Positive transcriptional feedback at the bvg locus controls expression of virulence factors in Bordetella pertussis.
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Base Sequence↗
Biomedical subjects
R Rappuoli
Publications and source records attributed to R Rappuoli.
Genetic characterization of Bordetella pertussis filamentous haemagglutinin: a protein processed from an unusually large precursor.
The nucleotide sequence of the structural gene for filamentous haemagglutinin (FHA), fhaB, a crucial adherence factor for Bordetella pertussis, has been determined. Its 10774 nucleotides are far more than necessary to encode the 220 kD biologically active, mature polypeptide product, suggesting a role for co- or post-translational processing. Fusion proteins derived from various portions of the fhaB open reading frame (ORF) were used to generate polyclonal antisera. Western immunoblot analysis of purified FHA and Bordetella sp. whole cell extracts with these antisera indicated that the 220 kD product is encoded by the 5' portion of the ORF and that the smaller polypeptide species are breakdown products of this polypeptide. These data, as well as N-terminal amino acid sequencing of the major polypeptide species, suggest a scheme for the proteolytic processing of an FHA precursor polypeptide.
Adhesins, Bacterial↗
Identification of a 35-kilodalton Mycobacterium tuberculosis protein containing B- and T-cell epitopes.
Screening of a Mycobacterium tuberculosis genomic DNA library in the lambda gt11 expression vector was carried out by using, as probes, sera from tuberculous patients and murine monoclonal antibody H61.3 recognizing a mycobacterial 35-kilodalton protein present only on the M. tuberculosis complex. The recombinant beta-galactosidase-fused protein present in the crude lysate induced the proliferation of T lymphocytes from patients with tuberculous pleuritis. As the recombinant insert contains an internal EcoRI restriction site, it was possible to identify two fragments, one proximal to the lacZ gene and 1.7 kilobases (kb) in size and the other distal to the lacZ gene and 2.2 kb in size. Southern blot analysis showed that both of them hybridized with the genomic DNA from M. tuberculosis and M. bovis but not with the DNA from other mycobacterial species. To perform extensive immunological studies, the amount of beta-galactosidase-fused protein being very low, we fused the 1.7-kb fragment to the N-terminal part of the gene coding for the DNA polymerase of bacteriophage MS2 in the expression vector pEx34. The fusion protein was partially purified, and subsequent Western blotting (immunoblotting) and T-cell proliferation experiments confirmed the presence of B- and T-cell mycobacterial epitopes. Furthermore, to isolate the chromosomal region containing the 35-kilodalton gene, we constructed another mycobacterial genomic library in the lambda 2001 vector by cloning 15 to 20 kb of foreign DNA. Screening of this library was carried out by using 1.7- and 2.2-kb recombinant fragments as probes. Restriction maps of some clones isolated were determined.
Antigens, Bacterial↗
Biochemical and biological activities of recombinant S1 subunit of pertussis toxin.
degP-deficient strains of Escherichia coli grown in M-9 medium supplemented with ZnCl2 expressed the recombinant S1 subunit of pertussis toxin (rS1) in a form electrophoretically identical to the authentic S1 subunit. Subcellular fractionation showed that the full-length form of rS1 was membrane associated, while proteolytic fragments of rS1 were present in the periplasm. rS1 was extracted from outer membrane preparations with 8 M urea and purified by gel filtration chromatography. Purified rS1 ADP-ribosylated transducin at a similar molar efficiency relative to authentic pertussis toxin and, when associated with the native B oligomer of pertussis toxin, elicited Chinese hamster ovary cell clustering.
Cells, Cultured↗
Histidine 21 is at the NAD+ binding site of diphtheria toxin.
Treatment of fragment A chain of diphtheria toxin (DT-A) with diethylpyrocarbonate modifies His-21, the single histidine residue present in the chain, without alteration of other residues. Parallel to histidine modification, NAD+ binding and the NAD-glycohydrolase and ADP-ribosyltransferase activities of DT-A are lost. Both NAD+ and adenosine are very effective in protecting DT-A from histidine modification and in preserving its biological properties, while adenine is ineffective. Reversal of histidine modification with hydroxylamine restores both NAD+ binding and enzymatic activities of the toxin. The possible role of His-21 in the activity of diphtheria toxin is discussed in relation to the available three-dimensional structure of the related toxin produced by Pseudomonas aeruginosa.
Adenine↗
Human T cell clones define S1 subunit as the most immunogenic moiety of pertussis toxin and determine its epitope map.
Human T lymphocyte clones specific for pertussis toxin (PT) were used to analyze the fine specificity of the response to PT, the basic component of new acellular vaccines against whooping cough. The majority (83%) of the clones specific for PT recognized S1, the subunit that in animal models has been shown to be highly immunogenic. To map T cell epitopes on S1, 18 S1-specific clones were tested for recognition of recombinant fragments representing NH2-terminal and COOH-terminal deletions of S1 and two recombinant S1 subunits containing amino acid substitutions. This approach led to the identification of three regions of the protein as the sequences containing T cell antigenic sites: 1-42, 181-211, and 212-235. Synthetic peptides were eventually used for a finer localization of the T cell epitopes. Two peptides, one of 13 residues (27-39) at the NH2 terminus and one of 24 residues (171-194) at the COOH terminus, stimulated proliferation of three and four clones, respectively. Both peptides are recognized in association with HLA DR1 molecules. These results stress the role of S1 in the immune response to PT and provide data useful for the development of a recombinant or synthetic antipertussis vaccine containing T cell epitopes from S1.
Amino Acid Sequence↗
Selective immunotoxins prepared with mutant diphtheria toxins coupled to monoclonal antibodies.
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Animals↗
Studies of the antigenic structure of two cross-reacting proteins, pertussis and cholera toxins, using synthetic peptides.
Peptide fragments of pertussis toxin subunit 1 (PT-S1) have been synthesized in order to investigate their antigenic and immunogenic activity, and to evaluate their possible use as components of a new vaccine. Two peptides (sequence 73-82, EAERAGRGTG and sequence 107-116, YVDTYGDNAG) were selected for their predictable exposure on the surface of the molecule, and a third (8-18, YRYDSRPPEDV) for its homology with the sequence 6-16 of cholera toxin subunit A (CT-A 6-16) (YRADSRPPDEI). Antipeptide polyclonal antibodies produced in rabbits, were tested in different immunoassays for their ability to interact with toxin proteins. All of them proved interactive with recombinant PT-S1 (rPT-S1); CT-A interact not only, as expected, with anti 8-18 antibodies, due to the high homology between the two toxins in this region, but also, unexpectedly, with anti 107-116 antibodies, in spite of the lack of homology of this peptide with the entire CT. We also found a direct cross-reactivity between the two toxins: anti PT and anti rPT-S1 antibodies interacted with CT-A, whereas anti CT antibodies did not recognize PT. Antipertussis antibodies also recognized the peptide 8-18, which therefore represents at least a part of an antigenic determinant of the toxin, while no interaction could be evidenced between anti-cholera antibodies and any of the peptides, thus demonstrating important differences in the antigenic structures of the two toxins. None of the antipeptide antibodies examined showed protective activity against the toxins in a Chinese hamster ovary (CHO) cell test.
Amino Acid Sequence↗
Sequences required for expression of Bordetella pertussis virulence factors share homology with prokaryotic signal transduction proteins.
The bvg locus of Bordetella pertussis is required for coordinate regulation of several factors associated with virulence. The control system is modulated by various environmental signals, including low temperature, MgSO4, and nicotinic acid. The nucleotide sequence of the bvg region has been determined and three open reading frames, bvgA, bvgB, and bvgC, are present. Twelve-base-pair linker insertion mutations in any of these open reading frames result in a Bvg- phenotype. The predicted protein products of bvgA and bvgC share homology with a family of prokaryotic regulatory proteins that respond to environmental stimuli and are members of two-component sensory transduction systems. We propose a model in which BvgB and the N-terminal portion of BvgC are localized in the periplasm. Environmental signals are recognized, transduced to the cytoplasmic portion of BvgC, and then transmitted to BvgA, a positive regulator of transcription.
Amino Acid Sequence↗
Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence.
Filamentous hemagglutinin is a surface-associated adherence protein of Bordetella pertussis, which is a component of some new acellular pertussis vaccines. The nucleotide sequence of an open reading frame that encompasses the filamentous hemagglutinin structural gene, fhaB, suggests that proteolytic processing is necessary to generate the mature 220-kDa filamentous hemagglutinin product. An Arg-Gly-Asp (RGD) tripeptide is found within filamentous hemagglutinin that may be involved in its adherence properties. An internal in-frame deletion in fhaB, encompassing the RGD region, causes loss of B. pertussis-binding to ciliated eukaryotic cells, confirming a potential role for this protein in host-cell binding and infection.
Amino Acid Sequence↗
Genetics of pertussis toxin.
Pertussis toxin (PT) is the major virulence factor of Bordetella pertussis. The cloning and nucleotide sequencing of the PT genes from B. pertussis, Bordetella parapertussis and Bordetella bronchiseptica has elucidated the evolution of the Bordetella species and allowed considerable advances towards the understanding of their gene expression and the development of safer vaccines against pertussis.
Base Sequence↗
Families of bacterial signal-transducing proteins.
Bacteria can respond to a variety of environmental stimuli by means of systems generally composed of two proteins. The first protein (sensor or transmitter) is usually a transmembrane protein with cytoplasmic and extracytoplasmic domains. The extracytoplasmic domain (sensor) senses the environment and transfers the signal through the transmembrane domain to the cytoplasmic domain (transmitter), which has kinase activity. The second protein is located in the cytoplasm and contains an amino-terminal domain (receiver), which can be phosphorylated by the transmitter, and a carboxy-terminal region (regulator), which regulates gene expression by binding to DNA. The transmitter and receiver modules (the kinase and its target) are conserved in all signal-transducing systems and are the 'core structure' of this two-component system. The sensors and the regulators vary according to the stimuli they respond to and the DNA structure they interact with. On the basis of their sequence homology, the proteins belonging to such two-component systems can be classified into different families, which are summarized in this review.
Base Sequence↗
Phase variants of Bordetella bronchiseptica arise by spontaneous deletions in the vir locus.
Bordetella bronchiseptica is a common respiratory tract pathogen of many mammalian species. Nucleotide sequences from the locus involved in coordinate regulation of B. pertussis virulence factors, vir, were shown to have a high degree of homology to chromosomal DNA from virulent (Vir+) and avirulent (Vir-) strains of B. bronchiseptica. Small deletions, 50 bp to 500 bp, within the vir locus were found in some of the Vir- phase variants. The vir locus and the adjacent 5' portion of the fhaB structural gene were cloned from the parental Vir+ B. bronchiseptica strain on a 23.5 kb BamHI fragment. Restriction enzyme mapping of the cloned B. bronchiseptica vir locus revealed similarities with and differences from the previously cloned B. pertussis vir locus. The cloned B. bronchiseptica vir locus complemented spontaneous Vir- variants of Bordetella pertussis and B. bronchiseptica as well as vir::Tn5 mutants of B. pertussis. Comparison of various functions of the vir loci of B. bronchiseptica and B. pertussis revealed some interesting differences in the coordinate regulation of virulence factors.
Bordetella↗
Photolabeling of Glu-129 of the S-1 subunit of pertussis toxin with NAD.
UV irradiation was shown to induce efficient transfer of radiolabel from nicotinamide-labeled NAD to a recombinant protein (C180 peptide) containing the catalytic region of the S-1 subunit of pertussis toxin. Incorporation of label from [3H-nicotinamide]NAD was efficient (0.5 to 0.6 mol/mol of protein) relative to incorporation from [32P-adenylate]NAD (0.2 mol/mol of protein). Label from [3H-nicotinamide]NAD was specifically associated with Glu-129. Replacement of Glu-129 with glycine or aspartic acid made the protein refractory to photolabeling with [3H-nicotinamide]NAD, whereas replacement of a nearby glutamic acid, Glu-139, with serine did not. Photolabeling of the C180 peptide with NAD is similar to that observed with diphtheria toxin and exotoxin A of Pseudomonas aeruginosa, in which the nicotinamide portion of NAD is transferred to Glu-148 and Glu-553, respectively, in the two toxins. These results implicate Glu-129 of the S-1 subunit as an active-site residue and a potentially important site for genetic modification of pertussis toxin for development of an acellular vaccine against Bordetella pertussis.
Cyanogen Bromide↗
Pertussis toxin promoter sequences involved in modulation.
Previous analysis of the pertussis toxin (PT) promoter has shown that expression of PT requires a trans-activating factor encoded by the vir locus and a 170-base-pair DNA sequence upstream from the transcription start site containing a 21-base-pair direct repeat sequence crucial trans-activation (R. Gross and R. Rappuoli, Proc. Natl. Acad. Sci. USA 85:3913-3917, 1988). In this paper we extend the analysis to the modulative response to environmental stimuli. We show that modulation acts at the transcriptional level and occurs only in phase I bacteria. Modulation also requires a functional vir locus and the same promoter region of 170 base pairs. We show that, in addition to the previously identified direct repeat, even the sequences downstream from position -117 are required for trans-activation and modulation and that the deletion of four cytosine residues at position -31 causes the inactivation of the promoter. The kinetics of the change in transcription show that the PT promoter can be shut off very rapidly by adding 50 mM MgSO4 to the medium, whereas resumption of transcription after removal of the modulative agents from the medium is slow.
Base Composition↗
Dissecting human T cell responses against Bordetella species.
To identify the minimal structures that may be important for the creation of a synthetic and/or recombinant vaccine against whooping cough, human T cell clones were obtained against Bordetella antigens. Cloned peripheral blood T lymphocytes from an immune donor were grown in IL-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 12 clones obtained into four groups with the following specificity: (1) filamentous hemagglutinin (FHA); (2) B. pertussis-specific antigens; (3) virulence-associated Bordetella-specific antigens; and (4) nonvirulence-associated Bordetella-specific antigens. Using two new B. pertussis deletion mutants, clone 6 (representative of cluster 1) was found to recognize the COOH terminus of FHA. Furthermore, three out of four clones of cluster 3 were specifically stimulated by the soluble 69-kD protein from the outer membrane of B. pertussis. Surprisingly, none of the twelve clones obtained by stimulation in vitro with whole inactivated bacteria recognized pertussis toxin (PT), which is believed to be the most important protein to be included in an acellular vaccine. 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. Thus, PT does not seem to be the most representative antigen on the whole inactivated bacteria, although T cell memory against PT exists in a donor who had the disease several years ago.
Adult↗
A C terminus cysteine of diphtheria toxin B chain involved in immunotoxin cell penetration and cytotoxicity.
The role of diphtheria toxin (DT) B-chain subdomains in DT cytotoxicity and immunotoxin mechanism of action has been investigated. OKT3 (mAb to the CD3 surface Ag of human T lymphocytes) was conjugated to DT or the DT mutant CRM 1001, which has a cys----tyr substitution at position 471 of the B chain. OKT3-CRM 1001 immunotoxin was about 1400-fold less cytotoxic for CD3 Jurkat cells than OKT3-DT and had a 12-fold slower kinetics of protein synthesis inactivation, CRM 1001 killed DT-sensitive Vero cells at a 5000-fold higher concentration than DT. Its cell surface-binding activity was comparable to DT. Based on kinetics of cell inactivation, toxicity determination at low extracellular pH and Triton X-114 distribution, it was concluded that CRM 1001 is defective in at least one crucial step of toxin penetration and is unable to cross cell membranes as efficiently as DT. The substituted cysteine appears to be important for DT translocating functions. Data on the function of DT B-chain subdomains are relevant for the study of whole toxin conjugates and their mechanism of action.
Animals↗
Molecular epidemiology of the 1984-1986 outbreak of diphtheria in Sweden.
Despite mass vaccination against diphtheria, many people have antibody titers below the protective level of 0.01 IU per milliliter. A recent outbreak of diphtheria in Sweden caused 17 clinical cases of diphtheria in the city of Göteborg; three of the patients died. A satellite outbreak occurred in Stockholm after a few months' delay. Using a new genetic probe, we analyzed 36 strains of Corynebacterium diphtheriae isolated in Sweden and Denmark during the period 1976 to 1986. Although the 36 strains can be classified in 17 different groups of C. diphtheriae (several of them containing toxigenic strains), all the clinical and fatal cases of diphtheria were caused by isolates from the same group, strongly suggesting that the outbreak in Sweden was caused by a single strain that possibly had a virulence factor separate from toxigenicity. This strain may have been imported into Sweden from Denmark, since it was isolated for the first time in Copenhagen in 1983, before the outbreak in Sweden.
Corynebacterium diphtheriae↗