A particular class of virulence factors: calmodulin-activated bacterial adenylate cyclases.
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Adenylate Cyclase Toxin↗
Biomedical subjects
M Mock
Publications and source records attributed to M Mock.
Bacillus anthracis pXO1 virulence plasmid encodes a type 1 DNA topoisomerase.
The virulence plasmid pXO1 is responsible for toxin production in Bacillus anthracis. A DNA fragment from pXO1 was isolated and was shown, by sequence analysis, to contain part of a type 1 DNA topoisomerase gene. Attempts to clone the entire wild-type gene, designated topX, in Escherichia coli, were unsuccessful. In order to obtain the complete gene, it was first insertionally inactivated and then cloned in the mutated form. The deduced amino acid sequence of Topo X1 shows similarities to that of the two E. coli type 1 DNA topoisomerases. The N-terminal two-thirds of the putative B. anthracis protein exhibits strongest sequence similarity to topoisomerase III, whereas the C-terminal portion contains cysteine residues that could form three zinc-binding domains, as they do in topoisomerase I. The suggested active-site tyrosine is conserved in all three proteins. The regulation of expression from the topX promoter is modified by addition of a gyrase inhibiting antibiotic. The Topo X1 protein is likely to be involved in the stability of pXO1.
Amino Acid Sequence↗
The three Bacillus anthracis toxin genes are coordinately regulated by bicarbonate and temperature.
The two Bacillus anthracis toxins are composed of three proteins, protective antigen, lethal factor, and edema factor. The structural genes for these three components are located on the virulence plasmid pXO1. We constructed transcriptional fusions between the regulatory region of each of these genes and lacZ. Each construct was then inserted as a single copy at the corresponding toxin gene locus on pXO1, resulting in three isogenic strains. Two environmental factors, bicarbonate and temperature, were found to induce beta-galactosidase synthesis in each recombinant strain. Furthermore, the transcription of the three toxin genes appears to be coordinately regulated.
Antigens, Bacterial↗
Calmodulin-activated bacterial adenylate cyclases as virulence factors.
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.
Adenylyl Cyclases↗
Construction of Bacillus anthracis mutant strains producing a single toxin component.
The two protein exotoxins secreted by Bacillus anthracis are composed of three distinct components: protective antigen (PA), lethal factor (LF), and (o)edema factor (EF). We have developed a genetic strategy that permits us selectively to inactivate each of the genes coding for PA, EF or LF. This strategy involved the deletion of a portion of the structural gene and the insertion of an antibiotic resistance cassette. With this technique, double mutant strains of B. anthracis producing only one toxin component have been constructed. Characterization of the mutant strains indicated that they produced the expected single toxin protein. Using a simple, two-step protocol, we have purified PA, LF and EF to homogeneity from culture supernatants. These three mutant strains are potentially powerful tools for studying the individual effect of each toxin component in vitro and in vivo.
Animals↗
Regulation of pag gene expression in Bacillus anthracis: use of a pag-lacZ transcriptional fusion.
The production of protective antigen (PA), the common component of the two anthrax toxins, is influenced by the environment. In order to examine factors involved in its regulation, a transcriptional fusion between the promoter region of the PA gene (pag) and the lacZ gene was constructed and introduced into Bacillus anthracis Sterne. Activity of the pag promoter was followed by measuring beta-galactosidase activities under various growth and medium conditions. Expression from the pag promoter was observed throughout exponential-phase and was maximal in early stationary phase. The activity of the pag promoter was stimulated by the addition of glucose in the medium.
Antigens, Bacterial↗
Structural and ligand-binding properties of a truncated form of Bacillus anthracis adenylate cyclase and of a catalytically inactive variant in which glutamine substitutes for lysine-346.
A truncated, 541-residue-long, Bacillus anthracis adenylate cyclase was expressed in Escherichia coli. The purified protein (CYA 62) exhibited catalytic and CaM-binding properties identical with those of the wild-type enzyme secreted by B. anthracis. The analysis of the secondary structure of the CYA 62 protein by Fourier transform infrared spectroscopy and circular dichroism revealed the dominance of beta-type structure. The protein shows a relatively low thermal stability with the midpoint denaturation temperature at 45 degrees C. A catalytically inactive variant of CYA 62 in which Gln substituted for Lys-346 (CYA 62 K346Q) was comparatively analyzed for its secondary structure and thermal stability, as well as ligand-binding properties with fluorescent derivatives of ATP and calmodulin. The K346Q variant of CYA 62 has a similar secondary structure and comparable calmodulin binding properties to those of the parent protein and exhibits only slightly reduced thermal stability (the apparent midpoint denaturation temperature is at 43 degrees C). Despite these similarities, the binding of 3'-anthraniloyl-2'-deoxy-ATP (a fluorescent ATP analogue) to the modified protein is severely impaired, from which we conclude that the prime function of Lys-346 in the wild-type enzyme from B. anthracis is to ensure tight binding of the nucleotide substrate to the active site.
Adenylyl Cyclases↗
Contribution of individual toxin components to virulence of Bacillus anthracis.
Three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF; a calmodulin-dependent adenylate cyclase), compose the lethal (PA + LF) and edema (PA + EF) toxins secreted by Bacillus anthracis. Mutant strains, each deficient in the production of one toxin component, were constructed, and their virulence was then studied. A kanamycin resistance cassette was inserted in each cya (encoding EF) and lef (encoding LF) gene, and the constructs were separately introduced into B. anthracis Sterne on a mobilizable shuttle plasmid. An EF- strain and an LF- strain were then isolated after homologous recombination with the resident toxin-encoding plasmid, pXO1. Spores from these mutants and from a previously constructed PA- mutant were used to inoculate mice, and the lethality and local edema formation were monitored. LF- or PA- mutants were not lethal even at high inocula, whereas the EF- mutant induced lethal infections. This indicates that LF in combination with PA is a key virulence factor required for lethality. Skin edema formation was observed with the LF- mutant, which produces only the combination of PA and EF. However, EF- and LF- mutants were significantly less efficient at inducing, respectively, lethality and edema than was the parental Sterne strain. These results suggest that the three toxin components might act synergistically in vivo to cause lethality and edema formation.
Animals↗
Structural and functional relationships between Pasteurella multocida and enterobacterial adenylate cyclases.
The Pasteurella multocida adenylate cyclase gene has been cloned and expressed in Escherichia coli. The primary structure of the protein (838 amino acids) deduced from the corresponding nucleotide sequence was compared with that of E. coli. The two enzymes have similar molecular sizes and, based on sequence conservation at the protein level, are likely to be organized in two functional domains: the amino-terminal catalytic domain and the carboxy-terminal regulatory domain. It was shown that P. multocida adenylate cyclase synthesizes increased levels of cyclic AMP in E. coli strains deficient in the catabolite gene activator protein compared with wild-type strains. This increase does not occur in strains deficient in both the catabolite gene activator protein and enzyme III-glucose, indicating that a protein similar to E. coli enzyme III-glucose is involved in the regulation of P. multocida adenylate cyclase. It also indicates that the underlying process leading to enterobacterial adenylate cyclase activation has been conserved through evolution.
Adenylyl Cyclases↗
Binding of 3'-anthraniloyl-2'-deoxy-ATP to calmodulin-activated adenylate cyclase from Bordetella pertussis and Bacillus anthracis.
3'-Anthraniloyl-2'-deoxyadenosine 5'-triphosphate (Ant-dATP), a fluorescent analogue of ATP, was tested as a probe for the nucleotide-binding site of calmodulin (CaM)-activated adenylate cyclases from Bordetella pertussis (BPCYA47) and Bacillus anthracis (BACYA62). Ant-dATP competitively inhibited both bacterial enzymes expressed in Escherichia coli (ki approximately 10 microM). Binding of the analogue to adenylate cyclase was monitored by equilibrium dialysis and by an increase in its fluorescence emission at 420 nm upon excitation at 330 nm. Whereas the fluorescence of Ant-dATP was little influenced by divalent cations, CaM, or adenylate cyclase alone, the Ca2+.CaM.cyclase complex increased up to 4 times the quantum yield of Ant-dATP. Binding of the analogue to the catalytic site of BPCYA47 and BACYA62 was specific as shown by its displacement with ATP or 3'-dATP. Our results substantiate the role of CaM in favoring substrate binding to CaM-activated enzymes.
Adenosine Triphosphate↗
Characterization of ATP and calmodulin-binding properties of a truncated form of Bacillus anthracis adenylate cyclase.
The Bacillus anthracis cya gene encodes a calmodulin-dependent adenylate cyclase. A deletion cya gene product obtained by removing 261 codons at the 5' end was expressed in a protease-deficient lon- E. coli strain and purified to homogeneity. This truncated enzyme (CYA 62) exhibits catalytic and calmodulin-binding properties similar to the properties of wild-type adenylate cyclase from B. anthracis culture supernatants, i.e., a kcat of 1100 s-1 at 30 degrees C and pH 8, an apparent Km for ATP of 0.25 mM, and a Kd for bovine brain calmodulin of 23 nM. The calmodulin-binding domain of the CYA 62 truncated enzyme was labeled with a cleavable radioactive photoaffinity cross-linker coupled to calmodulin. The labeled CYA 62 protein was then cleaved with cyanogen bromide and N-chlorosuccinimide. We show that the calmodulin-binding domain of B. anthracis adenylate cyclase is located within the last 150 amino acid residues of the protein. A further deletion at the 3' end of the CYA 62 coding sequence yielded an adenylate cyclase species (CYA 57) lacking 127 C-terminal amino residues. CYA 57, still sensitive to activation by high concentrations of calmodulin, exhibits less than 0.1% of the specific activity of CYA 62. Binding of 3'dATP (a competitive inhibitor) to CYA 62 was determined by equilibrium dialysis. In the absence of calmodulin, binding of the ATP analogue to this truncated protein was severely impaired, which explains, at least in part, the absolute requirement for calmodulin for the catalytic activity of B. anthracis adenylate cyclase.
Adenosine Triphosphate↗
Construction and characterization of a protective antigen-deficient Bacillus anthracis strain.
The pag gene coding for protective antigen (PA), one of the three toxin components of Bacillus anthracis, has been cloned into the mobilizable shuttle vector pAT187 and transferred by conjugation from Escherichia coli to B. anthracis. Using this strategy, an insertionally mutated pag gene constructed and characterized in E. coli, was introduced into B. anthracis Sterne strain. This transconjugant was used to select a recombinant clone (RP8) carrying the inactivated pag gene on the toxin-encoding plasmid, pXO1. Strain RP8 was deficient for PA while still producing the two other toxin components, i.e. lethal factor (LF) and edema factor (EF). In contrast to spores from the wild-type Sterne strain, spores prepared from RP8 were totally non-lethal in mice. These results clearly establish the central role played by PA in B. anthracis pathogenicity.
Animals↗
Identification of a common domain in calmodulin-activated eukaryotic and bacterial adenylate cyclases.
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.
Adenylyl Cyclases↗
Age variation in the association between angiographic coronary disease and angina from the Coronary Artery Surgery Study (CASS)
Between July 1974 and May 1979, 19,153 non-randomized patients without prior cardiac surgery and with chest pain were studied angiographically and enrolled in the Coronary Artery Surgery Study (CASS). The primary question addressed by this paper is: for fixed levels of coronary artery disease, are the presence and severity of angina pectoris greater for older than for younger patients? For those with the same extent of disease, older patients were more likely to have angina and to exhibit more severe symptoms. After adjustment for covariates using logistic regression analysis, age was found to be an important, independent predictor of the presence and severity of angina. There are many possible explanations for these findings, although physiologic factors related to aging, the disease process, and deconditioning associated with an increased sedentary life style seem most reasonable. Another possibility has to do with referral patterns for study and the prevalence of angina in the different age groups.
Adult↗
Immunity protein is not required for the bactericidal activity of colicin E3.
Plasmid pLAX3, carrying the colicin E3 gene, was used to direct the in vitro synthesis of a colicin E3* molecule totally devoid of its immunity protein. We established that this molecule is able to kill sensitive Escherichia coli cells in the total absence of immunity protein. Therefore, all of the information required for colicin E3 action is located on the colicin polypeptide itself. Furthermore, our studies indicated that immunity protein protects the C-terminal enzymatic part of native colicin E3 protein against proteolytic degradation before or during its translocation across the cell envelope. These results are discussed in relation to the mode of entry of colicin E3 into bacterial cells.
Bacterial Proteins↗
Structural homology between virulence-associated bacterial adenylate cyclases.
The primary structure of the calmodulin-sensitive adenylate cyclase toxin from Bacillus anthracis has been determined from the corresponding nucleotide sequence and compared to that of the homologous toxin secreted by Bordetella pertussis. The cya gene of Bacillus anthracis encodes an 800 amino acid (aa) protein beginning with an N-terminal signal peptide. The central part of the B. anthracis adenylate cyclase includes a region of striking homology with the N-terminal part of the B. pertussis enzyme. In this region a particularly well conserved 24-aa peptide and two other less homologous peptides have been identified. These data corroborate the immunological relatedness of the two enzymes and suggest that the two prokaryotic calmodulin-sensitive adenylate cyclases originate from a common ancestor.
Adenylyl Cyclases↗
Cloning and expression of the calmodulin-sensitive Bacillus anthracis adenylate cyclase in Escherichia coli.
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.
Animals↗
DNA sequence analysis of three missense mutations affecting colicin E3 bactericidal activity.
We have determined the nucleotide sequence changes caused by three missense mutations leading to the production of inactive colicin E3 proteins. The ceaC1 mutation, affecting the translocation of colicin E3 through bacterial membranes, is caused by a serine to phenylalanine change at position 37 within the glycine-rich region at the N-terminal part of colicin E3. This confirms previous results suggesting a role for this domain in colicin uptake by sensitive cells. The ceaC2 and ceaC3 mutations, abolishing colicin E3 RNase activity, affect the C-terminal enzymatic domain of the molecule. In the ceaC2 mutant, serine at position 529 was converted to leucine. The ceaC3 mutation replaced a glycine residue at position 524 with an aspartic acid residue. The two mutations ceaC2 and ceaC3 yield information on the amino acid residues involved in the RNase activity of colicin E3.
Amino Acid Sequence↗