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R Rappuoli

Publications and source records attributed to R Rappuoli.

At least 271 records · Page 15Linked to original sources
PubMed1988

On the membrane translocation of diphtheria toxin: at low pH the toxin induces ion channels on cells.

Diphtheria toxin (DT) in acidic media forms ion-conducting channels across the plasma membrane and inhibits protein synthesis of both highly and poorly DT-sensitive cell lines. This results in loss of cell potassium and in entry of both sodium and protons with a concomitant rapid lowering of membrane potential. The pH dependency of the permeability changes is similar to that of the inhibition of cell protein synthesis. DT-induced ion channels close when the pH of the external medium is returned to neutrality and cells recover their normal monovalent cation content. Similar permeability changes were induced by two DT mutants defective either in enzymatic activity or in cell binding, but not with a mutant defective in membrane translocation. The implication of these findings for the mechanism of DT membrane translocation is discussed.

Animals↗
PubMed1988

Positive regulation of pertussis toxin expression.

Although the genus Bordetella contains several closely related species, pertussis toxin (PT) is produced only by phase I Bordetella pertussis. In this work we have studied the regulation of expression of the PT operon and investigated why PT is produced by phase I and not by phase III B. pertussis despite the presence of the PT genes. We have constructed a vector for Bordetella species that contains the PT promoter fused to the coding region of the chloramphenicol acetyltransferase (CAT) gene, and we have used it to identify the regulatory elements involved in the transcription of the PT operon. Efficient transcription of these genes requires at least two features: (i) the 170-base-pair DNA sequence upstream from the start site of transcription and (ii) a trans-activating factor encoded by the vir locus. Bordetella parapertussis and Bordetella bronchiseptica, although endowed with a functional trans-activating system, do not produce PT because of mutations within their PT promoter regions. In contrast, phase III Bordetella species do not show any trans activity.

Base Sequence↗
PubMed1988

Subunit S1 of pertussis toxin: mapping of the regions essential for ADP-ribosyltransferase activity.

The toxicity of pertussis toxin is mediated by the ADP-ribosyltransferase activity of subunit S1. To understand the structure-function relationship of subunit S1 and guide the construction of nontoxic molecules suitable for vaccines, we constructed and expressed in Escherichia coli a series of amino-terminal and carboxyl-terminal deletion mutants as well as a number of molecules containing amino acid substitutions. The shortest peptide still retaining enzymatic activity contains amino acids 2-179. Within this region we identified three mutants in which amino acid substitutions abolish the enzymatic activity. Mutation of amino acids 8 and 9 or 50 and 53, located within the region of the S1 subunit of pertussis toxin homologous to cholera toxin, causes loss of enzymatic activity. Outside this homology region, substitution of Glu-129 with glycine or aspartic acid also eliminates the enzymatic activity of the S1 subunit. In this respect, Glu-129 resembles the glutamic acid that is crucial for the catalytic activity of diphtheria and Pseudomonas toxins. Once introduced into the Bordetella pertussis chromosome, the above mutations should lead to the synthesis of nontoxic pertussis toxin molecules suitable for vaccine production.

Amino Acid Sequence↗
PubMed1988

Cloning of a novel pilin-like gene from Bordetella pertussis: homology to the fim2 gene.

A search for pilin genes in a Bordetella pertussis (Bp) genomic library has led to the identification of several clones which hybridize to synthetic oligonucleotides with sequences derived from amino acid sequences of Bp fimbrial subunits. One of these clones (corresponding to a gene we have named fimX) contains an open reading frame encoding a protein with a molecular weight of about 20 kD and a sequence similar but not identical to the fimbrial subunit fim2 and to other fimbrial protein sequences. In this communication we present the cloning and nucleotide sequence of the fimX gene and its homology to the fim2 gene. A genomic analysis on the positional relationship between the two genes is also presented.

Amino Acid Sequence↗
PubMed1988

Cytoskeletal alterations as a parameter for assessment of toxicity.

1. Some environmental substances, drugs and pollutants affect the growth of cultured cells, and produce cytoskeletal alterations. 2. These have been used as parameters for toxicity assessment of cholera toxin and pertussis toxin in Chinese Hamster Ovary cells. 3. Cholera toxin stabilized microtubules and had no effect on microfilaments and intermediate filaments. 4. Pertussis toxin affected microfilaments but appeared to have no effect on microtubules and intermediate filaments.

Animals↗
PubMed1988

Engineering bacterial toxin for the development of new vaccine against pertussis.

Bordetella pertussis is the causative agent of whooping cough. The cellular pertussis vaccine introduced in the forties is highly effective and is widely used, but its reactogenicity has led to public concern regarding its safety. The attempts to reduce the side effects associated with pertussis immunization have led to the preparation of acellular B. pertussis products: one composed of detoxified pertussis toxin (PT) and filamentous haemagglutinin (FHA), another one composed only of detoxified PT and a third vaccine composed of detoxified PT, FHA and serotypes 1, 3 of fimbriae. In our laboratories we have approached the study of pertussis toxin, the molecule present in all the proposed acellular pertussis vaccines and one of the main virulence factors of B. Pertussis, with the aim of producing new acellular pertussis vaccines by using recombinant DNA techniques.

Antibodies, Monoclonal↗
PubMed1988

Human T-cell immunity against Bordetella pertussis analyzed at clonal level.

Cloned peripheral blood T lymphocytes from an immune donor were grown in interleukin 2 and tested for proliferation in response to inactivated Bordetella species (B. pertussis, B. parapertussis and B. bronchiseptica) and mutants deficient for the expression of virulence-associated antigens. All the T-cell clones obtained were CD4+8- and recognized specifically the Bordetella antigens when presented by autologous B cells. On the basis of the responsiveness to the whole inactivated bacteria, it was possible to cluster the twelve clones obtained into four groups with the following specificity: 1) filamentous hemagglutinin (FHA): 2) B. pertussis specific antigens; 3) virulence-associated Bordetella specific antigens; 4) non-virulence-associated Bordetella specific antigens. Employing two new B. pertussis deletion mutants, clone 6 (representative of cluster 1), was found to recognize the C-terminus of FHA. Furthermore, 3 out of 4 clones of cluster 3 were specifically stimulated by the soluble 69,000 MW protein from the outer membrane of B. pertussis. Surprisingly, none of the twelve clones obtained by stimulation in vitro with whole inactivated bacteria recognized PT. Thus, PT does not seem to be the most representative antigen on the whole inactivated bacteria. However, when a new generation of clones was obtained using soluble PT as the in vitro stimulus, it was observed that 11 clones of this group recognized this antigen. Furthermore, the majority of them was against the subunit S1 of PT. Therefore, we can conclude that a T-cell memory against PT exists in a donor who has had pertussis several years before. In conclusion, these results provide useful information in the attempt to obtain a simplified acellular vaccine for whooping cough.

Antigens, Bacterial↗
PubMed1987

Lipid interaction of diphtheria toxin and mutants with altered fragment B. 1. Liposome aggregation and fusion.

The interaction of diphtheria toxin and its cross-reacting mutants crm 45,228 and 1001 with small unilamellar vesicles has been followed by a turbidity assay, electron microscopy, fluorescence energy transfer and membrane permeability. All toxins at pH lower than 6 induce the aggregation and fusion of liposomes containing negatively charged phospholipids; crm 45 and crm 1001 are less potent than diphtheria toxin. Isolated diphtheria toxin fragment B is very effective while isolated fragment A is ineffective. Liposome fusion induced by the toxins at low pH occurs without release of the internal content implying that fusion does not involve vesicle breakage and resealing. The pH dependence of the membrane interaction of diphtheria toxin monitored by turbidity is in close agreement with that monitored by fluorescence energy transfer. It shows that diphtheria toxin can alter the lipid bilayer structure in the pH interval 5-6. This pH range occurs in endosomes and suggests that histidyl and carboxyl residues are likely to be involved in the conformational change of diphtheria toxin triggered by acidic pH.

Diphtheria Toxin↗
PubMed1987

Lipid interaction of diphtheria toxin and mutants with altered fragment B. 2. Hydrophobic photolabelling and cell intoxication.

The membrane insertion of diphtheria toxin and of its B chain mutants crm 45, crm 228 and crm 1001 has been followed by hydrophobic photolabelling with photoactivatable phosphatidylcholine analogues. It was found that diphtheria toxin binds to the lipid bilayer surface at neutral pH while at low pH both its A and B chains also interact with the hydrocarbon chains of phospholipids. The pH dependence of photolabelling of the two protomers is different: the pKa of fragment B is around 5.9 while that of fragment A is around 5.2. The latter value correlates with the pH of half-maximal intoxication of cells incubated with the toxin in acidic mediums. These results suggest that fragment B penetrates into the bilayer first and assists the insertion of fragment A and that the lipid insertion of fragment B is not the rate-controlling step in the process of membrane translocation of diphtheria toxin. crm 45 behaves as diphtheria toxin in the photolabelling assay but, nonetheless, it is found to be three orders of magnitude less toxic than diphtheria toxin on acid-treated cells, suggesting that the 12-kDa COOH-terminal segment of diphtheria toxin is important not only for its binding to the cell receptor but also for the membrane translocation of the toxin. It is suggested that crm 1001 is non-toxic because of a defect in its membrane translocation which occurs at a lower extent and at a lower pH than that of the native toxin; as a consequence crm 1001 may be unable to escape from the endosome lumen into the cytoplasm before the fusion of the endosome with lysosomes.

Animals↗
PubMed1987

Conformational changes in diphtheria toxoids. Analysis with monoclonal antibodies.

Monoclonal antibodies (Mab) were raised against CRM197, a non-toxic mutant of diphtheria toxin (DT). The ability of four Mabs to bind DT and the six functional mutants CRM197, CRM176, CRM228, CRM1001, CRM45 and CRM30 was assessed by immunoblotting and by a radioimmunoassay in which the protein antigen in solution competes with labeled CRM197 for the Mab binding site. The results show that the peptides recognized by Mab11.3, Mab53 and Mab23 are accessible in the mutant molecules in solution but not when they are part of the native DT structure, which could therefore be described for this purpose as 'closed' in contrast with an 'open' conformation of CRM197, CRM176 and CRM228. In particular, the behaviour of Mab53 indicates that the single amino acid substitutions in the A fragments of CRM197 and CRM176 also affect the conformation of their B fragments.

Amino Acid Sequence↗
PubMed1987

Diphtheria toxin and its mutant crm 197 differ in their interaction with lipids.

The interaction of diphtheria toxin and its enzymatically deficient mutants crm 176 and crm 197 with liposomes has been studied by turbidity measurement and hydrophobic photolabelling with photoactivatable phosphatidylcholines. Diphtheria toxin and crm 176 at neutral pH bind to the surface of lipid bilayers while crm 197 also appears to interact with the fatty acid chains of phospholipids. All proteins undergo a change in conformation over the same range of acidic pH and become able to insert in the lipid bilayer. The tighter lipid interaction of crm 197 may account for its higher cell association constant. The possibility is discussed that the binding of diphtheria toxin to cells is mediated by both a protein receptor and an interaction with the head group of phospholipids.

Diphtheria Toxin↗
PubMed1987

Evolutionary relationships in the genus Bordetella.

The nucleotide sequence of the pertussis toxin operon of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica, has shown that the last two species contain many common mutations and are likely to derive from a common ancestor (Aricò and Rappuoli, 1987). To elucidate further the evolutionary relationships between the Bordetella species, we have cloned and sequenced the promoter region and the gene coding for the S1 subunit of pertussis toxin from additional B. pertussis strains, such as the type strain BP 18323 and two recent clinical isolates, namely strain BP 13456 from Sweden and strain BP SA1 from Italy. While the strains BP SA1 and BP 13456 are shown to differ from the published B. pertussis sequences by only one base pair, the type strain BP 18323 contains a total of 11 base-pair substitutions. Remarkably, 9 of the 11 substitutions found in BP 18323 are also common to B. parapertussis and B. bronchiseptica, strongly suggesting that this strain derives from the same ancestor as B. parapertussis and B. bronchiseptica. Computer analysis of the sequence data allows the construction of an evolutionary 'tree' showing that the B. pertussis strains are very homogeneous and significantly distant from B. parapertussis and B. bronchiseptica. Therefore the proposed conversion from B. parapertussis to B. pertussis appears highly improbable.

Amino Acid Sequence↗
PubMed1987

Expression and immunological properties of the five subunits of pertussis toxin.

Pertussis toxin, a protein composed of five different subunits, is responsible for the pathogenicity of Bordetella pertussis and is the main component of a new vaccine against whooping cough. The genes coding for the five subunits, recently cloned and sequenced, are organized as an operon. We approached the problem of expression of the five genes in Escherichia coli and, although we obtained high levels of transcription of the native pertussis toxin genes, the amount of proteins produced was very low or undetectable. To obtain suitable expression of each of the five subunits, we fused their genes to the gene coding for the DNA polymerase of MS2 in the expression vector pEx31. A total of 5 to 30 mg of purified fusion proteins could be obtained from 1 liter of culture. The purified fusion proteins were used to immunize rabbits to obtain sera against each of the five subunits. These sera, although able to recognize the toxin in an enzyme-linked immunosorbent assay and the corresponding subunits in Western blots, were not able to protect CHO cells from the action of pertussis toxin. Mice immunized with the five subunits were not protected from an intracerebral challenge with B. pertussis. Subunits S2 and S3, which are 67% homologous, were shown to cross-react immunologically. The fused subunit S1 was able to ADP-ribosylate transducin as efficiently as the native pertussis toxin.

Adenosine Diphosphate Ribose↗
PubMed1987

Expression of the S-1 catalytic subunit of pertussis toxin in Escherichia coli.

The S-1 subunit of pertussis toxin was expressed as a fusion protein in a strain of Escherichia coli deficient in protein degradation. The fusion protein reacted with anti-pertussis toxin antibody, and, like authentic pertussis toxin, it ADP-ribosylated a 41,000-molecular-weight membrane protein from human erythrocytes.

Bordetella pertussis↗
PubMed1987

DNA element of Corynebacterium diphtheriae with properties of an insertion sequence and usefulness for epidemiological studies.

The segment of DNA which is inserted within the tox gene of bacteriophage gamma and is responsible for its Tox- phenotype was found to be present and repeated approximately 30 times in the chromosome of Corynebacterium diphtheriae Belfanti 1030. Other C. diphtheriae strains contained a variable number of copies (1 to 25) of the same element. Sequence analysis showed that this repeated and interspersed DNA element was flanked by 9-base-pair direct repeats and that the 5' and 3' ends of the insertion contained sequences forming an imperfect inverted repeat. Therefore, the DNA segment here described has most of the typical structural features of a bacterial insertion sequence element. We show that different C. diphtheriae isolates derived from the same outbreak of diphtheria have an identical genomic distribution of this DNA element and that such DNA can be useful for epidemiological studies.

Base Sequence↗
PubMed1987

High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli.

ABM508 is a recombinant fusion protein consisting of the N-terminal 485 amino acids of diphtheria toxin joined to alpha-melanocyte-stimulating hormone. When expressed in Escherichia coli under the control of the tox promoter and signal sequence, ABM508 is severely degraded. When overexpressed from a thermoinducible lambda pR promoter fusion, ABM508 is largely insoluble. We compared the expression of ABM508 (501 amino acids) to a full-length mutant form of the toxin (CRM197; 535 amino acids) and found that CRM197 showed minimal proteolysis. Thus, the removal of the C-terminal 50 amino acids of the toxin destabilizes the protein, making it a target for proteases. Proteolysis of ABM508 could be reduced by removal of the tox signal sequence (thereby directing the protein to the cytoplasm) and growth in lon and htpR mutant strains of E. coli. We also showed that the solubility of tox gene products expressed in E. coli was directly related to the growth temperature of the culture. Thus, a fragment A fusion protein (223 amino acids), ABM508, and CRM197 were found in soluble extracts when expressed at 30 degrees C but could not be released by the same procedures after growth at 42 degrees C. On the basis of these observations, we fused the coding sequences for mature ABM508 to the trc promoter (inducible at 30 degrees C by isopropyl-beta-D-thiogalactoside) and expressed this construct in a lon htpR strain of E. coli. This plasmid made 10 mg of soluble tox protein per liter of culture (7.7% of the total cell protein) or 14 times more than our previous maximal level. Extracts from lon htpR cells harboring this plasmid had high levels of ADP-ribosyltransferase activity, and although proteolysis still occurred, the major tox product corresponded to full-length ABM508.

Amino Acid Sequence↗