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<i>Science Translational Medicine</i> Podcast: 13 July 2011
No full textA conversation with Rino Rappuoli about engineering a single chimeric vaccine antigen that protects against all strains of meningococcus B.</jats:p
LIPOTEICHOIC ACID AND FLAGELLIN INDUCE UP-REGULATION OF ART1 INAIRWAY EPITHELIAL CELLS
No full textMono ADP-ribosylation, like phosporylation is a post-translational modification that alters key cellular events. This reaction is catalyzed by ADP-ribosyltransferases (ARTs), a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. The family of known ARTs consists of seven members, except for ART1 and 5, which transfer ADP-ribose unit from NAD to arginine, the amino-acid acceptor of the other ARTs is still unknown. We have shown that apical surface of ciliated and intermediate epithelial cells purified from bronchoalveolar lavage express ART1, 3 and 4. This is consistent with the possibility that these ecto-enzymes may have a role in inflammatory responses in lung. Indeed, A549 cells, which retain features of type II alveolar epithelial cells, represent a good model to study ART functions in epithelia. Using an arginine-specific enzymatic assay, we have identified an ART activity, on the surface of A549 cells. Possible candidates for catalyzing the arginine-specific ADP-ribosylation are ART1 and ART5 that recognize arginine as ADP-ribose acceptor. By reverse transcription-PCR we detected only ART1 mRNA and western blot of A549 membrane proteins with α-ART1 polyclonal antibodies further showed the expression of ART1. Since airway epithelial cells can interact with respiratory pathogens or their toxins, we investigated whether ART1 activity and expression is induced by bacterial components of Gram+ and Gram- bacteria. While lipopolysaccharide, peptidoglycan and Poly-(I:C) had no effect on transferase activity, lipoteichoic acid or flagelllin exerted a 3.7- and a 2.7-fold increase respectively of the transferase activity over the basal levels after 24 h treatment. The same effectors up-regulated ART1 expression, as shown by western blot analysis of cell membrane proteins suggesting as the up-regulation of ART1 is not a general event simply associated with cell activation
Promoter of the Pertussis Toxin Operon and Production of Pertussis Toxin
No full textPertussis toxin (PT), the major virulence factor of Bordetella pertussis, is composed of five different subunits whose genes are organized as an operon. We report the mapping of the promoter region of the PT operon and show that this promoter is only weakly active in Escherichia coli. Bordetella parapertussis and Bordetella bronchiseptica, which do not produce any PT, are shown to have a weaker promoter sequence for this operon and not to produce any detectable PT mRNA. We show that transcription of the PT operon in B. pertussis was constant throughout until the late stationary phase, when transcription significantly decreased. Analysis of the transposon Tn5 mutant BP347 showed that the product of the vir locus was required for transcription of the PT operon. Characterization of the Tn5 mutant BP356 showed that subunit S3 was required for the release of PT into the extracellular medium
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
Auto ADP-ribosylation of NarE, a Neisseria meningitidis ADP-ribosyltransferase, regulates its catalytic activities
No full textNarE is an arginine-specific mono-ADPribosyltransferase
identified in Neisseria meningitidis
that requires the presence of iron in a structured
cluster for its enzymatic activities. In this study, we
show that NarE can perform auto-ADP-ribosylation.
This automodification occurred in a time- and NADconcentration-
dependent manner; was inhibited by novobiocin,
an ADP-ribosyltransferase inhibitor; and did
not occur when NarE was heat inactivated. No reduction
in incorporation was evidenced in the presence of
high concentrations of ATP, GTP, ADP-ribose, or nicotinamide,
which inhibits NAD-glycohydrolase, impeding
the formation of free ADP-ribose. Based on the
electrophoretic profile of NarE on auto-ADP-ribosylation
and on the results of mutagenesis and mass
spectrometry analysis, the auto-ADP-ribosylation appeared
to be restricted to the addition of a single
ADP-ribose. Chemical stability experiments showed
that the ADP-ribosyl linkage was sensitive to hydroxylamine,
which breaks ADP-ribose-arginine bonds. Sitedirected
mutagenesis suggested that the auto-ADP-ribosylation
site occurred preferentially on the R7 residue,
which is located in the region I of the ADP-ribosyltransferase
family. After auto-ADP-ribosylation, NarE
showed a reduction in ADP-ribosyltransferase activity,
while NAD-glycohydrolase activity was increased. Overall,
our findings provide evidence for a novel intramolecular
mechanism used by NarE to regulate its enzymatic
activities.—Picchianti, M., Del Vecchio, M., Di
Marcello, F., Biagini, M., Veggi, D., Norais N., Rappuoli,
R., Pizza, M., Balducci, E. Auto ADP-ribosylation
of NarE, a Neisseria meningitidis ADP-ribosyltransferase,
regulates its catalytic activities. FASEB J
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 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
Brevetto depositato presso il Ministero del Commercio degli Stati Uniti: “Dosage and composition for nasal delivery and method of use of the same”. N° A-6874, 514-002.000, H88
No full textResponse of the bvg regulon of Bordetella pertussis to different temperatures and short-term temperature shifts
No full textBordetella pertussis produces a number of virulence factors whose expression is coordinately regulated by the bvgAS locus. Transcription of virulence genes is repressed by environmental factors such as low temperature (25°C) and chemical stimuli. Temperature shift of bacterial cultures from 25°C to 37°C activates two classes of bvg-regulated virulence genes: the early genes, which are activated within 10 min, and late genes, which require 2-4 h for activation. During the interval between the activation of the early and late genes, the intracellular concentration of BvgA increases 50-fold. It has been proposed that this increased concentration may be required for the activation of the late genes. Here we have analysed the response of the bvg locus to intermediate temperatures and to repeated temperature shifts. Temperature shifts of B. pertussis cultures from 22°C to 28°C, 32°C or 35°C resulted in the synthesis of low, intermediate, and high amounts of BvgA. This implied that the intracellular concentration of BvgA is temperature-dependent. We have also observed that the amount of virulence factors produced correlates with the BvgA concentration. When bacteria grown at 37°C were shifted to 22°C, transcription from the adenylate cyclase toxin haemolysis promoter (P(AC)) was repressed after 30 min, while transcription from the bvg (P1,) and filamentous haemagglutinin (P(FHA)) promoters was repressed after 2 h. During this time, the amount of BvgA did not decrease. A subsequent temperature shift from 22°C to 37°C induced transcription from the P1 and P(FHA) promoters after 10 min and transcription from the P(AC) promoter after 20 min. This result shows that in the presence of a high concentration of BvgA, the time lag between temperature shift and late promoter transcription is reduced from 2-4 h to 20 min. The above data support the proposal that the concentration of BvgA plays a role in activating expression of the late genes
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