Immune response induced by N-lauroylsarcosine extracted outer-membrane proteins of an isolate of Edwardsiella ictaluri in channel catfish

A virulent isolate of Edwardsiella ictaluri (AL-93-75), the causative agent of enteric septicaemia of catfish (ESC), was used to derive a lipopolysaccharide-reduced N-lauroylsarcosine outer-membrane protein (OMP) fraction vaccine. The OMP fraction was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and compared to whole-cell lysate, purified lipopolysaccharide (LPS) and a crude cell-wall fraction. The OMP fraction contained less than 2% (W/V) LPS. SDS-PAGE showed that whole cell lysates contained 27 proteins from 107 to 14.3 kDa, whereas OMP contained nine proteins from 97 to 14.3 kDa, LPS contained two proteins at 45 and 37 kDa bands and a smear of bands below 14.3 kDa, and cell wall fraction contained 21 proteins from 97 to 8 kDa. Channel catfish, Ictalurus punctatus, were vaccinated with 12.5 microg/100 microl OMP and immunogenicity was confirmed by subsequent Western blots. Blots showed that 97, 80, and 19 kDa proteins were immunogenic. Rapid enzyme-linked immunosorbent assays (ELISA) demonstrated that OMP produced a weak, but observable antibody response by 21 days post injection. OMP concentrations of 3.13, 6.25, 12.5, 25, and 50 microg/100 microl total protein were tested for protective immunity. Marginal protection by relative percent survival (RPS) was only seen for fish injected with 12.5 microg/100 microl with RPSs between 55-67.5%. A booster dose of 12.5 microg/100 microl OMP did not significantly enhance protection.     

Asymmetric pore occupancy in crystal structure of OmpF porin from Salmonella typhi

OmpF is a major general diffusion porin of Salmonella typhi, a Gram-negative bacterium, which is an obligatory human pathogen causing typhoid. The structure of S. typhi Ty21a OmpF (PDB Id: 3NSG) determined at 2.8 ? resolution by X-ray crystallography shows a 16-stranded β-barrel with three β-barrel monomers associated to form a trimer. The packing observed in S. typhi Ty21a rfOmpF crystals has not been observed earlier in other porin structures. The variations seen in the loop regions provide a starting point for using the S. typhi OmpF for structure-based multi-valent vaccine design. Along one side of the S. typhi Ty21a OmpF pore there exists a staircase arrangement of basic residues (20R, 60R, 62K, 65R, 77R, 130R and 16K), which also contribute, to the electrostatic potential in the pore. This structure suggests the presence of asymmetric electrostatics in the porin oligomer. Moreover, antibiotic translocation, permeability and reduced uptake in the case of mutants can be understood based on the structure paving the way for designing new antibiotics.     

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.     

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, properties and physiological regulation of a putative outer-membrane porin for methanol in Methylophilus methylotrophus

A major outer-membrane protein was purified and partially characterised from the methylotrophic bacterium Methylophilus methylotrophus. The protein had a subunit M_r of 38 000 and was similar in terms of its biochemical properties to the recently characterised amide-urea porin (FmdC) from the same organism. Expression of the protein, as determined by SDS-PAGE and Western blotting of cells grown in continuous culture under various nutrient limitations, varied in a similar manner to that of methanol dehydrogenase and was maximal under methanol limitation. It was concluded that the protein is probably an outer-membrane porin for methanol.     

Measurements of lipopolysaccharide (endotoxin) in meningococcal protein and polysaccharide preparations for vaccine usage

Lipopolysaccharide (LPS, i.e. endotoxin) present in meningococcal outer-membrane protein and polysaccharide preparations made for vaccine use was quantitated by a silver-stain method following SDS-PAGE. The reactivities of LPS in the preparations were also measured by rabbit pyrogenicity and Limulus amoebocyte lysate (LAL) assay. Although rabbit pyrogenicity and LAL assay are more sensitive than the silver stain method, the latter provided an actual amount of LPS present in the protein or in the polysaccharide. For a meningococcal protein preparation, rabbit pyrogenicity showed about one-tenth, and even less by LAL assay, of the actual amount of LPS. This is because protein-bound LPS in meningococcal protein preparations is about 10-fold less active in causing fever in rabbits, and 20- to 40-fold less active in the gelation of LAL than the same amount of a purified free LPS which is generally used as a reference in quantitating LPS in these two assays. As for the small amount of LPS present in a meningococcal polysaccharide preparation, similar LPS content was obtained when measured by the three methods suggesting that the LPS is not bound to the polysaccharide in contrast to that in the proteins mentioned above. The purified meningococcal LPS was pyrogenic in rabbits at 1 ng/kg.     

A major outer-membrane protein functions as a porin in Haemophilus influenzae

Porins are pore-forming outer-membrane proteins which serve as a non-specific pathway for the entry of hydrophilic molecules into Gram-negative bacteria. We studied four strains of Haemophilus influenzae that had decreased permeability to chloramphenicol associated with diminished quantities of a 40 kDa major outer-membrane protein. Isogenic pairs of organisms containing and lacking this protein were compared. The latter strains grew more slowly and were less permeable to sucrose and raffinose. They were also more resistant to multiple hydrophilic antibiotics than an isogenic strain containing the 40 kDa protein and were less permeable to penicillin G and chloramphenicol. We conclude that the 40 kDa outer-membrane protein functions as a porin in H. influenzae.     

Lipopolysaccharide tightly bound to porin monomers and trimers from Escherichia coli K-12.

Lipopolysaccharide (LPS) bound to isolated porin was detected on polyacrylamide gels by using a carbohydrate-specific silver stain and on Western blots by using anti-lipid A monoclonal antibodies. Porin was isolated from Escherichia coli JF733 (Ra chemotype) and D21f2 (Re chemotype). Isolated porin was separated from loosely associated LPS by polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). Unheated porin traveled on gels as aggregates, presumably trimers, with an apparent molecular weight of 78,000 to 83,000. After heating to 100 degrees C for 2 min in SDS, the porin traveled as a monomer with a molecular weight of 36,000. The unheated, high-molecular-weight trimer band reacted in the gel with the carbohydrate-specific silver stain, while the heated monomer band showed no staining. In contrast, lipid A-specific monoclonal antibodies showed reactivity on Western blots to the 36,000-molecular-weight band but not to the trimer. Finally, both monomer and trimer bands were isolated from gels and rerun by SDS-PAGE. LPS was released from the trimer preparation when the sample was heated, but the monomer band that was formed by heating the trimer isolate still reacted with anti-lipid A antibodies. Quantitative Limulus amebocyte lysate analysis revealed an approximately equal molar ratio of LPS to protein in the electroeluted porin monomer. Thus, some but not all of the LPS could be released from trimer complexes by boiling in SDS. The isolated monomer did not release more LPS on boiling in SDS a second time but still had LPS tightly bound, as detected by lipid A-specific monoclonal antibodies.     

Purification of Aeromonas hydrophila major outer-membrane proteins: N-terminal sequence analysis and channel-forming properties

Four outer-membrane proteins of Aeromonas hydrophila were purified and their N-terminal sequences and channel-forming properties were determined. Three could be matched with proteins from other species. One was a maltoporin, as its level increased when cells were grown in maltose-containing media, and the channel it formed was blocked by maltose. Another was like OmpF and OmpC of Escherichia coli, except that its channel fluctuated much more rapidly. The third protein, which was produced in low-phosphate medium, exhibited several properties of the general anion porin PhoE. The fourth showed no similarity to any known proteins. It had a unique N-terminus and it formed small sharply-defined cation-selective channels. Two other proteins which corresponded to OmpW of Vibrio cholerae and E. coli OmpA were partly characterized.     

HEp-2 adhesion and the expression of a 94 kDa outer-membrane protein by strains of Escherichia coli belonging to enteropathogenic serogroups

Sixty strains of Escherichia coli belonging to enteropathogenic serogroups (EPEC) were examined for the ability to adhere to HEp-2 cells, the possession of the genes encoding EPEC adherence factor (EAF) and the ability to express an outer-membrane protein (OMP) of 94 kDa thought to be involved in bacterial adhesion to eukaryotic cells. An absolute correlation was found between HEp-2 adhesion and the possession of the genes encoding EAF. An OMP of 94 kDa was observed in the SDS-PAGE profile of most adhesive strains. In some strains this protein was prone to proteolytic degradation. An antiserum raised to a HEp-2 adhesive strain of EPEC did not react with the 94 kDa OMP of all EPEC which were EAF-positive and HEp-2 adhesive, indicating some interstrain antigenic variation of this protein. Although this 94 kDa protein was surface-exposed, specific antibodies binding to the 94 kDa protein in situ in the outer membrane did not interfere with adhesion of EPEC to HEp-2 cells. Therefore, these studies question the value of this protein as a potential vaccine component.     

Porins and lipopolysaccharide of Escherichia coli ATCC 25922 and isogenic rough mutants

Abstract The lipopolysaccharide and porin profile of Escherichia coli ATCC 25922, a smooth strain commonly used in antibiotic susceptibility testing, and five isogenic rough mutants was examined. The lipopolysaccharide of the parent strain had the characteristic ladder pattern on polyacrylamide gels, while that of the mutants appeared similar to chemotypes Ra and Rc of Salmonella typhimurium with some changes in chemical composition. Of the porins, OmpC appeared markedly reduced in the parent strain while OmpF appeared markedly reduced in the mutants. In addition, a new outer-membrane protein of size intermediate to that of OmpC and OmpF was detected in all mutants. Neither parent nor mutants were susceptible to the LPS core-specific P1 phage or the porin-specific PA2 and K20 phages.     

Immunization with outer-membrane protein(s) of Bacteroides fragilis: an experimental study in mice

A crude outer-membrane protein (OMP) preparation from a strain of Bacteroides fragilis, grown in supplemented brain-heart infusion broth, was tested for its protective effect against subcutaneous infection in mice. Immunization with six doses, each of 100, 150 or 200 g OMP preparation, gave some protection: abscesses completely disappeared 15 to 22 days after immunization. In non-immunized animals and animals immunized with doses of 10, 20, 40 or 80 g each, well demarcated abscesses were seen beyond day 22 post-immunization. Although crude OMP elicited good antibody response, with maximum titres on day 4 post-immunization, high titres could not be associated with healing of the abscesses.     

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.     

Salmonella typhi Ty2 OmpC Porin Induces Bactericidal Activity on U937 Monocytes

The immunogenic effect of Salmonella typhi OmpC porin during typhoid fever in humans was evaluated in vitro. Peripheral blood mononuclear cells from 17 patients were challenged with outer membrane preparations from Escherichia coli UH302 and UH302/pSTP2K2 strains, both lacking E. coli OmpF and OmpC porins, although UH302/pSTP2K2 expressed a plasmid-encoded S. typhi Ty2 OmpC. The mononuclear cell supernatants, immunized in vitro with OmpC antigen, derived from 10 out of 17 patients activated U937 bactericidal capacity. In contrast, the supernatants from the immunization with outer membrane preparation lacking S. typhi Ty2 OmpC induced a significantly reduced bactericidal capacity of U937 cells. This procedure should prove useful for in vitro characterization of cellular immunogens from exclusive human pathogens.     

A comparative analysis of the spatial structure of nonspecific porins from <Emphasis Type="Italic">Yersinia ruckeri</Emphasis> using optical spectroscopy and molecular modeling

The spatial organization of outer-membrane porins is studied by optical spectroscopy and molecular modeling. It was found that the OmpF and OmpC porins from Yеrsiniа ruckeri are β-structured membrane proteins typical of the pore-forming proteins of other Gram-negative bacteria. The spatial structures of monomers and trimers of the OmpC and OmpF porins from Y. ruckeri are simulated using methods of structural bioinformatics. It was found that the structural stability of the more thermostable OmpF trimer is sustained by a greater number of hydrogen bonds and hydrophobic interactions. The main differences of the spatial structures of the test porins are observed in the structure of their outer loops. There are three tryptophan residues in the molecules of the OmpC and OmpF porins of Y. ruckeri. It is demonstrated by moleculardynamics methods that after thermal denaturation the solvent accessibility of the Trp212 residue in OmpF porin increased by two times, while the solvent accessibility of a Trp184 residue in OmpC porin was not increased. It is hypothesized that the red-shifted tryptophan fluorescence spectrum of OmpF porin during thermal denaturation is due to the behavior of the Trp212 residue.     

IgA-driven T cell-mediated anti-bacterial immunity in man after live oral Ty 21a vaccine

Cellular immunity against Salmonella typhi was observed by using a direct anti-bacterial in vitro assay in volunteers orally vaccinated with the live S. typhi mutant strain Ty 21a. With this experimental approach, it was demonstrated that Ty 21a vaccine also induces cellular immunity against S. paratyphi A and B. Interestingly, the mechanism involved in cellular immunity against bacteria seems to be of an antibody-dependent cellular cytotoxicity (ADCC) type, with IgA acting as the humoral arm and CD4+ T lymphocytes as the cellular one. In accordance with the increase in IgA-driven ADCC against S. typhi, a major rise in IgA against O and H antigens was observed in the serum of vaccinees in parallel to an increase in IgG of identical specificity. Furthermore, a Ty 21 vaccine induced cellular activity against flagellar antigens. These results indicate that IgA-ADCC by T lymphocytes against bacteria can originate from local stimulation of the gut mucosal immune system. This cellular defense mechanism might be at the origin of the protection induced by Ty 21a vaccine.     

Molecular modelling of epitope presentation using membrane protein OmpC

Three-dimensional models of the chimeric S. typhi OmpC protein carrying an epitope from rotavirus VP4 capsid protein on either of two exposed loops (fourth and sixth) were constructed separately, using computer-aided homology modelling. The theoretical model of S. typhi OmpC was used as a template. The monomers were initially energy minimized. The trimers were generated for both the chimeric S. typhi OmpC proteins and the structures were optimized after several cycles of minimization. The surface accessibility calculations for the resulting models show that epitope recognition should be more effective in the fourth loop than in the sixth loop, in accordance with the experimental results on the immunogenic nature of the rotaviral epitope inserted into the two putative loops of S. typhi OmpC.     

Native and chemically modified porin channels from Salmonella typhi Ty2 in planar lipid bilayers

Native porins, from Salmonella typhi Ty2 outer membrane, and porins alkylated with pyridoxal phosphate (Plp) were studied in planar lipid bilayers. The conductance of bilayers exposed to native or chemically modified porins increases in discrete jumps. Conductance histograms for native porins displayed two major peaks at 1.7 and 6.7 nS (in 0.5 M KCl). On the other hand, Plp-treated porins exhibited a single major peak at 1 nS. The relation between bilayer conductance and native porin concentration was linear. However, this relation became logarithmic in the presence of modified porins. The results support the notion that alkaline reduction of S. typhi Ty2 porins with Plp dissociates porin channel trimers in a reversible fashion.     

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.