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W Cieplak

Publications and source records attributed to W Cieplak.

At least 37 records · Page 2Linked to original sources
PubMed1992

Translocation of Campylobacter jejuni across human polarized epithelial cell monolayer cultures.

The ability of Campylobacter jejuni isolates to translocate across an epithelial cell barrier was investigated by using polarized Caco-2 cell monolayers grown on microporous membrane filters. The 4 C. jejuni isolates tested all traversed the Caco-2 cell monolayers and displayed similar translocation kinetics. The number of bacteria crossing the polarized cell monolayers continued to increase with time until 4 h after inoculation, at which time a maximum rate of translocation was observed. Transmission electron microscopy revealed that C. jejuni translocated across polarized Caco-2 cell monolayers by passing both through and between cells. Chloramphenicol, an inhibitor of bacterial protein synthesis, reduced the translocation of C. jejuni. Bacterial attachment, internalization, and translocation were inhibited at low temperature. These data indicate that adherence, penetration, and translocation of C. jejuni require active bacterial and target cell processes and further suggest a role for cellular translocation in the pathogenesis of C. jejuni-mediated enteritis.

Campylobacter jejuni↗
PubMed1992

Factors that influence the interaction of Campylobacter jejuni with cultured mammalian cells.

Although Campylobacter jejuni is now recognised as a common enteric pathogen, the mechanisms by which this organism produces enteritis remain ill-defined. It has been proposed that its abilities to adhere to and enter epithelial cells represent properties essential to virulence. However, the characteristics of these interactions and factors that may influence the association of C. jejuni with epithelial cells are incompletely described. We have determined that the ability of C. jejuni to bind to epithelial cell lines in vitro is significantly affected by the growth temperature and growth stage of the bacteria, but not by growth-medium composition. Binding of C. jejuni to cultured cells is not affected by temperature or phylogenetic origin of the target cell, and exhibits a non-uniform or patchy distribution. In contrast, internalisation is markedly diminished at low temperature, appears to involve active invagination of the target cell membrane via pseudopod formation, and is maximal when cells of human origin are employed.

Animals↗
PubMed1992

Evidence for proteolytic cleavage of the 120-kilodalton outer membrane protein of rickettsiae: identification of an avirulent mutant deficient in processing.

The 120-kDa rickettsial outer membrane protein (rOmpB) is encoded by a gene with the capacity to encode a protein of approximately 168 kDa. The carboxy-terminal end of the molecule is apparently cleaved to yield 120- and 32-kDa products. Both polypeptides are surface exposed and remain associated with the outer membrane of intact rickettsiae. All species of rickettsiae examined display similar cleavage of rOmpB. Comparison of diverse species of rickettsiae demonstrate a conserved N terminus of the 32-kDa fragment, with a predicted procaryotic secretory signal peptide immediately upstream of the proposed cleavage site. Coprecipitation of the 120-kDa rOmpB protein and the 32-kDa peptide by monoclonal antibodies specific for the 120-kDa portion of the molecule suggests that the two fragments remain noncovalently associated on the surface of rickettsiae. Analysis of an avirulent mutant of Rickettsia rickettsii revealed reduced amounts of the 120- and 32-kDa fragments, but with a correspondingly larger rOmpB protein that displayed properties expected of the putative precursor. This avirulent mutant grows intracellularly but fails to cause the lysis of infected cells that is typical of R. rickettsii. DNA sequence analysis of the region of the gene encoding the cleavage site of the avirulent strain revealed no difference from the sequence obtained from virulent R. rickettsii. The 168-kDa putative precursor of the avirulent strain of R. rickettsii was not extracted from the surface by dilute buffers, as is the 120-kDa protein of virulent R. rickettsii or R. prowazekii. These latter results suggest that the 32-kDa C-terminal region of the molecule may serve as a membrane anchor domain.

Amino Acid Sequence↗
PubMed1992

Altered synthetic response of Campylobacter jejuni to cocultivation with human epithelial cells is associated with enhanced internalization.

Campylobacter jejuni has been shown to bind to and enter epithelial cells in culture. The interaction of C. jejuni with INT 407 epithelial cells was examined to determine whether bacterial protein synthesis is required for either binding or internalization. Chloramphenicol, a selective inhibitor of bacterial protein synthesis, significantly reduced the internalization, but not binding, of C. jejuni compared with untreated controls as determined by protection from gentamicin. Electrophoretic analysis of metabolically labeled proteins revealed that C. jejuni cultured with INT 407 cells synthesized 14 proteins that were not detected in organisms cultured in medium alone. The inhibitory effect of chloramphenicol on internalization was reduced by preincubation of C. jejuni with INT 407 cells. The results indicate that C. jejuni, like some other enteric pathogens, engages in a directed response to cocultivation with epithelial cells by synthesizing one or more proteins that facilitate internalization and suggest that this phenomenon is relevant to the pathogenesis of enteritis caused by C. jejuni.

Bacterial Adhesion↗
PubMed1992

Group B streptococcal opacity variants.

Colony opacity variants were detected for type III group B streptococci (GBS). Transparent colonies predominate in the parent GBS, with occasional colonies having opaque portions. Two stable opaque variants (1.1 and 1.5) were compared with three transparent clones (1.2, 1.3, and 1.4). All grew well on blood agar and on GC medium, but variant 1.1 failed to grow on Todd-Hewitt medium. Scanning and transmission electron microscopy demonstrated that colony opacity correlated with bacterial aggregation status, with opaque variants forming longer and more organized chains. Opaque-transparent switches were observed in both directions for most variants, with transparent to opaque noted most frequently, but 1.5 did not switch at all. Switching of the opacity phenotype was observed both in vitro and in neonatal mice. Relationships between colony opacity and several cell surface phenomena were explored. (i) Opaque variant 1.1 had two surface proteins (46 and 75 kDa) that were either unique or greatly overexpressed. (ii) Variant 1.1 was deficient in type III polysaccharide, while 1.5 lacked group B antigen. Diminished capsular polysaccharide of variant 1.1 was reflected in reduced negative electrophoretic mobility and in increased buoyant density. (iii) Transparent variant colonies growing closest to a penicillin disk were opaque, but colonial variants did not differ in their sensitivity to penicillin. These data indicate that GBS can exist in both opaque and transparent forms, with opaque appearance occurring by multiple routes. Opaque variants grow poorly on Todd-Hewitt medium generally used for isolation of GBS, so any possible relationships between opacity variation and pathogenesis of GBS infection are unknown.

Bacterial Capsules↗
PubMed1991

The 120 kilodalton outer membrane protein (rOmp B) of Rickettsia rickettsii is encoded by an unusually long open reading frame: evidence for protein processing from a large precursor.

A Rickettsia rickettsii outer surface membrane protein (rOmp B), of an apparent molecular mass of 120 kilodaltons, is a major surface antigen of the Rickettsiae that displays genus, species, and sub-species specific antigenic determinants. The 5' portion of this gene was found to be unstable in plasmids, but was stably cloned in a lambda vector. The nucleotide sequence of the 5' terminus has been determined, thus completing the DNA sequence of the entire gene. Genetic analysis revealed an unusually large open reading frame with the capacity to encode a product much larger than the mature protein. A 32 kilodalton peptide from purified rickettsiae was isolated and the amino terminus was sequenced, which revealed that the peptide is encoded by the 3' portion of this large open reading frame. This suggests a role for post-translational processing of rOmp B from a large precursor molecule.

Amino Acid Sequence↗
PubMed1991

Effect of site-directed mutagenic alterations on ADP-ribosyltransferase activity of the A subunit of Escherichia coli heat-labile enterotoxin.

Previous studies of the S1 subunit of pertussis toxin, an NAD(+)-dependent ADP-ribosyltransferase, suggested that a small amino-terminal region of amino acid sequence similarity to the active fragments of both cholera toxin and Escherichia coli heat-labile enterotoxin represents a region containing critical active-site residues that might be involved in the binding of the substrate NAD+. Other studies of two other bacterial toxins possessing ADP-ribosyltransferase activity, diphtheria toxin and Pseudomonas exotoxin A, have revealed the presence of essential glutamic acid residues vicinal to the active site. To help determine the relevance of these observations to activities of the enterotoxins, the A-subunit gene of the E. coli heat-labile enterotoxin was subjected to site-specific mutagenesis in the region encoding the amino-terminal region of similarity to the S1 subunit of pertussis toxin delineated by residues 6 through 17 and at two glutamic acid residues, 110 and 112, that are conserved in the active domains of all of the heat-labile enterotoxin variants and in cholera toxin. Mutant proteins in which arginine 7 was either deleted or replaced with lysine exhibited undetectable levels of ADP-ribosyltransferase activity. However, limited trypsinolysis of the arginine 7 mutants yielded fragmentation kinetics that were different from that yielded by the wild-type recombinant subunit or the authentic A subunit. In contrast, mutant proteins in which glutamic acid residues at either position 110 or 112 were replaced with aspartic acid responded like the wild-type subunit upon limited trypsinolysis, while exhibiting severely depressed, but detectable, ADP-ribosyltransferase activity. The latter results may indicate that either glutamic acid 110 or glutamic acid 112 of the A subunit of heat-labile enterotoxin is analogous to those active-site glutamic acids identified in several other ADP-ribosylating toxins.

Adenosine Diphosphate Ribose↗
PubMed1991

The molecular engineering of pertussis toxoid.

The demand for a safer pertussis vaccine has led to the development of acellular vaccine products. We have sought to manufacture a component vaccine based upon the genetic inactivation of pertussis toxin derived by recombinant DNA technology and protein engineering. Rational site-directed mutagenesis of the S1 subunit of pertussis toxin has resulted in an enzymatically-deactivated polypeptide which retains its immunogenic potential. Mutagenic analysis of the other subunits of this toxin has permitted a delineation of the structural determinants involved in its recognition of cellular receptors. The in vitro assembly of holotoxin species possessing selectively engineered subunits may facilitate the production of a molecularly-defined genetic toxoid for pertussis prophylaxis.

Genetic Engineering↗
PubMed1990

Photolabelling of mutant forms of the S1 subunit of pertussis toxin with NAD+.

The S1 subunit of pertussis toxin catalyses the hydrolysis of NAD+ (NAD+ glycohydrolysis) and the NAD(+)-dependent ADP-ribosylation of guanine-nucleotide-binding proteins. Recently, the S1 subunit of pertussis toxin was shown to be photolabelled by using radiolabelled NAD+ and u.v.; the primary labelled residue was Glu-129, thereby implicating this residue in the binding of NAD+. Studies from various laboratories have shown that the N-terminal portion of the S1 subunit, which shows sequence similarity to cholera toxin and Escherichia coli heat-labile toxin, is important to the maintenance of both glycohydrolase and transferase activity. In the present study the photolabelling technique was applied to the analysis of a series of recombinant-derived S1 molecules that possessed deletions or substitutions near the N-terminus of the S1 molecule. The results revealed a positive correlation between the extent of photolabelling with NAD+ and the magnitude of specific NAD+ glycohydrolase activity exhibited by the mutants. Enzyme kinetic analyses of the N-terminal mutants also identified a mutant with substantially reduced activity, a depressed photolabelling efficiency and a markedly increased Km for NAD+. The results support a direct role for the N-terminal region of the S1 subunit in the binding of NAD+, thereby providing a rationale for the effect of mutations in this region on enzymic activity.

Macromolecular Substances↗
PubMed1990

The role of cysteine 41 in the enzymatic activities of the pertussis toxin S1 subunit as investigated by site-directed mutagenesis.

The S1 subunit (Mr 28,000) of pertussis toxin expresses thiol-dependent enzymatic ADP-ribosyltransferase and NAD-glycohydrolase activities. Site-directed mutagenesis experiments were performed on the codon for Cys-41 of this subunit to investigate the role of this residue in both enzymatic activities. Deletion of Cys-41 caused a decrease in both activities below detectable levels, whereas replacement of this residue by serine, glycine, proline, or asparagine only slightly reduced the activities. The enzymatic activities of these mutants were thiol-independent. The deletion of Ser-40, adjacent to Cys-41, again caused reduction of the enzymatic activities to undetectable levels. Steady-state kinetic experiments showed that the kcat of the mutant protein in which Cys-41 was replaced by glycine was nearly identical to the kcat of the parent version. However, the Km for NAD of the mutant was significantly higher relative to that of the wild type version. These results indicate that the side-chain of Cys-41 is not essential for enzymatic activities and that Cys-41 is not involved in the rate of catalysis but is probably located at or close to the NAD-binding site. The introduction of a negative charge at position 41 through the replacement of Cys-41 by either aspartate or glutamate reduced the enzymatic activities to very low but measurable levels, suggesting a charge-charge repulsive interaction between these residues and possibly one or both of the phosphates of NAD. Cys-41 may therefore be located close to the phosphate subsite of the NAD-binding site.

Adenosine Triphosphate↗
PubMed1990

Pertussis toxin analog with reduced enzymatic and biological activities is a protective immunogen.

Bordetella pertussis TOX3201 has a 12-base-pair insertion in the S1 subunit gene of pertussis toxin (PTX), which encodes for a 4-amino-acid insertion between residues 107 and 108 of the mature S1 subunit (Black et al., Science 240:656-659, 1988). This mutant strain has been shown to secrete a holotoxin analog of PTX, designated CRM3201, with reduced ADP-ribosyltransferase activity. In the present study, we evaluated the biochemical, biological, and immunoprotective activities of purified CRM3201. Assay of enzymatic activities showed that CRM3201 had 20 to 30% of the ADP-ribosyltransferase activity and 55 to 60% of the NAD glycohydrolase activity of native PTX. CRM3201, however, had only 2 to 6% of the activity of PTX in clustering CHO cells, promoting leukocytosis, inducing histamine sensitization, and potentiating an anaphylactic response to bovine serum albumin. In contrast, activities associated with the B oligomer (binding to fetuin, hemagglutination of goose erythrocytes, and lymphocyte mitogen activity) were comparable to those of native PTX. Injection of BALB/c mice with CRM3201 mixed with Al(OH)3 elicited high titers of antibody to PTX (as measured by enzyme-linked immunosorbent assay), which neutralized a leukocytosis-promoting dose of PTX in these mice and neutralized PTX in a CHO cell assay. Passive transfer of the anti-CRM3201 antibody protected 20-day-old Swiss-Webster mice against a lethal aerosol challenge with B. pertussis 18323. Active immunization with CRM3201 significantly reduced lung colonization in adult BALB/c mice with a B. pertussis respiratory infection. These results demonstrate (i) that the reduced ADP-ribosyltransferase activity of CRM3201 is associated with reductions in certain biological and toxic activities of PTX (the enzymatic and biological activities are not, however, totally concordant); (ii) that CRM3201 possesses a functional B oligomer; and (iii) that CRM3201 can induce toxin-neutralizing antibodies which protect mice against a respiratory challenge with B. pertussis. Our studies with CRM3201 show that recombinant analogs of PTX have the potential to be developed into safe, protective immunogens for use in new acellular pertussis vaccines.

Anaphylaxis↗
PubMed1989

Effects of mutations on enzyme activity and immunoreactivity of the S1 subunit of pertussis toxin.

By introducing a series of six different substitutions at and around position 9, we investigated the structural requirements of the amino-terminal region of the S1 subunit of pertussis toxin for both enzyme activity and immunoreactivity. All mutant S1 analogs with a substitution at this location exhibited severely decreased ADP-ribosyltransferase activity (range, 400- to 2,500-fold). In contrast, alteration of arginine 58 had considerably less effect. The reactivity of the mutant molecules with monoclonal antibody 1B7 varied with the nature of the substitution. These findings indicate an absolute requirement for the presence of an arginine residue at position 9 for the maintenance of efficient ADP-ribosyltransferase activity and illustrate the specific participation of vicinal residues in the formation of the protective epitope.

Adenosine Diphosphate Ribose↗
PubMed1988

Specific cleavage of diphtheria toxin by human urokinase.

Diphtheria toxin must undergo a specific cleavage reaction and subsequent reduction to express the enzymatic ADP-ribosyltransferase activity that is responsible for its toxicity. In an effort to identify potential cellular enzymes that might be involved in this process we have found that a human urinary plasminogen activator, urokinase, is capable of specifically cleaving diphtheria toxin to yield an enzymatically active A fragment (more homogeneous than that produced by trypsin cleavage) and a B fragment (with an identical amino-terminal sequence to that produced by trypsin cleavage). The results raise the possibility that urokinase or urokinase-like enzymes play a role in diphtheria toxin-mediated intoxication.

Amino Acid Sequence↗
PubMed1988

Pertussis toxin S1 mutant with reduced enzyme activity and a conserved protective epitope.

Pertussis toxin (PTX) is a major virulence factor in whooping cough and can elicit protective antibodies. Amino acid residues 8 to 15 of PTX subunit S1 are important for the adenosine diphosphate-ribosyltransferase activity associated with the pathobiological effects of PTX. Furthermore, this region contains at least a portion of an epitope that elicits both toxin-neutralizing and protective antibody responses in mice. The gene encoding the S1 subunit was subjected to site-specific mutagenesis in this critical region. A mutant containing a single amino acid substitution (Arg9----Lys) had reduced enzymatic activity (approximately 0.02% of control) while retaining the protective epitope. This analog S1 molecule may provide the basis for a genetically detoxified PTX with potential for use as a component of an acellular vaccine against whooping cough.

Base Sequence↗
PubMed1988

Identification of a region in the S1 subunit of pertussis toxin that is required for enzymatic activity and that contributes to the formation of a neutralizing antigenic determinant.

The S1 subunit of pertussis toxin possesses two regions (homology boxes), each spanning 8 residues, that are nearly identical in sequence to similarly located regions in the enzymatically active A fragments of two other ADP-ribosylating toxins: cholera toxin and Escherichia coli heat-labile toxin. This observation suggests a functional role for one or both of these regions in enzymatic activity. We have examined the role of one of these regions, located near the amino terminus of the S1 subunit, by using a high-level recombinant expression system and progressive truncation of the gene sequence encoding the amino terminus of the molecule. A series of six truncated, recombinant proteins were produced at high levels in E. coli and examined for their enzymatic and antigenic properties. The three molecules that lacked most or all of the homology box delimited by amino acid residues 8 and 15 lacked detectable enzymatic activity. All of the three molecules in which the box was retained exhibited detectable activity. Only those recombinant molecules that possessed the homology box reacted with a neutralizing and passively protective monoclonal anti-S1 antibody. These findings identify the region of homology located near the amino terminus of S1 as an apparent enzymatic subsite and a potentially important antigenic determinant.

Adenosine Diphosphate Ribose↗
PubMed1987

Diphtheria toxin receptor. Identification of specific diphtheria toxin-binding proteins on the surface of Vero and BS-C-1 cells.

The biochemical characteristics of specific receptor molecules for diphtheria toxin on the surface of two toxin-sensitive cell lines (Vero and BS-C-1) were examined. Diphtheria toxin was found to bind to a number of different proteins in Nonidet P-40 solubilized extracts of 125I-labeled cells. In contrast, permitting diphtheria toxin to bind first to labeled intact cells, which were subsequently solubilized and subjected to immunoprecipitation with anti-diphtheria toxin, resulted in a far more restricted profile of diphtheria toxin-binding proteins that possessed Mrs in the range of 10,000-20,000. Direct chemical cross-linking of radioiodinated diphtheria toxin to cell surface proteins resulted in the appearance of several predominant bands possessing Mrs of approximately 80,000. The Mr approximately 80,000 complexes were shown to be composed of radiolabeled diphtheria toxin (Mr 60,000) and unlabeled Mr approximately 20,000 cellular proteins. These complexes were judged to be a result of specific binding in that their appearance could be preferentially inhibited by the addition of a 100-fold excess of unlabeled diphtheria toxin. The formation of the Mr approximately 80,000 complexes was sensitive to prior trypsin treatment of the cells and to known inhibitors of diphtheria toxin binding. Furthermore, prior incubation of the cells with diphtheria toxin at 37 degrees C ("down regulation") markedly and specifically reduced the subsequent formation of the Mr approximately 80,000 cross-linked complexes, and these down-regulated cells were less sensitive to diphtheria toxin in cytotoxicity assays. Further incubation of down-regulated cells at 37 degrees C restored their ability to form Mr approximately 80,000 complexes; this regeneration requires protein synthesis and restores the cells' sensitivity to diphtheria toxin-mediated cytotoxicity. These results strongly suggest that a Mr 10,000-20,000 cell surface protein is, or constitutes a portion of, the functional diphtheria toxin receptor.

Animals↗