Conformational epitope mapping of OmpC, a major cell surface antigen from Salmonella typhi

The outer membrane protein OmpC, a trimer made of 16 stranded beta-barrel monomers, is a major cell surface antigen from the human pathogen Salmonella typhi. The relative stability of the epitopes recognising a Salmonella specific MAb (referred as MPN5) and an Enterobacteria specific MAb (referred as P7D8) and the role of the trimeric organisation has been probed using gel electrophoresis and monoclonal antibodies. The assembly of the trimer and the stability of the beta-barrel are found to be important for epitope presentation. The Salmonella specific conformational epitope is found to be more stable than the Enterobacteria specific one. The important residues of the Salmonella specific (Asp 25 of loop 1, Asp 340 of loop 8, Lys 334 of loop 8, and Tyr 210 of loop 5) and the Enterobacteria specific (Asp 25 of loop 1, Tyr 210 of loop 5, and Lys 152 of loop 4) conformational epitope have been identified using monoclonal antibodies, chemical modification, and solid phase binding methods.     

Homology model of surface antigen OmpC from Salmonella typhi and its functional implications

Homology based 3D structural model of the immunodominant major surface antigen OmpC from Salmonella typhi, an obligatory human pathogen, was built to understand the possible unique conformational features of its antigenic loops with respect to other immunologically cross reacting porins. The homology model was built based on the known crystal structures of the E. coli porins OmpF and PhoE. Structure based sequence alignment helped to define the structurally conserved regions (SCRs). The SCR regions of OmpC were modelled using the coordinates of corresponding regions from reference proteins. Surface exposed variable regions were modelled based on the sequence similarity and loop search in PDB. Structural refinement based on symmetry restrained energy minimization resulted in an agreeable model for the trimer of OmpC. The resulting model was compared with other porin structures, having b-barrel fold with 16 transmembrane beta-strands, and found that the variable regions are unique in terms of sequence and structure. A ranking of the loops taking into account the antigenic index, the sequence variability, the surface accessibility in the context of the trimer, and the structural variability suggests that loop 4 (151-172), loop 5 (194-218) and loop 6 (237-264) are the best ranked B-cell epitopes. The model provides possible explanations for the functional and unique immunological properties associated with the surface exposed regions and outlines the implications for structure based experimental design.     

Overexpression, refolding, and purification of the major immunodominant outer membrane porin OmpC from Salmonella typhi: characterization of refolded OmpC

The major immunodominant integral outer membrane protein C (OmpC) from Salmonella typhi Ty21a was overexpressed, without the signal peptide, in Escherichia coli. The protein aggregates as inclusion bodies (IBs) in the cytoplasm. OmpC from IBs was solubilized with 4 M urea and refolded. This involved rapid dilution of unfolded OmpC into a refolding buffer containing polyoxyethylene-9-lauryl ether (C(12)E(9)) and glycerol. The refolded OmpC (rfOmpC) was shown to be structurally similar to the native OmpC by SDS-PAGE, Western blotting, tryptic digestion, ultrafiltration, circular dichroism, and fluorescence spectroscopic techniques. Crystals of rfOmpC were obtained in preliminary crystallization trials. The rfOmpC also sets a stage for rational design by recombinant DNA technology for vaccine design and high resolution structure determination.     

Crystallization of the immunodominant outer membrane protein OmpC; the first protein crystals from Salmonella typhi, a human pathogen.

OmpC, a surface antigen of Salmonella typhi was crystallized after several attempts, using PEG 3350. Well shaped hexagonal crystals were grown from vapor diffusion method using octyl glucoside and C12E9 as detergents. Crystals are sensitive to X-ray and diffract weakly up to 7 A. Porin isoforms, due to the bound lipopolysaccharides, could be the cause for poor diffraction. Crystal quality depends largely on the purification method, and in case of LPS contamination, the genetic background of the bacteria. Crystallization and initial data collection suggest optimum conditions and the method of choice for OmpC crystallization.     

Purification and partial characterization of a major outer-membrane protein of Fusobacterium nucleatum

The major outer-membrane proteins of 40-41 kDa were identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in Fusobacterium nucleatum strains ATCC 10953, ATCC 25586, F3, F6 and Fev1. The proteins were purified by preparative gel electrophoresis. Their behaviour in gel filtration and gel electrophoresis, their sensitivity to proteolytic enzymes, and their amino acid composition were investigated. The purified proteins were partly sequenced from the N-terminal end. A 36.5 kDa portion was protected against extrinsic proteolytic (trypsin, chymotrypsin or pronase) digestion of whole cells. This polypeptide was isolated and partially sequenced from the N-terminal end. From these data and data from extrinsic iodination it was concluded that the N-terminal end of the protein is probably exposed on the surface of the cell. A database search revealed amino acid sequence similarity in an Ala-Pro-rich region of outer-membrane protein A (OmpA) in other Gram-negative bacteria.     

Functional assay of Salmonella typhi OmpC using reconstituted large unilamellar vesicles: a general method for characterization of outer membrane proteins

The immunodominant trimeric beta-barrel outer membrane protein OmpC from Salmonella typhi, the causative agent of typhoid, has been functionally characterized here. The activity in the vesicle environment was studied in vitro using OmpC reconstituted into proteoliposomes. Passage of polysaccharides and polyethyleneglycols through OmpC has been examined to determine the permeability properties. The relative rate of neutral solute flux yields a radius of 1.1 nm for the S. typhi OmpC pore. This is almost double the pore size of Escherichia coli. This provides an example of large pore size present in the porins that form trimers as in the general bacterial porin family. The method used in this study provides a good membrane model for functional studies of porins.     

Purification and structural studies of a major scrapie prion protein

Scrapie is a degenerative, neurological disorder caused by a slow infectious agent or prion. Extensively purified preparations of prions were denatured by boiling in sodium dodecyl sulfate and the major protein component (PrP 27-30) was isolated by preparative HPLC size exclusion chromatography after proteinase K digestion. The purified PrP 27-30 molecules were not infectious. Ultraviolet absorption spectra of purified PrP 27-30 demonstrated the absence of covalently linked polynucleotides. Amino acid composition studies showed that PrP 27-30 contains at least 17 naturally occurring amino acids. A single N-terminal amino acid sequence for PrP 27-30 was obtained; the sequence is N-Gly-Gln-Gly-Gly-Gly-Thr-His-Asn-Gln-Trp-Asn-Lys-Pro-Ser-Lys and it does not share homology with any known proteins. The same amino acid sequence was found when an extensively purified preparation of prions aggregated into rods and containing approximately 10(9.5) ID50 U/ml was sequenced directly. Knowledge of the amino acid sequence should permit determination of the genetic origin and replication mechanism of prions.     

Purification, characterization, and localization of a major membrane protein antigen from Porphyromonas (bacteroides) gingivalis.

Humans and rats infected with P. gingivalis develop a strong immune response to a 75 kDa major membrane component of P. gingivalis and hence knowledge of the nature of this molecule may aid in understanding the host response to P. gingivalis during infection. Purification of the 75 kDa protein was achieved by repeated precipitation from a crude sonicate of P. gingivalis 2561 at pH 5.0. Homogeneity of the purified 75 kDa protein was confirmed by SDS-PAGE and Western immunoblot analysis using monoclonal and polyclonal antibodies. The purified protein revealed an apparent molecular mass of 300 kDa in native form. Although most of the strains of P. gingivalis tested showed a major membrane protein band in the range of 61 to 78 kDa, on Western immunoblot analysis only the strains which have proteins in the range of 75 to 78 kDa were reactive with anti-75 kDa protein polyclonal antibodies. Affinity purified polyclonal antibodies were used to localized 75 kDa protein on the cell surface of P. gingivalis 2561 by immunogold electron microscopy. Immunolabeling of the 75 kDa protein demonstrated specific localization of the protein along the outer cell membrane, but not on the fimbriae. Furthermore, immunogold labeling of the 75 kDa protein on the thin sections showed that the 75 kDa component was present on not only the outer membrane, but also on the cell membrane, and on membrane bound organelles. Localization of this protein suggests that the 75 kDa component is a membrane-associated protein.     

Cloning, protein expression and display of synthetic multi-epitope mycobacterial antigens on Salmonella typhi Ty21a cell surface

Expressing proteins of interest as fusion to proteins of bacterial envelope is a powerful technique for biotechnological and medical applications. The synthetic gene (VacII) encoding for T-cell epitopes of selected genes of Mycobacterium tuberculosis namely, ESAT6, MTP40, 38 kDa, and MPT64 was fused with N- terminus of Pseudomonas syringae ice nucleation protein (INP) outer membrane protein. The fused genes were cloned into a bacterial expression vector pKK223-3. The recombinant protein was purified by Ni-NAT column. VacII gene was displayed on the cell surface of Salmonella typhi Ty21a using N-terminal region of ice nucleation proteins (INP) as an anchoring motif. Glycine method confirmed that VacII was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INP- VacII fusion protein of the expected size (52 kDa).     

Purification of the 25-kDa Vibrio cholerae major outer-membrane protein and the molecular cloning of its gene: ompV

The 25-kDa peptidoglycan-associated outer-membrane protein and most likely porin of Vibrio cholerae is a major immunogenic species. It has been purified by ion-exchange elution on hydroxyapatite followed by gel filtration on Bio-Gel P150 both in the presence of sodium dodecyl sulfate. This protein, of greater than 90% purity as judged by Western blotting, has been used to raise antibodies in rabbits. The antisera were then used to screen V. cholerae gene banks, constructed in Escherichia coli K12, and this has enabled us to isolate several colonies harbouring the cloned gene. The plasmids in these colonies have been designated pPM451, pPM455 and pPM472. These plasmids have a 5.3 X 10(3)-base BamHI fragment of V. cholerae DNA in common. Restriction endonuclease mapping of these plasmids has been performed and the protein identified both by Western blot analysis and in E. coli K12 minicells. The protein is not efficiently expressed in E. coli K12. It is proposed to use the name ompV to describe the structural gene, present in the cloned DNA, for this V. cholerae outer membrane protein.     

Immunomodulatory role of thyroid hormones: effect on humoral immune response to Salmonella typhi O antigen

Effect of long term administration of thyroid hormones and the removal of thyroid on humoral antibody response to S. typhi O antigen was studied in male albino rats of HM strain. Humoral antibody response to S. typhi O antigen was enhanced in animals pretreated with T3 or T4 at a dose of 10 micrograms daily for 15 days. Administration of thyroid hormones simultaneously along with antigen, resulted in suppression of antibody response. In thyroidectomized animals, the antibody titer to S. typhi O antigen was decreased; this decrease in antibody titer was restored to normal level following hormone supplementation. Thyroidectomy significantly depressed TLC as well. Total leucocyte count and absolute lymphocyte count were increased following hormone treatment. Present findings, thus show the lymphoproliferative response of thyroid hormones and their effect on antibody response suggesting the immunomodulatory role of thyroid hormones.     

Chromogenic detection of antigen in bacteriophage plaques: a microplaque method applicable to large-scale screening

We have developed a simple and rapid (24 h) enzyme-linked immuno-detection method to screen for rare antigen-positive phage among large numbers of antigen-negative ones. Horse-radish peroxidase-antibody conjugate, incorporated into the soft agar layer of a plaque assay system, is precipitated locally by antigen produced during plaque formation, and is detected by standard chromogenic methods. The method has been used to screen plaques of bacteriophage beta tox+ for the presence of diphtheria toxin and related cross-reacting material. When phage were plated on very dense bacterial lawns, they formed minute plaques (microplaques). Because of the high local concentration of antigen generated by lysis of the dense lawn, the microplaques gave more intense chromogenic signals than larger plaques formed on less dense Corynebacterium diphtheriae lawns. Thus, antigen-positive microplaques could be easily recognized even in the presence of very large numbers of antigen-negatives. In a reconstruction experiment, small numbers of antigen-positive phage were detected with high efficiency (greater than 75%) against a background of 3.8 X 10(4) antigen-negatives/cm2 of agar surface (equivalent to 2.4 X 10(6) plaques/9 cm petri plate). This screening method should facilitate isolation of phage mutants affecting production of given antigens and may be of particular value in detecting specific genes cloned into phage vectors.     

Role of the major capsid protein of phage T4 in DNA packaging from structure-function and site-directed mutagenesis studies

Heat cleavage of asp-pro peptide bonds was used to probe the primary structures of the Phage T4 major capsid protein precursor, gp23, its mature capsid form gp23*, and a DNA-dependent ATPase, called capsizyme. This analysis suggests that capsizyme is a gp23** resulting from the N-terminal processing found in gp23* as well as shortening at the C-terminus. Photoaffinity labeling with Azido-ATP and BrU-DNA, followed by heat cleavage, suggests binding sites for these compounds toward the C-terminus of gp23**, suggesting localization of functions within the gp23 primary sequence. Site-directed mutagenesis experiments were targeted therefore to the C-terminal end of g23 as well as to its processing sites. N-terminal processing site modification supports the consensus gp21 proteinase cleavage rule, whereas mutagenesis at the C-terminus suggests that the C-terminal alteration is unlikely to result from a gp21-morphogenesis proteinase cleavage. Amino acid replacements in gp23 at newly introduced amber sites reveal a new g23 mutant phenotype, defective partially DNA-filled heads, in support of the hypothesis that gp23 and its products function directly in the DNA packaging mechanism.     

Purification and partial characterization of a 65-kDa platelet aggregation-associated protein antigen from the surface of Streptococcus sanguis

Cells of Streptococcus sanguis express a collagen-like immunodeterminant (class II antigen) on their cell walls that induces aggregation of platelets in plasma. These platelet aggregation-associated proteins (PAAPs) are recovered in cell-free preparations obtained from cells of S. sanguis after 5 min of sonic or limited trypsin treatment. Pretreatment of platelet-rich plasma with these soluble preparations selectively inhibits platelet aggregation in response to S. sanguis cells. A PAAP antigen was isolated and purified from minimal tryptic digests of S. sanguis cells using (i) immunoaffinity chromatography or (ii) gel filtration and ion-exchange chromatography. A monospecific rabbit antiserum was prepared against PAAP (from procedure ii) and used to verify identity with PAAP fragments in different preparations. Criteria of purity included single precipitins in immunoelectrophoresis and crossed immunoelectrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western immunoblot, and COOH (Lys)- and NH2 (Pro)-terminal analyses. The 65-kDa (p65) antigen isolated by immunoaffinity chromatography had 50-fold greater specific inhibitory activity in S. sanguis-induced PRP aggregation than the original tryptic digest and about 1.4 times that recovered by sequential column chromatography. Amino acids of the p65 PAAP fragment constituted 89.5% of the total dry weight, with glycine, lysine, and glutamic acid predominant. Lesser amounts of proline were also noted. Monosaccharides, including glucose and galactose, comprised 4.0% of the total. A platelet interactive determinant of p65 was localized to a 23-kDa tryptic fragment after further trypsin treatment. Amino acids of this 23-kDa fragment constituted 99.8% of the total dry weight. In their native state on the cell wall of platelet-interactive strains of S. sanguis, platelet aggregation-associated proteins are probably assembled on fibrils as polyvalent agonists.     

Release of Vi antigens from Salmonella typhi: implications for virulence and diagnosis

The release of Vi antigens from three clinical isolates of Salmonella typhi was measured by a Vi-specific monoclonal antibody. Large quantities of Vi antigens were detected in the culture supernates from all three strains using either passive latex agglutination or rocket immunoelectrophoresis. Vi antigens were also detected in broth cultures of S. typhi containing about 10(5) cells/ml using the sandwich enzyme linked immunosorbent assay. The significance of this finding in relationship to the virulence and the diagnosis of S. typhi was discussed.     

Release of Vi antigens from Salmonella typhi: implications for virulence and diagnosis

Abstract The release of Vi antigens from three clinical isolates of Salmonella typhi was measured by a Vi-specific monoclonal antibody. Large quantities of Vi antigens were detected in the culture supernates from all three strains using either passive latex agglutination or rocket immunoelectrophoresis. Vi antigens were also detected in broth cultures of S. typhi containing about 10⁵ cells/ml using the sandwich enzyme linked immunosorbent assay. The significance of this finding in relationship to the virulence and the diagnosis of S. typhi was discussed.     

Gene fusions using the ompA gene coding for a major outer-membrane protein of Escherichia coli K12

It has been shown previously that fragments of the Escherichia coli major outer membrane protein OmpA lacking CO2H-terminal parts can be incorporated into this membrane in vivo [Bremer et al. (1982) Eur. J. Biochem. 122, 223-231]. The possibility that these fragments can be used, via gene fusions, as vehicles to transport other proteins to the outer membrane has been investigated. To test whether fragments of a certain size were optimal for this purpose a set of plasmids was prepared encoding 160, 193, 228, 274, and 280 NH2-terminal amino acids of the 325-residue OmpA protein. The 160-residue fragment was not assembled into the outer membrane whereas the others were all incorporated with equal efficiencies. Thus, if any kind of OmpA-associated stop transfer is required during export the corresponding signal might be present between residues 160 and 193 but not CO2H-terminal to 193. The ompA gene was fused to the gene (tet) specifying tetracycline resistance and the gene for the major antigen (vp1) of foot-and-mouth disease virus. In the former case a 584-residue chimeric protein is encoded consisting NH2-terminally of 228 OmpA residues followed by 356 CO2H-terminal residues of the 396-residue 'tetracycline resistance protein'. In the other case the same part of OmpA is followed by 250 CO2H-terminal residues of the 213-residue Vp1 plus 107 residues partly derived from another viral protein and from the vector. Full expression of both hybrids proved to be lethal. Lipophilic sequences bordered by basic residues, present in the non-OmpA parts of both hybrids were considered as candidates for the lethal effect. A plasmid was constructed which codes for 280 OmpA residues followed by a 31-residue tail containing the sequence: -Phe-Val-Ile-Met-Val-Ile-Ala-Val-Ser-Cys-Lys-. Expression of this hybrid gene was lethal but by changing the reading frame for the tail to encode another, 30-residue sequence the deleterious effect was abolished. It is possible that the sequence incriminated acts as a stop signal for transfer through the plasma membrane thereby jamming export sites for other proteins and causing lethality. If so, OmpA appears to cross the plasma membrane completely during export.     

A structure-function analysis of NOD,a kinesin-like protein from Drosopbila melanogaster

We have analyzed a collection of 12 mutations in the Drosophila melanogaster nod locus, which encodes a kinesin-like protein involved in female meiotic chromosome segregation. The kinesin-like domain is at the N-terminus of the protein, while the C-terminal portion of the protein is unique. Four of the mutations are missense and affect highly conserved domains of the kinesin-like portion of the predicted protein, and thus demonstrate that the sequence conservation is biologically relevant. Surprisingly, two other mutations, which behave genetically as null alleles, are the result of mutations in the last exon of the nod gene. Thus, these two mutations affect the most C-terminal residues in the unique portion of the predicted protein. Based on these mutations, we suggest that this part of the protein may also be essential for wild-type function. The mutations were induced by either gamma-rays or ethyl methanesulfonate (EMS). All of the gamma-ray induced mutations were small or large chromosomal rearrangements, while all of the EMS mutations were G → A transitions. These findings are consistent with the biochemical basis of the mode of action of each mutagen.