1,721,387 research outputs found
ADITEC: joining forces for next-generation vaccines
No full textScientists sit poised at a singular moment in the history of vaccine research. Genomics and systems biology have fueled advances in our understanding of human immunology. Together with adjuvant development and structure-based design of immunogens, these next-generation technologies are transforming the field of vaccinology and shaping the future of medicine. However, the sophisticated science behind the development of modern vaccines and the resulting knotty ethical issues have become so complex that scientists and policy-makers need a new model for vaccine research. The European Commission-sponsored Advanced Immunization Technologies project--ADITEC--brings together some of the leading laboratories in the field to tackle the problems that no lab can tackle in isolatio
Differential response of the bvg virulence regulon of Bordetella pertussis to MgSO4 modulation
No full textMagnesium sulfate is known to repress the expression of the virulence factors of Bordetella pertussis that are coordinately regulated by the bvg locus. We have tested the time required by MgSO4 to repress the synthesis of several bvg-regulated mRNA species and found that the promoters of the virulence genes (pertussis toxin, adenylate cyclase, and filamentous hemagglutinin) are repressed in 6 min, while the autogenously regulated promoters of the bvg locus (P1, P3, and P4) are repressed only several hours later. These data show a differential behavior between regulated and autoregulated genes of the bvg regulon
The pertussis toxin liberation genes of Bordetella pertussis are transcriptionally linked to the pertussis toxin operon
No full textThe DNA sequence of the pertussis toxin operon (ptx) of Bordetella pertussis predicts that transcription of the operon ends downstream from the ptxS3 gene at a possible stem-loop structure. Secretion of the assembled pertussis toxin into the culture medium requires the expression of 8 genes arranged in an operon (ptl) and lying 55 bp downstream from the ptx operon. We have investigated the rule of this 55-bp intergenic region in transcriptional regulation of the ptl operon. Our results show that ptx transcripts transverse this DNA region and that the ptx and ptl operons are cotranscribed and coregulated by the P(TOX) promoter. Deletion of the 55-bp DNA region caused an increase in the amount of the ptl transcripts. It is likely that this DNA region is involved in regulation of the ptx-ptl expression
A common conserved amino acid motif module shared by bacterial and intercellular adhesins: Bacterial adherence mimicking cell-cell recognition?
No full textNot availabl
DNA topology affects transcriptional regulation of the pertussis toxin gene of Bordetella pertussis in Escherichia coli and in vitro
No full textThe bvg locus of Bordetella pertussis encodes an environmentally inducible operon essential for the expression of virulence genes. We show that in Escherichia coli, the P(TOX) promoter cloned in cis of the bvg locus is activated and environmentally regulated. Cotransformation of E. coli with the bvg locus cloned in a low-copy-number plasmid and with the P(TOX) promoter cloned in a high-copy-number plasmid can give rise to two different results. If the P(TOX) promoter is cloned in the pGem-3 vector, transcription is absent. If the P(TOX) promoter is cloned in the plasmid pKK232, containing the P(TOX) promoter between two ribosomal gene terminators of transcription, transcription occurs, although regulation of transcription is abolished. Under these conditions, the intracellular amount of RNA transcripts is increased by adding to the culture medium novobiocin, an inhibitor of bacterial gyrases. In vitro, the transcription of the P(TOX) promoter is activated on E. coli RNA polymerase supplemented with cell extracts from wild-type B. pertussis. Addition of DNA gyrase to the mixture dramatically reduces the amount of RNA synthesized. Our data show that the products of the bvg locus, BvgA and BvgS, are directly involved in the regulation of the P(TOX) promoter in E. coli and that DNA topology may play a role in the induction of transcription
THE REGULATION OF THE PERTUSSIS TOXIN GENE OF BORDETELLA-PERTUSSIS IN ESCHERICHIA-COLI AND IN-VITRO IS SENSITIVE TO DNA TOPOLOGY
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MOLECULAR CHARACTERISATION OF A NOVEL ADP-RIBOSYLATING PUTATIVE TOXIN OF NEISSERIA MENINGITIDIS
Full text linkMolecular characterisation of a novel ADP-ribosylating putative toxin of Neisseria meningitidis
VEGGIi D, *BALDUCCI E, MASIGNANI V, DI MARCELLO F, SAVINO S, ARICO’ B, COMANDUCCI M, PIZZA M, RAPPUOLI R
IRIS, Chiron SpA, Via Fiorentina 1, 53100 Siena Italy; *Dipartimento Scienze morfologiche e Biochimiche Comparate, Università degli Studi di Camerino, Camerino, Italy
Session: Surface antigens
Introduction: By computer analysis on the Neisseria meningitidis (serogroup B, MC 58 strain) genome sequence, a protein with a feature similar to known bacterial ADP-ribosylating toxins (CT produced by Vibrio cholerae, LT by Escherichia coli and PT by Bordetella pertussis) has been identified. Enzymatic assay has shown that this protein (NM-ADPRT) possesses both NAD glycohydrolase and ADP-ribosyltransferase activity. In this study we describe the identification of the putative catalytic residues, their site-directed mutagenesis, and the resulting activity of the mutants.
Materials and methods: The novel NM-ADPRT and the correspondent mutants, were expressed in E. coli as C-terminus His-tag protein fusions. Site-directed mutagenesis was performed using the Multi Site-Directed Mutagenesis Kit (QuikChange). Recombinant NM-ADPRT forms were purified from E. coli in their soluble form by metal chelate affinity chromatography.
Both the wild-type and the mutants were assayed for their ADP-ribosylation and NAD-glycohydolase activites, using [adenine –U-14C] NAD and agmatine as ADP-ribose acceptor. Antisera against NM-ADPRT and the mutant derivatives were obtained by immunization of CD1 mice. 20μg of each recombinant protein were given i.p. together with CFA for the first dose and IFA for the second (day 21) and the third (day 35) booster doses. Blood sample were taken on days 34 and 49. Immune sera were used in western blot and tested in a bactericidal assay.
Results and discussion: On the basis of sequence homology of NM-ADPRT with LT, CT and PT we have identified the putative residues involved in enzymatic activity. These residues have been changed by site-directed mutagenesis and the purified mutant toxins have been tested for both ADP-ribosylating and NAD-glycohydrolase activities. Interestingly, some of the mutants show reduced or abolished enzymatic activity indicating that the identified residues play a role in catalysis. Antisera against the wild-type and mutant toxins have bactericidal activity. The titers induced by two mutants were higher than those induced by the wild-type form. These data suggest that the mutations introduced could influence not only the enzymatic activity but also the in vivo stability of the toxin.
Conclusion: A novel ADP-ribosyltransferase has been identified in meningococcus B. Catalytic residues have been predicted by sequence homology and their role in catalysis has been confirmed by site-directed mutagenesis. These molecules are also able to induce a bactericidal response
DETERMINATION OF DIPHTHERIA-TOXIN NEUTRALIZING ANTIBODY-TITERS WITH A CELL PROTEIN-SYNTHESIS INHIBITION ASSAY
No full textA method which allows an accurate determination of very low titers of antibodies able to neutralize diphtheria toxin has been developed. The assay is based on the incubation of a reference amount of diphtheria toxin with different dilution of the serum and the evaluation of the residual toxicity of diphtheria toxin on Vero cells. The cells are seeded in 96-well plastic plates with the toxin-serum mixtures and the residual toxin activity is measured as the block of cell protein synthesis after incubation with [14C]leucine. Antitoxin titers as low as 0.002 IU can be reproducibly and accurately measured and the method gives a linear response in the range 0.002 to 8.0 IU/ml
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