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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cross-reactivity of exposed surface epitopes of outer membrane proteins from a spectrum of Haemophilus influenzae type b isolates that varied in their evolutionary distance from each other and in their outer membrane protein composition was analyzed by using an immunoblot assay. The results for outer membrane proteins a, n, and b/c were as follows. (i) A total of 13 of 14 strains possessing a protein a with similar mobilities on gels (i.e., the same apparent molecular weight) as protein a of strain Eag absorbed antibodies to protein a of strain Eag. These strains represented a broad spectrum on a scale of evolutionary distance. (ii) In contrast, only one of seven strains possessing a protein a with different mobilities absorbed these antibodies. (iii) Of five isolates close to strain Eag on the evolutionary scale, the four with a protein n with the same mobility as protein n of strain Eag absorbed antibodies to protein n of strain Eag. (iv) In contrast, of five isolates distant from strain Eag on the evolutionary scale, none absorbed antibodies to protein n, including one strain that had a protein n of the same mobility as that of strain Eag. (v) All strains that absorbed antibodies to protein b/c also absorbed antibodies to lipopolysaccharide, and the reverse of this was also true. Evolutionary distance and mobility of protein b/c on gels were not factors. Control experiments indicated that this result was an artifact due to the strong association of lipopolysaccharide with protein b/c on the gel and subsequent blot. The important conclusions from these experiments, especially pertinent for consideration of these proteins in either whole or peptide vaccines, are that proteins with apparently identical molecular weights can possess different surface-exposed epitopes, that proteins with different molecular weights can possess cross-reactive surface-exposed epitopes, and that some surface-exposed epitopes have been conserved even though the bacterium has undergone evolutionary divergence. In addition, experiments were also performed to determine whether H. influenzae type b strains maintained their integrity during the absorption step, i.e., incubation in antiserum. Strain Eag, which was used as a prototype type b strain, released a small proportion of its membrane (0.13%), but this did not result in exposure of epitopes that were usually buried. In contrast, strain S2, an unencapsulated mutant of strain Eag, was quite unstable, releasing three times as much membrane and a large proportion of its periplasmic proteins.
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PMID:Cross-reactivity of surface-exposed epitopes of outer membrane antigens of Haemophilus influenzae type b. 244 84

Protein a (46,000 molecular weight [46K]) was purified from outer membranes of Haemophilus influenzae type b by a relatively simple procedure. Spontaneously shed outer membranes from a 24-h, 12-liter culture of an unencapsulated variant of strain Eag were combined with outer membranes released from the cells by Tris buffer and extracted with the nonionic detergent octylpolyoxyethylene. The extract was then subjected to open column chromatography on Sephacryl S-200 and Trisacryl-carboxymethyl to yield 7.5 mg of protein a from 180 mg of outer membrane protein. Approximately 99% of the protein in this preparation was protein a; in addition, the preparation contained 1.25% (wt/wt) lipopolysaccharide and had a residual detergent/protein ratio of 1.6:1 (wt/wt). Antibodies to the preparation were induced in rabbits by using alum as an adjuvant. As determined by immunoblotting, the great preponderance of antibodies induced were specific for protein a. However, very low levels of antibodies to several other outer membrane components, which were not apparent on gels of the pure preparation of protein a, were also induced. Preimmune and postimmune sera, after depletion of antibodies to capsular polysaccharide and lipopolysaccharide, were tested for biological activity against H. influenzae type b. Compared with preimmune serum, postimmune serum was bactericidal in vitro against strain Eag (the only strain tested) and offered significant protection (P less than 0.01) to infant rats against infection by all four strains tested, two of which had a protein a that was larger (47K) than the 46K protein a in the preparation. These results indicate that protein a should be considered as a vaccine to prevent H. influenzae type b disease.
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PMID:Protection of infant rats from Haemophilus influenzae type b infection by antiserum to purified outer membrane protein a. 349 97

OmpT is a protease present in the outer membrane of Escherichia coli. The enzyme was overexpressed without its signal sequence in E. coli using a T7 system, resulting in the accumulation of OmpT as inclusion bodies. After solubilization of the inclusion bodies in urea, the protein could be folded in vitro by dilution in the presence of detergent n-dodecyl-N, N-dimethyl-1-ammonio-3-propanesulphonate. The addition of lipopolysaccharide to the protein was essential to obtain active enzyme. The correctly folded protein was purified to homogeneity by ion exchange chromatography with a 57% overall yield. Autoproteolysis between Lys217-Arg218 was a major problem during purification, but degradation could be abolished by introducing the mutations G216K and K217G. A novel fluorimetric assay using the internally quenched substrate Abz-Ala-Arg-Arg-Ala-Tyr(NO2)-NH2 (where Abz is o-aminobenzoyl and Tyr(NO2) is 3-nitrotyrosine) enabled the determination of the kinetic parameters. The wild-type enzyme has an affinity Km of 0.4 microM for the substrate and a turnover number kcat of 40 s-1. The Km and kcat for the double variant were 1.1 microM and 1.6 s-1, respectively. The pH profiles of the wild type and variant were identical, showing optimal activity at pH 6.5 and pKa values of 5.6 and 7.5, respectively. Circular dichroism spectra of both enzymes indicated a high content of beta-strand conformation, and on that basis a beta-barrel topology model is proposed.
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PMID:In vitro folding, purification and characterization of Escherichia coli outer membrane protease ompT. 1065 27

OmpT from Escherichia coli belongs to a family of highly homologous outer membrane proteases, known as omptins, which are implicated in the virulence of several pathogenic Gram-negative bacteria. Here we present the crystal structure of OmpT, which shows a 10-stranded antiparallel beta-barrel that protrudes far from the lipid bilayer into the extracellular space. We identified a putative binding site for lipopolysaccharide, a molecule that is essential for OmpT activity. The proteolytic site is located in a groove at the extracellular top of the vase-shaped beta-barrel. Based on the constellation of active site residues, we propose a novel proteolytic mechanism, involving a His-Asp dyad and an Asp-Asp couple that activate a putative nucleophilic water molecule. The active site is fully conserved within the omptin family. Therefore, the structure described here provides a sound basis for the design of drugs against omptin-mediated bacterial pathogenesis. Coordinates are in the Protein Data Bank (accession No. 1I78)
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PMID:Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site. 1156 68

OmpT is an integral outer membrane protease of Escherichia coli. Overexpression of OmpT in E. coli and subsequent in vitro folding of the produced inclusion bodies yielded protein with a native-like structure. However, enzymatically active protease was only obtained after addition of the outer membrane lipid lipopolysaccharide (LPS). OmpT is the first example of an enzyme that requires LPS for activity. In this study, we investigated the nature of this activation. Circular dichroism analysis showed that binding of LPS did not lead to large structural changes. Titration of OmpT with LPS and determining the resulting OmpT activity with a fluorimetric assay yielded a dissociation constant of 10-4 m for E. coli K-12 LPS. Determining the dissociation constants for different LPS chemotypes revealed that a fully acylated lipid A part is minimally required for activation of OmpT. The heptose-bound phosphates in the inner core region were also important for activation. The affinity for LPS was not dependent on the concentration of substrate, neither was affinity for the substrate influenced by the concentration of LPS. This indicated that LPS most likely does not act at the level of substrate binding. We hypothesize that LPS induces a subtle conformational change in the protein that is required for obtaining a native active site geometry.
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PMID:Lipopolysaccharide regions involved in the activation of Escherichia coli outer membrane protease OmpT. 1189 45

The O-antigen of lipopolysaccharide (LPS) is a virulence factor in enterobacterial infections, and the advantage of its genetic loss in the lethal pathogen Yersinia pestis has remained unresolved. Y. pestis and Salmonella enterica express beta-barrel surface proteases of the omptin family that activate human plasminogen. Plasminogen activation is central in pathogenesis of plague but has not, however, been found to be important in diarrhoeal disease. We observed that the presence of O-antigen repeats on wild-type or recombinant S. enterica, Yersinia pseudotuberculosis or Escherichia coli prevents plasminogen activation by PgtE of S. enterica and Pla of Y. pestis; the O-antigen did not affect incorporation of the omptins into the bacterial outer membrane. Purified His6-Pla was successfully reconstituted with rough LPS but remained inactive after reconstitution with smooth LPS. Expression of smooth LPS prevented Pla-mediated adhesion of recombinant E. coli to basement membrane as well as invasion into human endothelial cells. Similarly, the presence of an O-antigen prevented PgtE-mediated bacterial adhesion to basement membrane. Substitution of Arg-138 and Arg-171 of the motif for protein binding to lipid A 4'-phosphate abolished proteolytic activity but not membrane translocation of PgtE, indicating dependence of omptin activity on a specific interaction with lipid A. The results suggest that Pla and PgtE require LPS for activity and that the O-antigen sterically prevents recognition of large-molecular-weight substrates. Loss of O-antigen facilitates Pla functions and invasiveness of Y. pestis; on the other hand, smooth LPS renders plasminogen activator cryptic in S. enterica.
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PMID:Lack of O-antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica. 1465 23

The omptins are a family of enterobacterial surface proteases/adhesins that share high sequence identity and a conserved beta-barrel fold in the outer membrane. The omptins are multifunctional, and the individual omptins exhibit differing virulence-associated functions. The Pla plasminogen activator of Yersinia pestis contributes by several mechanisms to bacterial invasiveness and the systemic, uncontrolled proteolysis in plague. Pla proteolytically activates the human proenzyme plasminogen and inactivates the antiprotease alpha2-antiplasmin, and its binding to laminin localizes the uncontrolled plasmin activity onto basement membranes. These properties enhance bacterial migration through tissue barriers. Pla also degrades circulating complement proteins and functions in bacterial invasion into human epithelial cells. PgtE of Salmonella enterica and OmpT of Escherichia coli have been shown to degrade cationic antimicrobial peptides from epithelial cells or macrophages. PgtE and SopA of Shigella flexneri appear important in the intracellular phases of salmonellosis and shigellosis, whereas functions of OmpT have mainly been associated with protein degradation in E. coli cells. The differing virulence roles and functions have been attributed to minor sequence variations at the surface-exposed regions important for substrate recognition, to the dependence of omptin functions on lipopolysaccharide, and to the different regulation of omptin expression.
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PMID:The omptin family of enterobacterial surface proteases/adhesins: from housekeeping in Escherichia coli to systemic spread of Yersinia pestis. 1529 49

Yersinia pestis is a species that emerged recently from Yersinia pseudotuberculosis and gained an exceptional pathogenicity potential. Among the major genetic differences between the plague bacillus and its ancestor is the acquisition of the pPla plasmid, which has been associated with the increased virulence of Y. pestis. In a previous study, introduction of pPla into Y. pseudotuberculosis did not lead to any modification of the virulence of the host bacterium. However, it was subsequently demonstrated that the presence of smooth lipopolysaccharide (LPS) inhibits the activity of Pla. In this study, pPla was introduced into a Y. pseudotuberculosis strain expressing smooth LPS, and into a variant in which a mutation that abrogates the formation of O-antigen (O-Ag) repeats (as in natural isolates of Y. pestis) was generated. It was found that in both strains, Pla was synthesized, exported to the bacterial membrane and processed as in Y. pestis. However, the ability of Pla to activate plasminogen was weak and observed only at 37 degrees C in the smooth strain, while this activity was similar to that of Y. pestis and expressed at both 28 and 37 degrees C in the O-Ag mutant strain. Similarly, Pla-mediated inactivation of the antiprotease alpha2-antiplasmin was not detected in the smooth Y. pseudotuberculosis strain grown at 28 degrees C, but was expressed at both temperatures in the O-Ag mutant strain. Despite the more efficient activity of Pla, the Y. pseudotuberculosis O-Ag mutant strain exhibited a lower pathogenicity upon subcutaneous infection of mice. The results thus indicate that, although abrogation of O side chain synthesis in a Y. pseudotuberculosis strain harbouring pPla potentiates the two proteolytic activities of Pla, this is not sufficient to confer to Y. pseudotuberculosis a higher pathogenicity potential. These results also suggest that acquisition of pPla may not have been sufficient to confer an immediate higher pathogenic potential to the ancestor Y. pestis strain.
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PMID:Evaluation of O-antigen inactivation on Pla activity and virulence of Yersinia pseudotuberculosis harbouring the pPla plasmid. 1627 97

The beta-barrel outer membrane protease Pla from Yersinia pestis is an important virulence factor in plague and enables initiation of the bubonic plague. Pla is a multifunctional protease whose expression also enhances bacterial adherence to extracellular matrix. It has remained uncertain whether the increase in cellular adhesiveness results from modification of the bacterial surface by Pla, or whether the Pla molecule is an adhesin. Pla was purified as a His6-fusion protein from Escherichia coli and reconstituted with lipopolysaccharide to an enzymatically active form. Purified His6-Pla was coated onto fluorescent micro-particles (FMPs) that expressed plasminogen activity. Pla-coated FMPs also bound to laminin and to reconstituted basement membrane (Matrigel) immobilized on permanox slides, whereas only poor activity was seen with lipopolysaccharide-coated FMPs or bovine serum albumin-coated FMPs. The results show that the Pla molecule has intrinsic adhesive properties and that purified transmembrane proteins coated onto FMPs can be used for functional assays.
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PMID:Adhesive properties of the purified plasminogen activator Pla of Yersinia pestis. 1692 70

Escherichia coli OmpP is an F episome-encoded outer membrane protease that exhibits 71% amino acid sequence identity with OmpT. These two enzymes cleave substrate polypeptides primarily between pairs of basic amino acids. We found that, like OmpT, purified OmpP is active only in the presence of lipopolysaccharide. With optimal peptide substrates, OmpP exhibits high catalytic efficiency (k(cat)/K(m) = 3.0 x 10(6) M(-1)s(-1)). Analysis of the extended amino acid specificity of OmpP by substrate phage revealed that both Arg and Lys are strongly preferred at the P1 and P1' sites of the enzyme. In addition, Thr, Arg, or Ala is preferred at P2; Leu, Ala, or Glu is preferred at P4; and Arg is preferred at P3'. Notable differences in OmpP and OmpT specificities include the greater ability of OmpP to accept Lys at the P1 or P1', site as well as the prominence of Ser at P3 in OmpP substrates. Likewise, the OmpP P1 site could better accommodate Ser; as a result, OmpP was able to cleave a peptide substrate between Ser-Arg about 120 times more efficiently than was OmpT. Interestingly, OmpP and OmpT cleave peptides with three consecutive Arg residues at different sites, a difference in specificity that might be important in the inactivation of cationic antimicrobial peptides. Accordingly, we show that the presence of an F' episome results in increased resistance to the antimicrobial peptide protamine both in ompT mutants and in wild-type E. coli cells.
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PMID:Substrate specificity of the Escherichia coli outer membrane protease OmpP. 1708 56


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