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.

Cloning and expression of the hup encoding a histone-like protein of Campylobacter jejuni.

A Campylobacter jejuni gene, designated hup, that appears to encode a homolog of the histone-like DNA-binding protein, HU, has been cloned, sequenced and expressed in Escherichia coli. Immunoblotting and in vitro transcription/translation analyses revealed a 11-kDa protein that was produced by recombinant plasmids containing hup. The gene contains an open reading frame (ORF) sufficient to encode a protein of 98 amino acids (aa) with a calculated molecular mass of 10,267 Da and a predicted isoelectric point of 10.1. The deduced aa sequence of the protein, designated HCj, exhibits considerable sequence identity with members of the HU family of proteins from other eubacterial species. The transcription start point was identified by primer extension analysis and appropriately spaced promoter sequences were found which exhibit considerable similarity to E. coli and Bacillus promoters. Southern hybridization analyses indicate that C. jejuni has a single copy of hup.

Homology between Borrelia burgdorferi OspC and members of the family of Borrelia hermsii variable major proteins.

Synthesis of the Borrelia burgdorferi outer surface protein C (OspC) is quite variable. We have cloned and sequenced the ospC gene from B. burgdorferi isolate CA-11.2A, a clone in which ospC expression varies. The 5' flanking region of the gene contains at least two consensus promoter regions, as well as two large overlapping inverted repeats. Sequence comparison to other OspC proteins indicated that the CA-11.2A OspC is as closely related to OspC from two different genospecies of Lyme disease spirochetes as it is to OspC from the prototype B. burgdorferi strain, B31. Comparisons of the OspC amino acid (aa) sequence with those in aa sequence databases revealed partial identity with the variable major proteins Vmp3 and Vmp24 of B. hermsii, a causative agent of tick-borne relapsing fever. An ospC probe hybridized to B. hermsii restriction fragments and linear plasmids that also were recognized by the vmp3 and vmp24 probes. OspC and these Vmp appear to be related, but their synthesis is regulated differently in the two species of spirochetes. This represents a fascinating example of the evolution of the number, position, regulation and perhaps function of homologous genes in two related pathogens. These parameters may relate to characteristic properties of the pathogens and their separate tick vectors.

Formation of stable complexes between urokinase and a human urinary component.

Human urine was found to contain multiple species of urokinase (UK)-like plasminogen activator (PA) activity when subjected to concentration and/or dialysis and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. Untreated, freshly voided urine contained only Mr = 52,000 and 35,000 UK while dialyzed or undialyzed urine concentrates contained additional PA activity at Mr = 80,000 and 95,000. SDS-PAGE of incubation mixtures of radioiodinated UK (Mr = 52,000 or 35,000) and urine concentrates revealed the presence of radiolabeled complexes with Mrs of 80,000 and 95,000. The bond(s) involved in complex formation was also relatively resistant to heat and reduction. Treatment of radioiodinated UK with the serine proteinase inhibitor, p-nitrophenyl guanidinobenzoate, prior to incubation with dialyzed, concentrated urine prevented formation of the complexes. In addition, the enzymatic activity of the Mr = 80,000 and 95,000 species was unaffected by diisopropyl fluorophosphate. These results indicate that UK forms SDS-stable complexes with a urinary component that has a Mr of approximately 40,000. The results further suggest that these complexes express PA activity when analyzed by SDS-PAGE and zymography.

Identification of subpopulations of human urinary plasminogen activators.

Enzymes functioning as plasminogen activators in commercial urokinase preparations and individual human urine concentrates were subjected to affinity chromatography on columns of lentil lectin-sepharose, ricin-sepharose, wheat germ agglutinin-sepharose, lotus lectin-sepharose and concanavalin A-sepharose. Chromatography of the enzymes from both sources yielded similar results for all lectins except lentil lectin. Urokinase from several commercial sources was approximately 50% adherent to lentil lectin-sepharose while only 5-10% of the urinary plasminogen activators from individuals was adherent to this lectin. SDS-PAGE followed by zymography indicated that the observed differences between commercial and individual samples could be due to the presence in urine concentrates of subpopulations of plasminogen activators which were absent from the commercial samples.

Modulation of secreted plasminogen activator activity of human renal carcinoma cells by dimethylsulfoxide, butyrate and retinoate.

Dimethylsulfoxide, butyrate and retinoic acid, agents which induce differentiation of certain malignant cells, were examined for their effect on the activity of plasminogen activator (PA) of serumless conditioned medium (CM) of two human renal carcinoma cell lines. All three agents produced a decrease in PA activity. More than 90% of the PA was secreted in latent form, and this was not altered by the agents. Active PA components of Mr 52,000 and 93,000 were identified in cell secretions by zymography. In the presence of DMSO or butyrate the Mr 52,000 component was markedly reduced. Reversibility of the effect on PA was demonstrated for both DMSO and retinoic acid with activity of CM returning to control level after removal of the agent. That the effect was temporary agrees with most observations of the effects of these and similar agents on cells from solid tumors.

Nucleotide sequence and analysis of the gene in Borrelia burgdorferi encoding the immunogenic P39 antigen.

The P39 antigen is a specific, highly conserved, and immunogenic protein of Lyme disease spirochetes, Borrelia burgdorferi sensu lato. The nucleotide sequence of the gene encoding this protein was determined and found to be the first of two tandemly arranged open reading frames located on the spirochete's chromosome. These two open reading frames were designated bmpA for the gene encoding P39 and bmpB for the gene encoding the putative protein ORF2 encoded by the second open reading frame. The nucleic acid sequence identity for the two open reading frames was 62% while their deduced amino acid sequences were 52% identical. Comparison to sequence data bases demonstrated that the deduced amino acid sequences of both P39 and ORF2 were homologous to TmpC, a putative outer or cytoplasmic membrane lipoprotein of the syphilis spirochete, Treponema pallidum.

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.

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.

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.

Kinetic and antigenic characterization of altered protein synthesis by Campylobacter jejuni during cultivation with human epithelial cells.

Cultivation of Campylobacter jejuni with INT 407 cell monolayer cultures results in new or enhanced synthesis of a number of proteins compared with bacteria cultured in the absence of the epithelial cells. These proteins were detected within 60 min after the addition of the bacteria to the epithelial cell cultures, and their synthesis was temporally associated with an increase in C. jejuni internalization. A rabbit antiserum raised against bacteria that were cultivated with INT 407 cells recognized nine proteins that were not recognized by an antiserum against C. jejuni cultivated in medium alone. The former antiserum inhibited the internalization, but not the binding, of Campylobacter jejuni in a dose-dependent fashion. The results suggest that one or more of the proteins synthesized by C. jejuni in response to cocultivation with epithelial cells plays a role in facilitating internalization.

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.

Role of trypsin-like cleavage at arginine 192 in the enzymatic and cytotonic activities of Escherichia coli heat-labile enterotoxin.

Previous studies of cholera toxin and Escherichia coli heat-labile enterotoxin have suggested that proteolytic cleavage plays an important role in the expression of ADP-ribosyltransferase activity and toxicity. Specifically, several studies have implicated a trypsin-like cleavage at arginine 192, which lies within an exposed region subtended by a disulfide bond in the intact A subunit, in toxicity. To investigate the role of this modification in the enzymatic and cytotonic properties of heat-labile enterotoxin, the response of purified, recombinant A subunit to tryptic activation and the effect of substituting arginine 192 with glycine on the activities of the holotoxin were examined. The recombinant A subunit of heat-labile enterotoxin exhibited significant levels of ADP-ribosyltransferase activity that were only nominally increased (approximately twofold) by prior limited trypsinolysis. The enzymatic activity also did not appear to be affected by auto-ADP-ribosylation that occurs during the high-level synthesis of the recombinant A subunit in E. coli. A mutant form of the holotoxin containing the arginine 192-to-glycine substitution exhibited levels of cytotonic activity for CHO cells that were similar to that of the untreated, wild-type holotoxin but exhibited a marked delay in the ability to increase intracellular levels of cyclic AMP in Caco-2 cells. The results indicate that trypsin-like cleavage of the A subunit of E. coli heat-labile enterotoxin at arginine 192 is not requisite to the expression of enzymatic activity by the A subunit and further reveal that this modification, although it enhances the biological and enzymatic activities of the toxin, is not absolutely required for the enterotoxin to elicit cytotonic effects.

Species differences in the expression of caseinolytic proteinases and plasminogen activators by bone marrow cells.

Intact viable bone marrow cells from hamsters (Mesocricetus aruatus), rat (Rattus norvegicus), guinea pig (Cavia porcella) and rabbit (Oryctolagus cuniculus) were analyzed for caseinolytic proteinases and plasminogen activator activity. Species specific quantitative and qualitative differences in caseinolytic activity were detected. Quantitative and qualitative differences in plasminogen activator expression were observed. Cellular fractionation experiments revealed a heterogenous distribution of plasminogen activator activity among subpopulations of cells. The plasminogen activator activity associated with bone marrow cells behaved as ectoenzymes. These results indicated that different species may regulate bone marrow cell proteinase activity in a species-specific manner, compatible with their unique regulatory requirements.

Cloning, sequencing, and expression of a gene from Campylobacter jejuni encoding a protein (Omp18) with similarity to peptidoglycan-associated lipoproteins.

A Campylobacter jejuni genomic plasmid library was screened with antiserum generated against whole C. jejuni, revealing two immunoreactive clones. Sequence analysis of the recombinant plasmids revealed a common open reading frame of 498 nucleotides encoding a protein of 165 amino acids with a calculated molecular mass of 18,018 Da. The recombinant product partitioned to the outer membrane fractions of Escherichia coli transformants and has been designated Omp18. The deduced amino acid sequence of the cloned C. jejuni gene exhibits considerable similarity to peptidoglycan-associated lipoproteins from other gram-negative bacteria.

Increased plasma proteinase activity of mice bearing the BCL1 leukemia.

Plasma samples from mice bearing the BCL1 leukemia were shown to express elevated levels of neutral proteinase activity when assayed with radioiodinated casein as a substrate. Increased plasma proteinase activity reached levels 3-4-fold higher than controls. The increased levels of activity did not correlate with the degree of hepatosplenomegaly or the number of tumor cells in the blood. The onset of the increase in plasma activity correlated with the onset of the leukemic phase of the disease. These findings with the murine leukemia may be analogous to recent findings of abnormal proteinase activity in plasma from patients with acute leukemia.

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.

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.

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.