Biomedical subjects
Publications and source records attributed to A Ullmann.
A DNA fragment of Streptomyces coelicolor encoding the carboxy-terminal catalytic domain of adenylate cyclase was cloned, sequenced and expressed in an Escherichia coli cya-defective strain where it produced nanomole levels of cAMP. The amino acid sequence of the enzyme displays similarities with the Brevibacterium liquefaciens pyruvate regulated adenylate cyclase.
Bordetella pertussis and Bacillus anthracis each produce a virulence-associated, calmodulin-dependent adenylate cyclase toxin, which generates increased levels of cyclic AMP in eukaryotic cells. The two proteins share sequence similarities in their catalytic domains. The remaining regions display different structural and functional organizations that account for the differences both in interaction of the two toxins with target cells and in the resulting disease symptoms.
The adenylate cyclase toxin of Bordetella pertussis is a secreted multifunctional protein, endowed with calmodulin-activated catalytic, haemolytic and cytotoxic activities. Residues and domains involved in different functions have been localized and several permissive sites, able to accommodate insertion of peptides without impairing the different functions of the toxin, have been identified. A 400-bp region in the promoter upstream region of the cyaA gene, encoding the toxin, has been defined as the target of transcriptional activation.
The cyaA gene of Bordetella pertussis and of Bordetella bronchiseptica encodes a toxin which is a bifunctional protein exhibiting adenylate cyclase and haemolytic activities. In Bordetella, virulence factors are synthesized under the control of the bvg regulatory locus, in response to environmental signals. In Escherichia coli the cyaA gene is not expressed, nor is it activated by bvg indicating that the activation of cya by bvg is indirect. To characterize cis-acting regulatory regions required for the activation of the cyaA gene we constructed cyaA-lacZY fusions containing progressive deletions in the promoter upstream region and isolated promoter mutations by chemical and site-directed mutagenesis. Deletion analysis shows that a region extending from -569 to -136 bp upstream from the start site of transcription is required for transactivation by bvg, suggesting that multiple binding sites are involved in the activation of the cyaA promoter. No single or double mutations in the promoter upstream region were found which conferred inactive or bvg-independent Cya phenotype. A double mutation in positions +10 and +13, relative to the transcription start site, rendered the promoter bvg-independent and functional in E. coli. The constitutive mutations create a new transcription start site, 20 bp downstream from the wild-type site, by providing new -10 and -35 elements recognized by RNA polymerase alone.
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We developed an improved method of linker insertion mutagenesis for introducing 2 or 16 codons into the Bordetella pertussis cyaA gene which encodes a calmodulin-dependent adenylate cyclase. A recombinant kanamycin resistance cassette, containing oligonucleotide linkers, was cloned in plasmids which carried a truncated cyaA gene, fused at its 3' end to the 5' end of the Escherichia coli lacZ gene, specifying the alpha-peptide. This construction permitted a double selection for in-frame insertions by using screening for kanamycin resistance and for lactose-positive phenotype, resulting from alpha-complementation. We showed that most of the two-amino acid insertions within the N-terminal moiety of the catalytic domain of adenylate cyclase abolished enzymatic activity and/or altered the stability of the protein. All two-amino acid insertions within the C-terminal part of adenylate cyclase resulted in fully stable and active enzymes. These results confirm the modular structure of the catalytic domain of adenylate cyclase, previously proposed on the basis of proteolytic studies. Two-amino acid insertions between residues 247-248 and 335-336 were shown to affect the calmodulin responsiveness of adenylate cyclase, suggesting that the corresponding region in the enzyme is involved in the binding of calmodulin or in the process of calmodulin activation. In addition, we have identified within the primary structure of adenylate cyclase several permissive sites which tolerate 16-amino acid insertions without interfering with the catalytic activity or calmodulin binding. By inserting foreign antigenic determinants into these permissive sites the resulting recombinant adenylate cyclase toxin could be used to deliver specific epitopes into antigen-presenting cells.
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The Bordetella pertussis adenylate cyclase(Cya) toxin-encoding locus (cya) is composed of five genes. The cyaA gene encodes a virulence factor (CyaA), exhibiting adenylate cyclase, hemolytic and invasive activities. The cyaB, D and E gene products are necessary for CyaA transport, and the cyaC gene product is required to activate CyaA. We reconstructed, in Escherichia coli, the cya locus of B. pertussis by cloning the different genes on appropriate vectors under the control of strong promoters and E. coli-specific translation initiation signals. We show that in the absence of additional gene products, CyaA is synthesized at high levels, is endowed with adenylate cyclase activity, but is devoid of invasive and hemolytic activities. CyaC is sufficient to confer upon the adenylate cyclase holotoxin full invasive and partial hemolytic activities. Coexpression of the cyaB, D and E genes neither stimulates nor potentiates the activation brought about by CyaC. This reconstructed system should help to elucidate both the mechanism and the structural requirements of holotoxin activation.
In Bordetella pertussis virulence-associated genes, including adenylate cyclase toxin (Cya), are coordinately regulated in response to environmental signals by proteins coded by the bvg-locus. We have constructed cya-lac fusions in Escherichia coli and have shown that the cya operon is not expressed in E. coli, neither is it activated by bvg, when introduced in trans. The cya-lac fusion is fully active when returned to B. pertussis by homologous recombination and responds to bvg-dependent activation and environmental regulation. These results indicate that in B. pertussis the activation of the cya operon by bvg is indirect.
The Bordetella pertussis adenylate cyclase (cya) operon is composed of four open reading frames, cyaA, B, D and E (Glaser et al., 1988, EMBO J., 7, 3997-4004). The cyaA gene encodes a virulence factor, cyclolysin, a bifunctional protein exhibiting both adenylate cyclase and haemolytic activities while the cyaB, D and E gene products are necessary for cyclolysin transport. We show that the cyaA gene is activated by a promoter located 115 bp upstream from the translational start codon and that transcription is only activated in virulent strains. Termination of transcription occurs 3' to the cyaA structural gene, however there appears to be some read-through into the downstream genes, resulting in full length cyaABDE transcripts. We also identify a second start site of transcription 30 bp upstream from the cyaB gene, in the intergenic cyaA--cyaB region. Transcription is activated from this site in both Vir+ and Vir- strains. Thus, the expression of the virulence associated cyclolysin is positively controlled via a trans-acting protein encoded by the bvg locus while the transport genes show a lower level of constitutive expression which is independent of virulence control.
Urea, at concentrations which do not interfere with bacterial growth, specifically inhibits the expression of catabolite sensitive operons. To search for the target and the mechanism of urea action we measured lactose (lac) and tryptophanase (tna) specific mRNA synthesis in vivo and in vitro. We show that urea acts by two different mechanisms at these two catabolite sensitive operons, resembling the manner in which catabolite repression regulates lac and tna. At the lac promoter, urea abolishes transcription initiation or blocks an early step in mRNA elongation without interfering with the binding of RNA polymerase and catabolite gene activator protein (CAP). At the tna promoter, urea does not abolish transcription initiation but could interfere with tnaC translation.
The Bordetella pertussis cyaA gene encodes a virulence factor which is a bifunctional protein exhibiting calmodulin-sensitive adenylate cyclase and hemolytic activities (P. Glaser, H. Sakamoto, J. Bellahov, A. Ullmann, and A. Danchin, EMBO J. 7:3997-4004, 1988). We characterized the hemolytic and toxin activities of the 200-kilodalton (kDa) bifunctional (CyaA) protein and showed that, whether cell associated or secreted, the 200-kDa CyaA protein carries hemolytic and toxin functions. The catalytically active 45-kDa form of adenylate cyclase released by proteolytic digestion of the 200-kDa CyaA protein displayed neither hemolytic nor toxin activities. We constructed in-phase deletions in the 3' region of the cyaA gene, which presumably carries the hemolytic determinant, and showed that the resulting proteins exhibited wild-type adenylate cyclase activity and were secreted without processing into culture supernatants. The hemolytic activities of these mutant CyaA proteins were severely reduced, and their toxin activities were abolished. These results suggest that the structural integrity of the 200-kDa CyaA protein is necessary for toxin activity and that distinct structural determinants within the CyaA protein are involved in secretion, pore formation, and entry into target cells.
Bordetella pertussis and Bacillus anthracis, two taxonomically distinct bacteria, secrete adenylate cyclase toxins that are activated by the eukaryotic protein calmodulin. The two enzymes contain a well-conserved stretch of 24 amino acid residues [Escuyer et al. (1988) Gene 71, 293-298]. Antibodies have been obtained against two synthetic heptadecapeptides, covering part of the conserved sequences. The anti-peptide antibodies specifically reacted in Western blots with the rat brain adenylate cyclase as well as with the two bacterial enzymes. Anti-rat brain adenylate cyclase serum contained antibodies that were retained by the immobilized peptides, and the affinity-purified antibodies yielded the same recognition pattern of the eukaryotic enzyme as did the unfractionated serum. These results indicate that the eukaryotic adenylate cyclase contains an epitope closely related to that specified by the conserved bacterial sequence. The synthetic peptides and the bacterial adenylate cyclases appeared to compete for ATP (KD of the ATP-peptide complex ca. 0.2 mM), suggesting that the conserved sequence may be part of the substrate binding site in these two enzymes.
The calmodulin-sensitive adenylate cyclase of Bordetella pertussis, a 45 kd secreted protein, is synthesized as a 1706 amino acid precursor. We have shown that this precursor is a bifunctional protein, carrying both adenylate cyclase and haemolytic activities. The 1250 carboxy-terminal amino acids of the precursor showed 25% similarity with Escherichia coli alpha-haemolysin (HlyA) and 22% similarity with Pasteurella haemolytica leucotoxin. Three open reading frames were identified downstream from the cyaA gene: cyaB, cyaD and cyaE, coding for polypeptides of 712, 440 and 474 amino acid residues, respectively. As for E. coli alpha-haemolysin, secretion of B.pertussis adenylate cyclase and haemolysin requires the expression of additional genes. The gene products of cyaB and cyaD are highly similar to HlyB and HlyD, known to be necessary for the transport of HlyA across the cell envelope and for its release into the external medium. Complementation and functional studies indicate that the B.pertussis adenylate cyclase-haemolysin bifunctional protein is secreted by a mechanism similar to that described for E.coli alpha-haemolysin, requiring, in addition to the cyaB and cyaD gene products, the presence of a third gene product specified by the cyaE gene.
The adenylate cyclase gene of Bacillus anthracis, encoding the edema factor, a component of anthrax toxin, has been cloned and expressed in Escherichia coli. Clones were selected by their capacity to complement the cyclase deficiency (cya-) of an E. coli strain expressing the eukaryotic protein calmodulin, an essential activator of B. anthracis adenylate cyclase. The protein expressed in E. coli was shown to exhibit adenylate cyclase activity only in the presence of calmodulin. Experiments using a coupled in vitro transcription-translation system revealed that the protein synthesized from the cloned DNA fragment was enzymatically active, upon addition of calmodulin, and could be immunoprecipitated by antibodies directed against purified Bordetella pertussis adenylate cyclase toxin. This indicates that the two calmodulin-dependent adenylate cyclase toxins are immunologically related.
An engineered calmodulin differs from vertebrate calmodulin in its ability to activate Bordetella pertussis adenylate cyclase, and this difference has been utilized as the basis for a new purification protocol for the adenylate cyclase. VU-8 calmodulin, in which 3 glutamic acid residues (residues 82-84) have been substituted with 3 lysine residues, has a 1000-fold lower apparent affinity for the adenylate cyclase, compared to vertebrate calmodulin, and decreased maximal activity. Because of the relatively calcium-independent nature of the interaction between calmodulin and the cyclase, the use of calmodulin-Sepharose conjugates in the purification of the cyclase requires the use of chaotropic agents for elution. However, when immobilized VU-8 calmodulin was tested as a calcium-dependent, affinity-based, adsorption chromatography step in the purification of the cyclase from culture media or bacterial extracts, the enzyme bound to the column in a calcium-dependent manner, and a nearly homogeneous enzyme was obtained in high yield. These results demonstrate the feasibility of using engineered calmodulins that have selective differences in activity for the rational design of rapid purification protocols for calmodulin-binding proteins as well as indicate the importance of the conserved negative charge cluster at residues 82-84 of calmodulin for activation of this cyclase.
A prokaryotic adenylyl cyclase, secreted by Bordetella pertussis, shares a common functional property with eukaryotic adenylyl cyclases, i.e., regulation by the eukaryotic protein calmodulin. Making use of polyclonal antibodies raised against the bacterial adenylyl cyclase and the rat brain adenylyl cyclase catalytic component, respectively, we showed an immunological cross-reactivity between the two enzymes. Furthermore, B. pertussis adenylyl cyclase was inhibited and immunoprecipitated by the homologous and one of the heterologous immune sera. These results suggest an evolutionary relationship between the B. pertussis enzyme and its eukaryotic counterpart.
The adenylate cyclase toxin of the prokaryote Bordetella pertussis is stimulated by the eukaryotic regulatory protein, calmodulin. A general strategy, using the adenylate-cyclase-calmodulin interaction as a tool, has permitted cloning and expression of the toxin in Escherichia coli in the absence of any B. pertussis trans-activating factor. We show that the protein is synthesized in a large precursor form composed of 1706 amino acids. The calmodulin-stimulated catalytic activity resides in the amino-terminal 450 amino acids of the adenylate cyclase. The enzyme expressed in E. coli is recognized in Western blots by antibodies directed against purified B. pertussis adenylate cyclase, and its activity is inhibited by these antibodies.