combination with O157-specific MAbs, to identify or detect E. coli O157:H7 in food products or in human and veterinary clinical specimens.     

Opsonizing and bactericidal activity of antibodies against common antigen of Enterobacteriaceae

Domingue, Gerald J. (State University of New York at Buffalo, Buffalo, N.Y.), and Erwin Neter. Opsonizing and bactericidal activity of antibodies against common antigen of Enterobacteriaceae. J. Bacteriol. 91:129-133. 1966.-In addition to the well-known O, H, K, and Vi antigens, Enterobacteriaceae produce a common antigen, which was first identified by hemagglutination tests with Escherichia coli O14 antiserum. Studies on the biological significance of this antigen by in vitro phagocytic experiments have revealed that opsonization is markedly enhanced in the presence of the corresponding antibody, rabbit polymorphonuclear leukocytes, and normal rabbit serum. This effect is specific, because Pseudomonas aeruginosa, which is devoid of this antigen, is not opsonized under these conditions, and removal of the antibody by absorption markedly reduces the uptake of enteric bacteria containing the antigen. Opsonization thus represents another method for the study of this antigen-antibody system. Bactericidal tests have revealed that antibodies against this antigen, engendered in rabbits by different strains of enteric bacteria and various procedures, are bactericidal for E. coli O14 but not for other enteric bacteria, possibly due to previously demonstrated differences between the antigen moieties obtained from these microorganisms.     

Amlodipine: a cardiovascular drug with powerful antimicrobial property

Ten cardiovascular drugs were procured in pure form from their manufacturers in India and screened for antimicrobial property against fifteen known bacteria belonging to both gram-positive and gram-negative types. These bacteria were inhibited by the common antibiotics at 1-5 mg ml(-1) level through our earlier studies. Since most of the bacteria were moderate to highly responsive to amlodipine, this compound was further tested in vitro against 504 bacteria comprising 4 genera of gram-positive and 15 genera of gram-negative bacteria. Most of these were inhibited by the drug at 50-200 microg ml(-1) level and few strains were sensitive even at lower concentrations (10 microg ml(-1)). The bacteria could be arranged in the decreasing order of sensitivity towards amlodipine in the following manner: Staphylococcus aureus, Vibrio cholerae, Vibrio parahemolyticus, Shigella spp., Salmonella spp., Bacillus spp., whereas Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa were found to be resistant to the lower concentrations of the drug. Amlodipine was found to be bactericidal in nature when its mode of action was studied against S. aureus 6571, V. cholerae 14035 and Sh boydii 8 NCTC 254/66. The antibacterial activity of amlodipine could also be confirmed in vivo. When it was given to Swiss strain of white mice at different dosages (30 and 60 microg/mouse), it could significantly protect the animals challenged with 50 MLD of Salmonella typhimurium NCTC 74. According to Chi square test the in vivo data were highly significant (p<0.001).     

N-Acetyl-d-glucosamine-specific lectin purified from Vibrio cholerae 01

An N-acetyl-D-glucosamine-specific cell associated hemagglutinin (HA) was isolated and purified from a strain of Vibrio cholerae 01 by chitin affinity chromatography followed by separation on Bio Gel P-150. A single stained protein band of 47 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was observed with the purified HA. HA-antisera produced a single precipitin band against the purified HA in an immunodiffusion test without exhibiting any reactivity towards purified lipopolysaccharide (LPS). Purified HA, used as solid-phase antigen in an enzyme-linked immunosorbent assay (ELISA), reacted strongly with HA-antisera but cross-reacted negligibly with antisera raised against purified LPS. Hemagglutinating activity of the purified HA was highly sensitive to N-acetyl-D-glucosamine. The immunogold-labelling method using HA-antisera confirmed the location of the HA on the surface of the bacterial cells. The HA-antisera reacted with a protein component of the homologous outer membrane preparation. A significant inhibition was observed in the adhesive capability of the V. cholerae 01 strain to isolated rabbit intestinal epithelial cells (RIEC) in vitro when the later were pre-treated with the purified HA.     

Production of monoclonal antibody to bovine leukemia virus glycoprotein gp51 by in vitro immunization

The monoclonal antibody against glycoprotein gp51 of bovine leukemia virus (BLV) envelope antigen was produced by in vitro immunization. This monoclonal antibody reacted with viral antigen was observed at the 69 kilodalton (kDa) glycoprotein. However, this monoclonal antibody was not involved in neutralizing. It was shown that in comparison with in vivo immunization, in vitro immunization has some advantages, namely a short immunization period and a small antigen quantity.