Structure-function studies with Neisseria gonorrhoeae Opa outer membrane proteins expressed in Escherichia coli.
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Antigens, Bacterial↗
Biomedical subjects
M S Blake
Publications and source records attributed to M S Blake.
Protein B: an important human IgA-binding reagent.
Protein B had a much higher affinity for human IgA than Jacalin, increasing the sensitivity and specificity of the measurement of total human IgA. Protein B, used as a capturing agent, greatly enhanced the measurement of antigen-specific IgA as compared to alpha chain-specific antibodies.
Antibody Specificity↗
The pathogenesis of gonococcal urethritis in men: confocal and immunoelectron microscopic analysis of urethral exudates from men infected with Neisseria gonorrhoeae.
Confocal and immunoelectron microscopic analysis of urethral exudates from 12 men with gonococcal urethritis showed that Neisseria gonorrhoeae can invade urethral epithelial cells. Studies with acridine orange stain demonstrated that the majority of organisms within urethral epithelial cells were viable at the time of fixation. Three-dimensional modeling of an infected epithelial cell using image analysis of 21 digitized confocal sections stained with YOYO-1 and DiIC 18(3) revealed that gonococcal invasion of these cells occurred in a polar fashion, most likely at the epithelial luminal surface. Serial immunoelectron micrographs showed evidence of membrane fusion with pedestal formation between the gonococcus and the epithelial cell, gonococci within vacuoles, and occasional gonococci free in the cytoplasm. Immunoelectron microscopy studies showed ruptured vacuoles at the cell surface releasing organisms. These studies demonstrate that urethral epithelial cells are invaded by gonococci during the course of infection in males.
Epithelium↗
Production of Haemophilus influenzae type-b porin in Escherichia coli and its folding into the trimeric form.
The P2 protein from pathogenic Haemophilus influenzae type b (Hib) functions as a bacterial porin and is one of several immunogenic outer membrane proteins. The P2 gene was expressed in Escherichia coli and the recombinant P2 protein (re-P2) purified to facilitate functional and immunologic studies. P2 was obtained from Hib strain Eagan using PCR and the pET vectors (17b and 11a) were used to produce re-P2 at levels exceeding 30% of the total E. coli proteins. Since previous reports had indicated that P2 was toxic to E. coli, steps were taken to control the toxicity. The plasmid was stabilized by tightly controlling the synthesis of re-P2 prior to induction. Subsequent to induction, re-P2 was sequestered into inclusion bodies rather than to membrane compartments. The refolding of the denatured re-P2 into the trimeric form involved high salt and calcium ions. re-P2 was then purified to homogeneity using gel-filtration and ion-exchange chromatography.
Antigens, Bacterial↗
Vaccines for gonorrhea: where are we on the curve?
Human antibodies that bind the gonococcal outer membrane modulate gonorrheal transmission and disease. The effects of antibody binding can favor either the host or the bacteria, and depend on the antigen involved. An effective gonococcal vaccine is feasible, but only by the careful selection and formulation of gonococcal antigens that elicit only host-protective antibodies.
Bacterial Vaccines↗
Group A streptococcus-liposome ELISA antibody titers to group A polysaccharide and opsonophagocytic capabilities of the antibodies.
Antibodies reactive with group A streptococci (GAS) carbohydrate were studied by ELISA and in an indirect bactericidal assay. The ELISA used GAS carbohydrate covalently bound to phosphatidylethanolamine incorporated into liposomes so that both precipitating and nonprecipitating antibodies were measured. Sera from children from different geographic areas exhibited marked differences in levels of anti-GAS carbohydrate antibody, which increased with age. The antibodies were predominantly of IgG. In bactericidal assays, most of these sera promoted phagocytosis of several type-specific M-positive strains. Opsonization was also related to serum levels of anti-GAS carbohydrate antibodies. These opsonizing antibodies were depleted from the serum by absorption of the sera on an N-acetyl-D-glucosamine affinity column. Antibody eluted from this column could partially restore opsonization of GAS. Anti-GAS carbohydrate antibodies play a major role in these opsonophagocytosis assays.
Antibodies, Bacterial↗
Gonococcal opacity: lectin-like interactions between Opa proteins and lipooligosaccharide.
Previous evidence from our laboratory suggested that the tight intercellular adhesions between the outer membranes of gonococci displaying the opacity colony phenotype occurred because Opa proteins expressed on one gonococcus adhered to the lipooligosaccharide (LOS) of the opposing bacterium (M.S. Blake, p. 51-66, in G. G. Jackson and H. Thomas, ed., The Pathogenesis of Bacterial Infections, 1985, and M. S. Blake and E. C. Gotschlich, p. 377-400, in M. Inouye, ed., Bacterial Outer Membranes as Model Systems, 1986). A noncompetitive inhibition assay used previously to determine the carbohydrate structures recognized by the major hepatic asialoglycoprotein receptor was modified to determine the gonococcal LOS structures that bind Opa proteins (R. T. Lee, Targeted Diagn. Ther. Ser. 4:65-84, 1991). The LOS carbohydrates used in these assays were LOS structures purified from pyocin LOS mutants of Neisseria gonorrhoeae 1291 described by K. C. Dudas and M. A. Apicella (Infect. Immun. 56:499-504, 1988) and further characterized by C. M. John et al. (J. Biol. Chem. 266:19303-19311, 1991). Purified gonococcal Opa proteins were incubated with each of the parent and mutant LOS, and the amount of binding of Opa proteins was measured by a direct enzyme-linked immunosorbent assay using the Opa-specific monoclonal antibody 4B12. The affinities of the Opa proteins for each of the LOS were determined indirectly by measuring the concentrations of Opa proteins that noncompetitively inhibited 50% of the binding of LOS-specific monoclonal antibodies. This concentration is inversely proportional to the affinity of the inhibitor (R. T. Lee, Targeted Diagn. Ther. Ser. 4:65-84, 1991). Our data suggest that the gonococcal Opa proteins tested had the highest affinity for the Gal beta 1-4GlcNAc residue present on the gonococcal lactoneoseries LOS. This affinity was comparable to that reported for the binding of the major hepatic asialoglycoprotein receptor to glycoconjugates containing terminal galactose and N-acetylgalactosamine (R. T. Lee, Targeted Diagn. Ther. Ser. 4:65-84, 1991). After sialylation of the lactoneoseries LOS, presumably on the terminal galactose residue, the interaction with the Opa proteins was ablated. Therefore, the gonococcal Opa-LOS and mammalian epithelial cell asialoglycoprotein receptor-carbohydrate interactions have quite similar specificities.
Antigens, Bacterial↗
Neisseria gonorrhoeae utilizes and enhances the biosynthesis of the asialoglycoprotein receptor expressed on the surface of the hepatic HepG2 cell line.
One of the lipooligosaccharide (LOS) structures of Neisseria gonorrhoeae contains a terminal Gal(beta 1-4)GlcNAc residue which is a good candidate to serve as a ligand for human asialoglycoprotein receptors (ASGP-R). These receptors have been shown to be present on macrophages, sperm cells, and hepatocytes. The human tissue culture cell line used most often to study this receptor, HepG2, was used in our investigations only as a model. We also chose N. gonorrhoeae 1291 for these studies because, unlike many other gonococcal strains, this strain expresses one main species of LOS. The LOS structure expressed by this strain has also been fully characterized. Using well-established assays for the utilization of the ASGP-R, we found that incubation of HepG2 cells with gonococci expressing the terminal Gal(beta 1-4)GlcNAc asialo-LOS carbohydrate structure competitively inhibited the ASGP-R from binding to one of its well-known ligands, asialo-alpha-acid-1-glycoprotein. The inhibition was specific to the ASGP-R, since binding of two other ligands to their specific receptors in the same model cell system was not affected. Immunoblot analysis for the ASGP-R suggested that gonococci seemed to stimulate the HepG2 cells to increase the expression of the major (46-kDa) receptor species. This observation was confirmed both by functional analysis, which showed that the concentration of total receptor molecules, as well as surface receptors, was about 60% higher after incubation with gonococci than in control cells and by Northern (RNA) blot analysis using a cDNA probe of the major human H1 subunit. Poly(A) RNA purified from control and HepG2 cells exposed to gonococci indicated the presence of increased amounts of mRNA coding for the ASGP-R after incubation with gonococci. This result supports the idea that the molecular mechanism controlling the receptor level after gonococcal exposure is under transcriptional regulation.
Antigens, Bacterial↗
A lipooligosaccharide-binding site on HepG2 cells similar to the gonococcal opacity-associated surface protein Opa.
The lacto-N-neotetraose-containing lipooligosaccharide (LOS) present on the surface of most Neisseria gonorrhoeae organisms may serve many important functions in gonococcal pathogenesis. This surface glycolipid contains the cross-reactive epitope to human paragloboside and can be sialylated by gonococci grown in the presence of CMP-N-acetylneuraminic acid. Another possible role for this glycolipid could be to mimic human asialocarbohydrates and act as a ligand for asialoglycoprotein receptors contained on numerous human cells. The most noted of this large family of receptors is that expressed on the surface of hepatic cells. In a model cell system, using the hepatoma tissue culture cell line HepG2, we wanted to investigate if the presence of this asialoglycoprotein receptor influenced the adherence and/or invasion of gonococci expressing the lacto-N-neotetraose structure. Piliated variants of the gonococcal wild-type strain 1291 and its isogeneic LOS mutant 1291E were used in adherence-invasion assays. This gonococcal strain is somewhat unusual in that it expresses large amounts of predominantly one species of LOS, thus reducing the complexity of interpreting the data. The data from these assays suggested that the Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc carbohydrate structure on the wild-type LOS affected the adherence-invasion of gonococci into the HepG2 cells. In studies to determine whether the major hepatic asialoglycoprotein receptor was involved in these interactions, we found that the HepG2 cells contained two receptors which bound gonococcal LOS. One of these was the asialoglycoprotein receptor, and the data concerning this receptor will be reported elsewhere. The data on the second receptor are reported here. Purified, 125I-labeled gonococcal LOS was used to identify specific high-affinity LOS-binding sites. These binding experiments revealed one major binding site corresponding to a protein with a molecular mass of 70 kDa (p70). Several lines of evidence in this study suggested that the oligosaccharide region of LOS played an important role in LOS binding to the p70 of HepG2 cells. In addition, we show that this human LOS receptor has some similarities to the gonococcal Opa proteins.
Antigens, Bacterial↗
Serum resistance of Neisseria gonorrhoeae. Does it thwart the inflammatory response and facilitate the transmission of infection?
N. gonorrhoeae differentially subvert the effectiveness of complement (C) and alter the inflammatory responses elicited in human infection. Disseminated (DGI) isolates typically resist killing by normal serum (are serum-resistant), inactivate more C3b (to iC3b preferentially bound via amide linkages), generate less C5a, and result in less inflammation at local sites. Pelvic inflammatory disease isolates are serum-sensitive, inactivate less C3b (while maintaining active C3b via stable amide linkages), generate more C5a, and result in more inflammation at local sites. Sialylation of SS gonococci, presumed to occur in vivo, converts them to serum-resistant, but it does not change the patterns of C3b inactivation and therefore may not affect local inflammation. IgG antibody directed against gonococcal reduction modifiable protein (Rmp) blocks C-mediated killing of N. gonorrhoeae. Anti-Rmp blocking antibodies may harbor specificity for OmpA sequences shared with other neisserial species or Enterobacteriaceae or may be directed against unique Rmp upstream cysteine loop specific sequences, or both. Preexisting antibodies directed against Rmp facilitate transmission of gonococcal infection to exposed women; exclusion of highly immunogenic Rmp antigens from vaccine candidates may be important.
Antibodies, Bacterial↗
Expression of large amounts of neisserial porin proteins in Escherichia coli and refolding of the proteins into native trimers.
Porins from different neisserial strains and species have been shown to have differences in both primary amino acid sequence and biophysical characteristics as observed by functional assays. A closer examination of how the changes in the primary amino acid sequence of Neisseria porin molecules correlate with these observed biophysical changes has been impeded by the inability to easily manipulate the cloned porin genes by modern molecular techniques and then obtain enough of the expressed modified porin protein to purify and use in these biophysical functional assays. In this report, we describe a method by which the genes encoding three different porin proteins, lacking their neisserial promoter and signal sequences, were cloned into an expression plasmid and transformed into Escherichia coli. Upon induction, large amounts of the porin proteins were produced. The expressed porin proteins were then manipulated to regenerate their native trimer structure and purified by standard protein chemistry. Sufficient purified recombinant porin protein was obtained for further antigenic as well as biophysical characterization. This sets the stage for the biophysical characterization of these neisserial porin proteins in more detail.
Base Sequence↗
Processing of bone marrow for transplantation: development of a mononuclear cell enrichment protocol.
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Blood Component Removal↗
GD3/proteosome vaccines induce consistent IgM antibodies against the ganglioside GD3.
The gangliosides of melanoma and other tumours of neuroectodermal origin are suitable targets for immune intervention with tumour vaccines. The optimal vaccines in current use contain ganglioside plus bacillus Calmette-Guérin and induce considerable morbidity. We have screened a variety of new adjuvants in the mouse, and describe one antigen-delivery system, proteosomes, which is especially effective. Highly hydrophobic Neisserial outer membrane proteins (OMP) form multimolecular liposome-like vesicular structures termed proteosomes which can readily incorporate amphiphilic molecules such as GD3 ganglioside. The optimal GD3/proteosome vaccine formulation for induction of GD3 antibodies in the mouse is determined. Interestingly, the use of potent immunological adjuvants in addition to proteosomes augments the IgM and IgG antibody titres against OMP in these vaccines but GD3 antibody titres are unaffected. The application of proteosomes to enhance the immune response to GD3 extends the concept of the proteosome immunopotentiating system from lipopeptides to amphipathic carbohydrate epitopes such as cell-surface gangliosides. The demonstrated safety of meningococcal OMP in humans and the data in mice presented here suggest that proteosome vaccines have potential for augmenting the immunogenicity of amphipathic tumour antigens in humans.
Adjuvants, Immunologic↗
Variation in phenotypic expression of the Opa outer membrane protein and lipooligosaccharide of Neisseria meningitidis serogroup C causing periorbital cellulitis and bacteremia.
Expression of the Opa outer membrane protein varies widely among isolates of Neisseria meningitidis; the clinical significance of this variation is unclear. A sialic-acid acceptor is present in the lipooligosaccharide of some strains of Neisseria and has been shown to render Neisseria gonorrhoeae serum-resistant. We report the case of a patient who had an unusual clinical manifestation of infection due to N. meningitidis serogroup C, periorbital cellulitis with concomitant bacteremia. Clinical isolates from the blood and aspirate of the periorbital cellulitis were identical except for the phenotypic expression of the Opa outer membrane protein in the isolate from the periorbital cellulitis and in the lipooligosaccharide phenotype of the sialic-acid acceptor as defined by monoclonal antibodies. We discuss the laboratory and clinical implications of these findings.
Antigens, Bacterial↗
The major outer membrane protein of Haemophilus ducreyi is a member of the OmpA family of proteins.
Haemophilus ducreyi contains a major outer membrane protein (MOMP) whose apparent molecular weight is 39,000 to 42,000 for all strains tested. Two monoclonal antibodies (MAbs), designated 9D12 and 2C7, bound to the MOMP for all strains of H. ducreyi tested. As reported previously, MAb 9D12 was H. ducreyi specific (E. J. Hansen and T. A. Loftus, Infect. Immun. 44:196-198, 1984). MAb 2C7 bound to all members of the family Pasteurellaceae tested, suggesting that the MAbs bound to distinct epitopes on the MOMP. The MOMP was purified by extraction of whole cells with Zwittergent and ion-exchange chromatography. A peak eluted from a cation-exchange column contained three bands. All three species bound both MAbs, and the fraction yielded a single N-terminal amino acid sequence, suggesting that the bands represented different conformations of the MOMP. The MOMP was heat modifiable, contained two cysteine residues, and was cationic at pH 8.0, features not usually associated with classical porin proteins. The N-terminal amino acid sequence and total amino acid content of the MOMP were homologous to the OmpA proteins of members of the family Enterobacteriaceae and the OmpA-like protein of Actinobacillus actinomycetemcomitans. An OmpA-specific polyclonal serum bound to the MOMP, and MAb 2C7 bound to Haemophilus influenzae protein 5, an OmpA-like protein, indicating that the MOMP was antigenically related to OmpA. These data indicated that the most abundant protein in the outer membrane of H. ducreyi was not a classical porin and belonged to the OmpA family of proteins.
Amino Acid Sequence↗
Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus.
Campylobacter fetus strains possess regular paracrystalline surface layers (S-layers) composed of high-molecular-weight proteins and can change the size and crystalline structure of the predominant protein expressed. Polyclonal antisera demonstrate antigenic cross-reactivity among these proteins but suggest differences in epitopes. Monoclonal antibodies to the 97-kDa S-layer protein of Campylobacter fetus subsp. fetus strain 82-40LP showed three different reactivities. Monoclonal antibody 1D1 recognized 97-kDa S-layer proteins from all C. fetus strains studied; reactivity of monoclonal antibody 6E4 was similar except for epitopes in S-layer proteins from reptile strains and strains with type B lipopolysaccharide. Monoclonal antibody 2E11 only recognized epitopes on S-layer proteins from strains with type A lipopolysaccharide regardless of size. In vitro shift from a 97-kDa S-layer protein to a 127-kDa S-layer protein resulted in different reactivity, indicating that size change was accompanied by antigenic variation. To examine in vivo variation, heifers were genetically challenged with Campylobacter fetus subsp. venerealis strains and the S-layer proteins from sequential isolates were characterized. Analysis with monoclonal antibodies showed that antigenic reactivities of the S-layer proteins were varied, indicating that these proteins represent a system for antigenic variation.
Animals↗
Gonococcal porin vaccine evaluation: comparison of Por proteosomes, liposomes, and blebs isolated from rmp deletion mutants.
The gonococcal major outer membrane protein, Por (protein I), is a potential vaccine candidate. A large-scale animal trial compared the immunogenicity of Por inserted in liposomes (mimicking its in vivo structure) with previously used vaccines containing Por. Por was purified from Rmp (protein III)-negative gonococcal mutants and made into five different formulations: proteosomes, proteosomes absorbed to alum, liposomes, gonococcal membrane blebs, and gonococcal membrane blebs absorbed to alum. Por liposomes induced the greatest amount of Por antibodies; proteosomes and both preparations of blebs induced minimal amounts of Por antibodies. Proteosomes absorbed to alum induced slightly lower amounts of Por antibodies than did liposomes, especially when protein IA was used. Whole-organism absorption studies revealed that a significantly greater percentage of liposome-induced Por antibodies recognized exposed portions of the protein than did proteosome- or proteosome/alum-induced antibodies.
Adjuvants, Immunologic↗
Antibodies against IgA1 protease are stimulated both by clinical disease and asymptomatic carriage of serogroup A Neisseria meningitidis.
IgA1 protease was purified from a strain of serogroup A Neisseria meningitidis subgroup IV-1, representative of bacteria that caused an epidemic of meningococcal meningitis in The Gambia in 1982-1983. ELISAs and immunoblot assays were done using this protease as antigen with paired acute- and convalescent-phase sera from patients from that epidemic and from one in Finland caused by other serogroup A meningococci. Paired sera were also tested from healthy Gambians who were persistent nasopharyngeal carriers, persistent noncarriers, or persons who became carriers after the first serum sample was taken. The results correlated well between the two methods: Antibodies were stimulated by disease or acquisition of carriage, and they remained at a constant level upon continued carriage.
Antibodies, Bacterial↗