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N Guiso

Publications and source records attributed to N Guiso.

At least 73 records · Page 4Linked to original sources
PubMed1993

CyaC-mediated activation is important not only for toxic but also for protective activities of Bordetella pertussis adenylate cyclase-hemolysin.

Bordetella pertussis adenylate cyclase-hemolysin (AC-Hly), encoded by the cyaA gene, belongs to the RTX family of toxins with extensive glycine-rich repeats in the carboxy-terminal portion. AC-Hly possesses both adenylate cyclase toxic and hemolytic activities that depend on a posttranslational modification mediated by the product of the cyaC gene. An improved system for AC-Hly synthesis and activation in Escherichia coli was developed. The results show that with purified AC-Hly (i) increased expression of the cyaC gene leads to a higher proportion of activated AC-Hly, (ii) the increase in protective activity of the activated recombinant AC-Hly correlates with the increase in its invasive and hemolytic activities, and (iii) the activated recombinant AC-Hly, but not the nonactivated recombinant AC-Hly, is a protective antigen against B. pertussis infection in a murine respiratory model. This suggests that possibly an immunodominant epitope required for protective activity is linked to the CyaC-mediated modification. Surprisingly, the protective and hemolytic activities of activated recombinant AC-Hly were lower than those of AC-Hly produced by B. pertussis, while its invasive activity was higher. This indicates that the modification of AC-Hly in B. pertussis and that in E. coli may differ.

Adenylyl Cyclases↗
PubMed1993

A phase variant of Bordetella pertussis with a mutation in a new locus involved in the regulation of pertussis toxin and adenylate cyclase toxin expression.

A novel nonhemolytic phase variant of Bordetella pertussis was characterized. This strain is strongly impaired in the transcription of the pertussis and adenylate cyclase toxins, whereas other known virulence-related factors such as the filamentous hemagglutinin, the fimbriae, and the outer membrane protein pertactin are expressed and regulated normally. Complementation and allelic exchange experiments demonstrated that the mutation is localized neither in the bvg locus involved in virulence regulation nor in the genes responsible for synthesis and transport of the toxins pertussis and adenylate cyclase. Instead, the mutation impairing transcription of at least the two toxin genes is located in a new genetic locus, which acts together with the BvgA/S two-component regulatory system on the expression of a subset of virulence genes. Further analysis suggested that most presumably the mutation affects a sequence-specific DNA-binding protein which contributes to transcriptional activation. The mutant was nonlethal in a murine respiratory model, which corresponds well with the lack of expression of the toxins. However, the clearing rate of this mutant from the lungs of mice was much lower than that of a bvg mutant, suggesting that factors other than the toxins may play a role in the persistence of the bacteria in the respiratory tract of mice.

Adenylate Cyclase Toxin↗
PubMed1993

Comparison of polymerase chain reaction, culture, and western immunoblot serology for diagnosis of Bordetella pertussis infection.

Polymerase chain reaction (PCR) amplification of the pertussis toxin promoter region was used to detect Bordetella pertussis infection in nasopharyngeal aspirates collected from 24 infants and children infected with pertussis and 13 adult contacts during an epidemiological study. The sensitivity of this PCR assay was approximately one bacterium, and the assay was specific for B. pertussis in tests with other Bordetella species and other respiratory pathogens. The pertussis case definition required a cough with a duration of more than 21 days for infants and children and laboratory confirmation by serology as the primary detection method for infants, children, and adults. The sensitivity of PCR and culture on Bordet-Gengou agar medium was assessed with regard to the case definitions. In the group of infants and children (index cases), the sensitivities of the culture and the PCR were 54.1% (13 of 24) and 95.8% (23 of 24), respectively. In the adult group (household contacts), the sensitivities of the two methods were 15.4% (2 of 13) and 61.5% (8 of 13), respectively. PCR combined with pertussis-specific serology appears to be a useful tool for diagnosis of pertussis especially in epidemiological studies.

Adult↗
PubMed1992

Both adenylate cyclase and hemolytic activities are required by Bordetella pertussis to initiate infection.

Among virulence factors synthesized and secreted by Bordetella pertussis, pertussis toxin (PTX) and the bifunctional adenylate cyclase-hemolysin (AC-Hly) are able to invade mammalian cells and to impair intracellular functions. Moreover, both proteins are protective antigens in murine intracerebral and respiratory models. In order to study their in vivo properties, different B. pertussis mutants, deficient in AC-Hly expression or secretion, or producing modified AC-Hly devoid of either adenylate cyclase or hemolytic activities, were constructed and examined. The in vivo properties of the mutants were compared to PTX deficient strains, using the murine respiratory model. We show that lack of PTX as well as adenylate cyclase or hemolytic activities results in avirulence. Furthermore, we show that mutants lacking adenylate cyclase or hemolytic activities were unable to multiply as fast as the parental strains and PTX mutants during the first 5 days following infection. Thus, both adenylate cyclase and hemolytic activities are required by B. pertussis to initiate infection.

Adenylate Cyclase Toxin↗
PubMed1991

Protective activity of Bordetella adenylate cyclase-hemolysin against bacterial colonization.

Bordetella pertussis synthesizes several factors. It has been suggested that one of these factors, the adenylate cyclase-hemolysin (AC-Hly), directly penetrates target cells and impairs their normal functions by elevating intracellular cAMP. In the present study, we show that active immunization with purified B. pertussis AC-Hly or AC (a fragment of the AC-Hly molecule carrying only the adenylate cyclase activity but no toxin activity in vitro) protects mice against B. pertussis intranasal infection. Immunization with AC-Hly or AC significantly shortens the period of bacterial colonization of the mouse respiratory tract. Furthermore, B. parapertussis AC-Hly or AC are also protective antigens against B. parapertussis colonization; their protective activities are equivalent to that of the whole-cell vaccine. These results suggest that AC-Hly may play an important role in Bordetella pathogenesis, in a murine model. If this factor plays a similar role in the human disease, its use as a protective antigen could reduce not only the incidence of the disease, but also the asymptomatic human reservoir by limiting bacterial carriage.

Adenylyl Cyclases↗
PubMed1989

Enzyme immunoassays of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate using acetylcholinesterase.

The pure tetrameric form of acetylcholinesterase (EC 3.1.1.7) from the electric eel Electrophorus electricus has been covalently coupled to 2'-O-succinyl-cAMP tyrosine methyl ester and 2'-O-succinyl-cGMP. Both enzymatic conjugates have been used as tracers in a classical heterogeneous competitive enzyme immunoassay allowing the determination of cAMP and cGMP, respectively. The test was performed in 96-well microtiter plates coated with a mouse monoclonal anti-rabbit Immunoglobulin antibody in order to ensure separation between bound and free moieties of the tracer. Acetylcholinesterase activity bound to the solid phase was measured by colorimetric assay. When standards or samples were first acetylated by treatment with acetic anhydride, the sensitivity of both assays appeared very good since minimum detectable concentration close to 0.04 pmol/mL (2 fmol/well) could be calculated for each assay. Precision was also very satisfying since the coefficient of variation was less than 5% in the 0.2-10 pmol/mL range. Good correlation was noted between enzymoimmunological and radioimmunological measurements of cAMP performed for different biological samples (urine, serum, or tissue extracts).

Acetylcholinesterase↗
PubMed1989

Bordetella adenylate cyclase is a virulence associated factor and an immunoprotective antigen.

Bordetella pertussis and Bordetella parapertussis are both causative agents of whooping cough outbreaks. Although not expressing the pertussis toxin, B. parapertussis induces, in a murine model, an acute hemorrhagic edematous alveolitis, similar to that observed with B. pertussis. These data suggest that the pertussis toxin may only play an accessory role in the acute pulmonary syndrome observed during Bordetella infection. Both with B. pertussis and B. parapertussis, the ability to induce lethal pulmonary lesions is associated with enhanced in vitro adenylate cyclase expression and activity. We also demonstrate that passive immunization with specific anti-B. pertussis adenylate cyclase antibodies or active immunization with purified B. pertussis secreted adenylate cyclase protect mice against a lethal respiratory challenge with B. pertussis or B. parapertussis. Our results suggest that adenylate cyclase might be the primary cytotoxin responsible for mouse pulmonary lesions during respiratory tract infection with B. pertussis or with the related species B. parapertussis and is a protective antigen of B. pertussis.

Adenylyl Cyclases↗
PubMed1989

Purification, location, and immunological characterization of the iron-regulated high-molecular-weight proteins of the highly pathogenic yersiniae.

We have previously shown that under iron limitation, different Yersinia species synthesize new polypeptides. Two of them, the high-molecular-weight proteins (HMWPs), are expressed only by the highly pathogenic strains. In the present study, the HMWPs from Y. enterocolitica serovar O:8 were purified by gel filtration, and specific antibodies were obtained. Using these antibodies, we show that the two polypeptides were synthesized de novo during iron starvation and that they were found essentially in the bacterial outer membrane fractions, although the majority of the molecules were not exposed on the cell surface. We also demonstrate that the two proteins had common epitopes and that the HMWPs of the high-virulence-phenotype species Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica serovar O:8 (a strain different from the one used to purify the proteins) are antigenically related. The less pathogenic and nonpathogenic strains did not exhibit cross-reacting material, suggesting that these strains do not synthesize even an altered form of the HMWPs.

Animals↗
PubMed1986

Bordetella pertussis adenylate cyclase. Purification, characterization, and radioimmunoassay.

The extracellular adenylate cyclase of Bordetella pertussis was purified either as a free enzyme or as a complex with calmodulin. The purified enzyme has a specific activity of 1600 mumol of cAMP min-1 X mg-1 and exists under two molecular forms of 45 and 43 kDa which are apparently structurally related. Calmodulin increased considerably the resistance of adenylate cyclase to inactivation by trypsin. Although trypsin cleaved the adenylate cyclase-calmodulin complex, the digested fragments remained associated by noncovalent interactions in an active conformation. Specific mouse anti-adenylate cyclase antibodies inhibit adenylate cyclase activity and were used to develop a specific radioimmunoassay that allows detection of as little as 5 ng of adenylate cyclase in culture supernatants.

Adenylyl Cyclases↗
PubMed1986

Characterization of a beta-galactosidase hybrid protein carrying the catalytic domain of Escherichia coli adenylate cyclase.

A hybrid protein of Escherichia coli, exhibiting both adenylate cyclase and beta-galactosidase activities, was purified and characterized. This protein, obtained by genetic engineering, contained the first 556 amino acids of adenylate cyclase connected to the eighth-residue of beta-galactosidase through a pentapeptide Val-Gly-Asp-Pro-Val. The fusion protein was less stable than the native beta-galatosidase. Trypsin cleaved preferentially the adenylate cyclase moiety of the hybrid protein at a ratio of 1/50 (w/w). The kinetic properties of the hybrid protein were comparable, with a few exceptions, to those of native adenylate cyclase and beta-galactosidase. 'Truncated' adenylate cyclase was no longer sensitive to inhibition by excess ATP, which seems to indicate a second nucleotide binding site of wild-type adenylate cyclase. Photoirradiation of the hybrid protein with 8-azidoadenosine 5'-triphosphate inactivated the adenylate cyclase activity, leaving intact the beta-galactosidase activity. A radiolabeled ATP analog was incorporated after photoirradiation into the adenylate cyclase moiety of the fusion protein as shown by limited digestion with trypsin.

Adenosine Triphosphate↗
PubMed1986

Interactions of the Escherichia coli methionine repressor with the metF operator and with its corepressor, S-adenosylmethionine.

The metJ gene encoding the methionine aporepressor was placed under the control of a strong and inducible promoter, ptac. Bacterial strains carrying the recombinant plasmid pIP35 overproduced the regulatory protein by a factor of 200 over the wild type strain as determined by the immunoblot technique. The purified metJ gene product negatively controls the expression of the metF gene, in a cell-free system as shown by repression of beta-galactosidase synthesis under the control of the metF promoter. The metJ protein binds to a DNA fragment containing the potential operator of the metF gene with an affinity which is 10 times greater in the presence of S-adenosylmethionine than in its absence. Equilibrium dialysis experiments showed that the met aporepressor binds 2 mol of S-adenosylmethionine per mol of dimer with a dissociation constant of 200 microM.

Escherichia coli↗
PubMed1985

Evidence for the presence of cAMP, cAMP receptor and transcription termination factor rho in different gram-negative bacteria.

Cyclic AMP has been shown to be present in 12 different Gram-negative bacteria and the regulation of its concentration, as a function of growth conditions, is similar to that described for Escherichia coli K12. Antibodies raised against catabolite activator protein (CAP) and Rho protein of E. coli K12 were used to check for the occurrence of cross-reactive antigens. Using radioimmunological assays, immunoblotting techniques and biochemical criteria we showed a wide distribution of CAP and Rho, structurally and functionally closely related to the corresponding E. coli K12 proteins. These results suggest that transcription is similarly regulated by these factors in Gram-negative bacteria.

Cyclic AMP↗
PubMed1984

Inactivation and proteolysis of heat-sensitive adenylate kinase of Escherichia coli CR341 T28.

Adenylate kinase from Escherichia coli K12 (strains CR341 and CR341 T28, a temperature-sensitive mutant) was purified by a two-step chromatographic procedure. Denaturation by heat above 60 degrees C of pure or crude preparations of adenylate kinase from both strains of bacteria was shown to be "reversible" if the enzyme was converted to the random coiled state by guanidinium chloride after heat treatment. Like other small monomeric proteins, adenylate kinase refolded rapidly to the native active state by dilution of guanidinium chloride. Adenylate kinase from the mutant strain was irreversibly inactivated by exposure of crude extracts at 40 degrees C. This inactivation is due to proteolysis which follows thermal denaturation (or transconformation) of mutant adenylate kinase at 40 degrees C. ATP, P1, P5-di(adenosine 5')-pentaphosphate, and anti-adenylate kinase antibodies protected the thermosensitive adenylate kinase in crude extracts against denaturation and proteolysis at 40 degrees C.

Adenylate Kinase↗
PubMed1984

Identification of the Escherichia coli cya gene product as authentic adenylate cyclase.

The Escherichia coli cya gene has been fused in the same register with the lacZ gene. The corresponding hybrid cya-lacZ gene is expressed as a bifunctional protein that exhibits both adenylate cyclase and beta-galactosidase activities, thus proving that cya is the structural gene for adenylate cyclase. The hybrid protein was purified to homogeneity and has been used to raise antibodies that recognize wild-type adenylate cyclase. Finally, the protein has been submitted to amino acid sequence analysis. It has been found that the first ten amino acids fit the predicted sequence obtained from DNA sequence analysis, thus substantiating the prediction that the cya translation initiation codon is UUG .

Adenylyl Cyclases↗
PubMed1984

Antibodies against a fused 'lacZ-yeast mitochondrial intron' gene product allow identification of the mRNA maturase encoded by the fourth intron of the yeast cob-box gene.

Several missense or nonsense mutations have been localized in the fourth intron open reading frame (ORF) of the yeast mitochondrial cytochrome b gene. These results and the phenotypes of mutants strongly suggested that a mRNA maturase, controlling the expression of both cytochrome b and cytochrome oxidase subunit I (COXI) genes, is encoded in this ORF. To investigate more directly the biosynthesis of mRNA maturase we raised antibodies against a part of the putative ORF translation product. For that purpose we inserted a fragment of the ORF sequence, in phase, into the C-terminal EcoRI site of lacZ gene. The hybrid gene was then expressed in Escherichia coli under the control of either the wild-type lac promoter or the thermoregulated lambda system PR/cI857. The hybrid protein was partially purified and antibodies were raised against it. These antibodies recognized a mitochondrially coded protein, p27, in intron mutants, whereas no such protein was detected in the wild-type cell. These results demonstrate that the p27 protein, previously shown to be associated with the mRNA maturase activity, is actually translated from the intron ORF. The autoregulated mRNA maturase synthesis model is discussed in relation to these results.

Base Sequence↗
PubMed1984

Antibodies against synthetic oligopeptides allow identification of the mRNA-maturase encoded by the second intron of the yeast cob-box gene.

Genetic and biochemical evidence has strongly suggested that several introns located in yeast mitochondrial genes specifying apocytochrome b or cytochrome oxidase encode trans-acting proteins (termed mRNA-maturases) responsible for splicing the cognate intron and maturation of the mRNA. We have chemically synthesized three oligopeptides, predicted from the DNA sequence of the open reading frame (ORF) present in the second intron of the cob-box gene, and raised antibodies against them. These antibodies have allowed us to identify a protein of 42 kd as the product translated from the ORF of the wild-type intron. In two splicing-deficient mutants this protein is replaced by shorter polypeptides whose lengths and antigenic properties are in full agreement with the positions of TAA codons established by the DNA sequence of the intron's ORF.

Antibodies, Fungal↗
PubMed1983

Transcription termination factor rho of Escherichia coli K-12: some regulatory aspects of its expression and activity.

A highly sensitive radioimmunological assay of the transcription termination factor (rho) of Escherichia coli K-12 has been developed. This method allows to measure in crude bacterial extracts quantities as low as 1.8 X 10(-15) moles of rho. We studied, under various conditions of growth and in mutants lacking or overproducing the cyclic AMP receptor protein (CAP) or adenylate cyclase, the relationship between the level of rho and its poly(C)-dependent ATPase activity. We showed that neither growth conditions, nor the presence or absence of a functional cAMP-CAP complex affect the synthesis and the enzymatic activity of the rho protein.

Adenosine Triphosphatases↗