PubMed Health⌕ Search

Biomedical subjects

A Ullmann

Publications and source records attributed to A Ullmann.

At least 37 records · Page 2Linked to original sources
PubMed1997

Anti-viral protection conferred by recombinant adenylate cyclase toxins from Bordetella pertussis carrying a CD8+ T cell epitope from lymphocytic choriomeningitis virus.

The elucidation of the mechanisms of antigen presentation by major histocompatibility complex class I molecules has stimulated the search for nonreplicative vectors that could deliver CD8+ T cell epitopes to the cytosol of antigen-presenting cells to trigger the activation of specific cytotoxic T lymphocytes (CTLs) in vivo. In the present study, we investigated the potential ability of an invasive adenylate cyclase toxin from Bordetella pertussis, carrying a CD8+ T cell epitope from the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), to stimulate protective anti-viral immunity. Mice immunized with this recombinant toxin developed strong CTL responses against LCMV-infected target cells. Moreover, these mice were protected against an intracerebral challenge with a virulent strain of LCMV that killed all nonimmunized mice within 7 days. This protection was abolished after in vivo elimination of CD8+ T cells. A mutant toxin devoid of adenylate cyclase activity (i.e., cAMP synthesizing activity) was constructed by insertion of a dipeptide into the catalytic site of the molecule. This genetically detoxified invasive toxin carrying the LCMV epitope stimulated a strong CTL response against both peptide-coated and virus-infected target cells, and mice immunized with this molecule were fully protected against a lethal intracerebral LCMV challenge. To our knowledge, this study represents the first demonstration that a genetically detoxified bacterial toxin carrying a viral CTL epitope can stimulate efficient protective immunity.

Adenylyl Cyclases↗
PubMed1997

The Bordetella pertussis sigma subunit of RNA polymerase confers enhanced expression of fha in Escherichia coli.

We have cloned the rpoD gene coding for the major sigma factor of Bordetella pertussis. The deduced amino acid sequence reveals a protein of 733 residues which has extensive amino acid homology with the principal sigma factors of a number of divergent prokaryotes. It is larger than most sigma factors identified to date, having a molecular mass of 81.3 kDa. We have designated this factor sigma 80. In a heterologous complementation assay, B. pertussis rpoD was able to complement the Escherichia coli rpoD temperature-sensitive mutant UQ285. Furthermore, B. pertussis rpoD conferred better specificity to the E. coli RNA polymerase, allowing increased expression of the B. pertussis virulence-associated tha promoter, but could not activate the ptx and cya promoters in the E. coli UQ285 strains carrying the B. pertussis bvg locus. We discuss the implications of these results on the mechanisms involved in the activation of virulence-associated promoters.

Amino Acid Sequence↗
PubMed1997

Characterization of DNA binding sites for the BvgA protein of Bordetella pertussis.

Expression of virulence-associated genes in Bordetella pertussis is under the control of the pleiotropic regulator BvgA. Although previous studies have identified recognition sequences for BvgA in several promoter regions, their structures have not been clearly characterized. We show that the BvgA binding sites within the bvgp(1) and cyaA promoters consist of inverted repeats and suggest that inverted-repeat motifs may represent the recognition elements for DNA-BvgA interaction.

Bacterial Proteins↗
PubMed1996

Cellular responses of HPV-positive cancer cells to genotoxic anti-cancer agents: repression of E6/E7-oncogene expression and induction of apoptosis.

The E6 gene of tumor-associated types of human papillomaviruses codes for a functional antagonist of p53. Overexpression of E6 from heterologous promoters can block p53-mediated cellular responses to DNA damage, such as transcriptional stimulation of p53 target genes and cell-cycle arrest in G1. In contrast, genotoxic treatment of HPV-positive cancer cells, which express the E6 gene from chromosomally integrated viral copies, results in increased expression of the p53 target gene p21WAF1 and, in several cell lines, induction of G1 arrest. In the present study, we show that treatment with genotoxic agents, such as mitomycin C and cisplatin, leads to strong repression of viral E6/E7 oncogene expression in HPV16- and HPV18-positive cervical carcinoma cell lines. Kinetic analyses revealed that reduction of E6/E7 expression was not a prerequisite for induction of p21WAF1. We furthermore found that the apoptosis-promoting bax gene could be induced by genotoxic stress in some, but not all, HPV-positive cancer cell lines. Treatment with DNA-damaging agents eventually resulted in apoptotic cell death of HPV-positive cancer cells, irrespective of their capacity to induce the p53 target gene bax. These results support the notion that HPV-positive cancer cells can exhibit intact cellular responses to genotoxic stress, which may involve p53-dependent and -independent biochemical pathways. The ability of HPV-positive cancer cells to induce apoptotic cell death in response to DNA damage could provide a molecular explanation for the therapeutic effects of genotoxic agents in the treatment of cervical cancer.

Apoptosis↗
PubMed1996

In vivo induction of CTL responses by recombinant adenylate cyclase of Bordetella pertussis carrying viral CD8+ T cell epitopes.

Exogenous Ags enter the endosomal pathway and are presented to CD4+ T cells in association with class II molecules whereas endogenously synthesized Ags, such as viral proteins, are presented to CD8+ T cells in association with MHC class I molecules. Therefore, most CTL activation strategies use live vectors although an alternative possibility could be to deliver the epitope into the cytosol by targeting it to an invasive nonreplicative vector. The adenylate cyclase toxin of Bordetella pertussis is able to invade a large number of eukaryotic cells and to deliver its catalytic domain to the cytosol of the cells. In the present study, we have tested the in vivo immunogenicity of recombinant adenylate cyclase toxins expressing CTL epitopes either from the nucleoprotein of lymphocytic choriomeningitis virus or from the V3 region of HIV-1 gp120. BALB/c mice immunized with these toxins developed high specific CTL responses that were shown to be mediated by class I-restricted CD8+ CTL. The induction of CTL responses by recombinant toxins did not require CD4+ T cells and the cytotoxic activity persisted 2 mo after immunization. The activation of CTL responses by the recombinant adenylate cyclase toxin required the full-length invasive activity of the toxin but did not depend upon its catalytic adenylate cyclase activity as demonstrated with a genetically inactivated recombinant toxin expressing the lymphocytic choriomeningitis virus epitope. This genetically detoxified invasive toxin represents, therefore, an attractive new vector for CTL activation.

Adenylyl Cyclases↗
PubMed1996

Phosphorylated BvgA is sufficient for transcriptional activation of virulence-regulated genes in Bordetella pertussis.

In Bordetella pertussis the expression of virulence factors is coordinately regulated by the BvgS and BvgA proteins, members of the bacterial two-component signal transduction family, BvgS being the transmembrane sensor and BvgA the regulator. Activation of virulence gene expression requires phosphorylation of BvgA. On the basis of observed differences in the regulation of individual genes, the existence of accessory regulators has been postulated. They were supposed to be necessary for expression of genes encoding adenylate cyclase toxin (cya) and pertussis toxin (ptx), but not required for the expression of fha, encoding filamentous hemagglutinin. To clarify this issue we investigated the mechanism of activation of the BvgAS-controlled genes by performing in vitro run-off transcription experiments. We show, using purified RNA polymerase of B.pertussis, that phosphorylated BvgA is sufficient for transcriptional activation of the major virulence genes, thus providing good evidence that BvgA regulation operates directly with the transcription initiation machinery at the promoters of the virulence genes without a requirement for accessory activators. In addition, our results indicate that activation of the different promoters may involve distinct mechanisms. We suggest that the previously observed differences in regulation of individual virulence-associated genes reflect differences in the phosphorylation state of BvgA.

Bacterial Proteins↗
PubMed1996

Phosphorylation-dependent binding of BvgA to the upstream region of the cyaA gene of Bordetella pertussis.

In Bordetella pertussis, transcription of virulence-associated genes is regulated by the BvgS and BvgA proteins, members of the bacterial two-component signal-transduction family. BvgS is the transmembrane sensor and BvgA, in its phosphorylated form, is believed to be the key transcriptional activator in B. pertussis. However, the BvgA recognition sites in most virulence promoters have not yet been identified. To investigate the interaction of BvgA with the upstream region of cyaA, the gene encoding adenylate cyclase haemolysin, we have produced large amounts of BvgA in Escherichia coli. The protein was purified from inclusion bodies and then phosphorylated by acetyl phosphate. Using electrophoretic mobility-shift and footprinting assays, we provide evidence that BvgA cannot bind to the cyaA promoter unless it is phosphorylated. The phosphorylated form of BvgA (BvgA-P) is able to bind specifically to the upstream region of cyaA. Analysis of this region revealed that an unexpectedly large sequence, from -137 to -51, appears to be the target for BvgA-P binding, and probably contains multiple binding sites.

Adenylate Cyclase Toxin↗
PubMed1995

Hemolytic, but not cell-invasive activity, of adenylate cyclase toxin is selectively affected by differential fatty-acylation in Escherichia coli.

Adenylate cyclase toxin from Bordetella pertussis requires posttranslational acylation of lysine 983 for the ability to deliver its catalytic domain to the target cell interior and produce cyclic adenosine monophosphate (cell-invasive activity) and to form transmembrane channels (hemolytic activity). When the toxin is expressed in Escherichia coli, it has reduced hemolytic activity, but comparable cell-invasive activity to that of adenylate cyclase toxin from B. pertussis. In contrast to the native protein from B. pertussis, which is exclusively palmitoylated, recombinant toxin from E. coli is acylated at lysine 983 with about 87% palmitoylated and the remainder myristoylated. Furthermore, the recombinant toxin contains an additional palmitoylation on approximately two-thirds of the lysines at position 860. These observations suggest that the site and nature of posttranslational fatty-acylation can be dictated by the bacterial host used for expression and can have a significant, but selective, effect on protein function.

Acylation↗
PubMed1995

Functional p53 protein in human papillomavirus-positive cancer cells.

There is accumulating evidence that the p53 protein contributes to tumor suppression by stimulating the transcription of specific cellular genes, such as the cell cycle control gene WAF1/ClP1. p53-mediated transcriptional activation is inhibited in cotransfection assays by overexpressed E6 protein from cancer-associated human papillomavirus (HPV) types, pointing at a possible molecular mechanism by which these viruses contribute to malignant cell transformation. Here we analysed the transcriptional transactivation function of endogenous p53 protein in a series of cervical cancer cell lines, which express the E6 gene from integrated viral sequences. Transient and stable transfection analyses employing p53-responsive reporter constructs indicated that HPV-positive cervical cancer cells contained transactivating p53 protein. Treatment of HPV-positive cells with genotoxic agents, such as mitomycin C, cisplatin, or u.v. irradiation, resulted in an increase of nuclear p53 protein levels and enhanced binding of p53 to a p53-recognition site. These effects were accompanied by an increase of WAF1/ClP1 mRNA levels. In several HPV-positive cell lines, these molecular events were linked to a cell cycle arrest in G1. In contrast, cancer cells containing mutant p53 genes did not contain transactivating endogenous p53 protein and lacked the p53-mediated response to DNA damaging agents. These results indicate that the tumorigenic phenotype of HPV-positive cancer cell lines does not necessarily correlate with a lack of basal or DNA damage induced p53 activities and that therefore the presence of high risk HPV sequences is not functionally equivalent to the loss of p53 function through somatic mutations of the p53 gene.

Base Sequence↗
PubMed1995

Mutations which result in constitutive expression of the Bordetella pertussis filamentous haemagglutinin gene.

The expression of the filamentous haemagglutinin (FhaB) of Bordetella pertussis is positively regulated by the bvg locus which encodes a transcriptional activator, BvgA, and a transmembrane sensor protein, BvgS. The gene encoding FhaB, fhaB alone, is not expressed in Escherichia coli, but the introduction of the bvg locus in trans can restore fhaB expression. Using fhaB::lacZY fusions, we have isolated, in E. coli, partially bvg-independent constitutive mutants. The corresponding mutations have been localized to the upstream region of the fhaB promoter at position -15, -16 and -42 from the transcription initiation site. In the absence of the bvg locus, the strength of the mutated promoters was 15 and 200 times higher than the wild-type promoter in the absence of the bvg locus. The expression of these mutated promoters was still enhanced by the presence of the bvg locus.

Base Sequence↗
PubMed1995

Identification by in vitro complementation of regions required for cell-invasive activity of Bordetella pertussis adenylate cyclase toxin.

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein composed of an amino-terminal adenylate cyclase (AC) domain linked to a 1300-residue channel-forming RTX (repeats in toxin) haemolysin. The toxin delivers its AC domain into a variety of eukaryotic cells and impairs cellular functions by catalysing unregulated synthesis of cAMP from intracellular ATP. We have examined toxin activities of a set of deletion derivatives of CyaA. The results indicate that CyaA does not have a dedicated target cell-binding domain and that structural integrity and co-operation of all domains, as well as the post-translational fatty acylation mediated by an accessory protein CyaC, are all essential for target cell association and toxin activity of CyaA. When tested individually, all toxin derivatives were inactive and impaired in the tight association with the target cell surface. However, pairs of constructs with nonoverlapping deletions complemented each other in vitro and exhibited a partially restored cytotoxic activity. This suggests that at least a part of the active toxin may act in the form of dimers or higher oligomers. The complementation analysis revealed that the last 217 residues of CyaA, containing the unprocessed secretion signal, form an autonomous domain essential for toxin activity, and that the region from residue 624 to 780 may be directly involved in delivery of the AC toxin into cells.

Acylation↗
PubMed1995

A novel chromatin-forming histone H1 homologue is encoded by a dispensable and growth-regulated gene in Bordetella pertussis.

We report the identification of a protein homologous to a histone H1 in Bordetella pertussis. The B. pertussis histone homologue, BpH1, varies in size in different strains from 182 to 206 amino acids. The variability of the size of the protein is due to gene variability by insertion or deletion of DNA modules. Insertion of a kanamycin cassette into the bpH1 gene generates a BpH1 null mutant with phenotypic properties and growth rate similar to those of the wild-type strain, showing that this gene is dispensable. In vitro, the BpH1 protein prevents chromosomal DNA degradation from DNase I and constrains supercoiled DNA. Transcription of the bpH1 gene is activated during exponential growth of the bacteria, whereas it is repressed during the stationary phase of growth. It is proposed that BpH1 plays a role in chromatin formation and condensation during DNA replication and that repression of transcription depends upon a reduced rate of DNA replication.

Amino Acid Sequence↗
PubMed1995

Cell-invasive activity of epitope-tagged adenylate cyclase of Bordetella pertussis allows in vitro presentation of a foreign epitope to CD8+ cytotoxic T cells.

The adenylate cyclase (AC) toxin (CyaA) of Bordetella pertussis has an invasive catalytic domain (AC domain) which penetrates the cytoplasmic membrane of a variety of eukaryotic cells and intoxicates them by unregulated synthesis of cyclic AMP. Previous work led to identification of five permissive sites in the AC domain at which heterologous peptides are accommodated without affecting its enzymatic properties. We have constructed a set of CyaA toxins tagged at these permissive sites by insertion of a CD8+ T-cell epitope, RPQASGVYMGNLTAQ, from the nucleoprotein of lymphocytic choriomeningitis virus. Introduction of the epitope at any of the five sites did not affect the capacity of the toxin to deliver its AC domain into target cells. Moreover, the toxin with the inserted epitope was shown to sensitize target cells for lysis by epitope-specific CD8+ cytotoxic T lymphocytes in vitro, showing that the tagged AC was processed for presentation of the lymphocytic choriomeningitis virus epitope in association with the major histocompatibility complex class I molecules. This finding indicates that by virtue of delivery of foreign epitopes into the antigen-presenting cells, purpose-designed recombinant CyaAs may be useful for induction of specific major histocompatibility complex class I-restricted cell-mediated immunity also in vivo.

Adenylyl Cyclases↗
PubMed1994

Chemical fatty acylation confers hemolytic and toxic activities to adenylate cyclase protoxin of Bordetella pertussis.

Adenylate cyclase toxin (ACT), a virulence factor of Bordetella pertussis, acquires hemolytic and toxic activities after post-translational modification of the cyaA gene product, CyaA. The exact nature of this modification is unknown, but homology to the related repeat toxin alpha-hemolysin of Escherichia coli suggests that fatty acylation of a lysine residue may be involved. In the present study, we used an in vitro chemical approach to acylate unmodified, inactive adenylate cyclase protoxin by using a new water-soluble compound, acylpyrophosphate. We show that undirected transfer of lauric, myristic, or palmitic acid chains to the CyaA protoxin is able to confer both hemolytic and toxic activities to ACT. The chemically modified protoxin shows a specific requirement for Ca2+ ions for toxic activity, as does the wild type toxin. However, the toxic and hemolytic activities of chemically modified ACT are low in comparison to ACT modified in vivo, suggesting that in vitro fatty acylation of the protoxin involves random modification of nucleophilic residues present in the toxin in contrast to the in vivo modification of specific sites.

Acylation↗
PubMed1994

Adenylate cyclase toxin (CyaA) of Bordetella pertussis. Evidence for the formation of small ion-permeable channels and comparison with HlyA of Escherichia coli.

The interaction between the adenylate cyclase toxin (CyaA) of Bordetella pertussis and lipid was studied using the lipid bilayer assay. The addition of CyaA to the aqueous phase bathing lipid bilayer membranes composed of different lipids resulted in the increase of the membrane conductance. This increase was rather small for membranes formed of pure lipids as compared with lipid mixtures such as asolectin. The toxin formed in asolectin membranes small transient ion-permeable channels with a single-channel conductance of 27 pS in 1 M KCl, which is considerably smaller than that of the alpha-hemolysin (HlyA) of Escherichia coli (1500 pS). Experiments with different salts suggested that the CyaA-induced channels were exclusively cation-selective because of negative charges localized at the channel mouth. The single-channel conductance of channels initiated by CyaA was independent of whether the toxin was purified from B. pertussis or from recombinant E. coli. However, the channel-forming activity of the CyaA expressed in B. pertussis was substantially higher than that of the recombinant toxin. Experiments with mutant forms of CyaA suggested that both the activation of CyaA by CyaC and the hemeolytic part of the toxin, but not the repeats and the cyclase activity, are required for channel formation in lipid bilayer membranes.

Adenylate Cyclase Toxin↗
PubMed1994

Mutations in the Bordetella pertussis bvgS gene that confer altered expression of the fhaB gene in Escherichia coli.

The bvg locus of Bordetella pertussis, required for coordinate regulation of virulence genes in response to environmental signals, encodes two proteins, BvgS and BvgA, that belong to the bacterial two-component signal transduction systems. We have isolated spontaneous mutations of the bvg locus in Escherichia coli and analyzed their effects on the expression of fhaB::lacZY transcriptional fusions. The mutations, localized in the linker and transmitter domain of BvgS, result in increased activation of fhaB and/or in insensitivity to a modulating agent, nicotinic acid.

Amino Acid Sequence↗