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An intracellular target for Helicobacter pylori vacuolating toxin - Response from de Bernard and Arico
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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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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
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
Sequential activation and environmental regulation of virulence genes in Bordetella pertussis
No full textBacterial pathogens undergo profound physiological changes when they infect their host and require coordinated regulation of gene expression in response to the stress encountered during infection. In Bordetella pertussis, the human pathogen which causes whooping cough, virulence factors are synthesized in response to environmental signals under the control of the bvg regulatory locus. Here we demonstrate that the bvg locus is responsible for two events of gene activation. In the first step the bvg locus transactivates its own autoregulated promoter (P1) and the promoter of the adherence factor filamentous haemagglutinin (P(FHA)). The second step occurs several hours later and consists of the transactivation of adenylate cyclase and pertussis toxin genes. We provide evidence that the second step of transactivation requires overexpression of regulatory proteins. Our results imply that bacterial adhesion and tissue colonization-intoxication are two separate steps at the molecular level
The bvg‐dependent promoters show similar behaviour in different Bordetella species and share sequence homologies
No full textThe expression of the virulence‐associated genes in Bordetella species is co‐ordinately regulated by the gene products encoded by the bvg locus. In Bordetella pertussis the expression of this locus is regulated by the P1, P2, P3 and P4 promoters which are located in a 350bp DNA fragment also containing the PFHA promoter. Here we report the transcriptional regulation of the bvg locus and the fha gene in Bordetella paraper‐tussis and a sequence analysis of the byg‐regulated promoters. The Pp1, Pp2, Pp4 and PpFHA promoters are indistinguishable, both in transcription initiation sites and environmental regulation, from the corresponding promoters of B. pertussis, while the Pp3 promoter is not active. Sequence homologies from nine bvg‐regulated promoters show a conserved dinucleotide, 5′‐TG‐3′, at approximately one turn of helix upstream of the ‐10 5′‐A.AaTat‐3’region, and a 5′‐TTTCC‐3’sequence in the ‐90 region. Since the nucleotide sequence of the inactive Pp3 promoter shows several base substitutions with respect to the found sequence homologies, it is likely that some of these bases play an essential role in promoter activity. Copyright © 1991, Wiley Blackwell. All rights reserve
Positive transcriptional feedback at the bvg locus controls expression of virulence factors in Bordetella pertussis
No full textRegulation of the genes coding for virulence factors in Bordetella pertussis is controlled by the bvg locus, which encodes one putative sensory protein (BvgS) and one positive regulator of transcription (BvgA). We have studied the transcription of the bvg locus and found that this is controlled by a 350-base-pair DNA fragment, which contains five promoters, three of which transcribe the bvg locus, one transcribes an antisense RNA, and one transcribes a virulence-associated gene. Under noninducing conditions, only the promoter P2 is active and this is responsible for the production of low amounts of regulatory proteins. Upon induction, the other four promoters become active and, by a mechanism that may involve transcriptional and translational regulation, cause a 50-fold increase of the transcriptional activator BvgA. A model of the autoregulation of the bvg locus is presented
Mutations in the linker region of BvgS abolish response to environmental signals for the regulation of the virulence factors in Bordetella pertussis
No full textExpression of virulence factors of Bordetella pertussis is coordinately regulated by the products of the bvg locus, which codes for a sensory protein (BvgS) and a positive regulator of transcription (BvgA), a pair in the family of bacterial 'two-component' regulators. Transcription of the bvg-regulated promoters is repressed by modulating environmental factors such as 50 mM MgSO4, 10 mM nicotinic acid (NA) or low temperature (25°C). We have isolated a spontaneous mutant (SK170) which expresses virulence genes at either 25°C, or in the presence of 1-5 mM NA, or 10-50 mM MgSO4. Virulence factors in strain SK170 are still repressed by higher concentrations of NA (10 mM), or by a combination of low temperature (25°C) and one of the other modulating agents. From this strain, we have isolated a second mutant (SK180) that showed constitutive synthesis of the virulence factors under any growth regime. Nucleotide (nt) and deduced amino acid (aa) sequence analysis showed that SK170 contains a substitution at aa570 of BvgS and SKI80 contains an additional substitution at aa680. These substitutions are confined to a 161-aa sequence that links the transmembrane (TM) and kinase domains of BvgS. These mutations also alter the transcriptional autoregulation of the p1 and P2 promoters of the bvg locus. P1 which in the wild-type (wt) strain is repressed by modulating agents, is constitutively active in the mutant strains. On the contrary, P2, which is normally induced by all three modulating agents, is active in strain SK170 only in the presence of MgSO4 or NA, while in strain SK180 this promoter is repressed by modulating agents. The mutants exhibit elevated levels of the BvgA regulatory protein and have a virulent phenotype also in the presence of modulating agents. © 1994
Erratum: Positive transcriptional feedback at the bvg locus controls expression of virulence factors in Bordetella pertussis (Proc. Natl. Acad. Sci. USA (September 1990) 87 (6753-6757))
No full textTHERMOREGULATION CONTROLS TWO STEPS OF VIRULENCE GENE-EXPRESSION IN BORDETELLA-PERTUSSIS
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