Biomedical subjects

P Sebo

Publications and source records attributed to P Sebo.

32 records · Page 2Linked to original sources

PubMed1995

Hemolytic, but not cell-invasive activity, of adenylate cyclase toxin is selectively affected by differential fatty-acylation in Escherichia coli.

Adenylate cyclase toxin from Bordetella pertussis requires posttranslational acylation of lysine 983 for the ability to deliver its catalytic domain to the target cell interior and produce cyclic adenosine monophosphate (cell-invasive activity) and to form transmembrane channels (hemolytic activity). When the toxin is expressed in Escherichia coli, it has reduced hemolytic activity, but comparable cell-invasive activity to that of adenylate cyclase toxin from B. pertussis. In contrast to the native protein from B. pertussis, which is exclusively palmitoylated, recombinant toxin from E. coli is acylated at lysine 983 with about 87% palmitoylated and the remainder myristoylated. Furthermore, the recombinant toxin contains an additional palmitoylation on approximately two-thirds of the lysines at position 860. These observations suggest that the site and nature of posttranslational fatty-acylation can be dictated by the bacterial host used for expression and can have a significant, but selective, effect on protein function.

Acylation↗

PubMed1995

Identification by in vitro complementation of regions required for cell-invasive activity of Bordetella pertussis adenylate cyclase toxin.

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein composed of an amino-terminal adenylate cyclase (AC) domain linked to a 1300-residue channel-forming RTX (repeats in toxin) haemolysin. The toxin delivers its AC domain into a variety of eukaryotic cells and impairs cellular functions by catalysing unregulated synthesis of cAMP from intracellular ATP. We have examined toxin activities of a set of deletion derivatives of CyaA. The results indicate that CyaA does not have a dedicated target cell-binding domain and that structural integrity and co-operation of all domains, as well as the post-translational fatty acylation mediated by an accessory protein CyaC, are all essential for target cell association and toxin activity of CyaA. When tested individually, all toxin derivatives were inactive and impaired in the tight association with the target cell surface. However, pairs of constructs with nonoverlapping deletions complemented each other in vitro and exhibited a partially restored cytotoxic activity. This suggests that at least a part of the active toxin may act in the form of dimers or higher oligomers. The complementation analysis revealed that the last 217 residues of CyaA, containing the unprocessed secretion signal, form an autonomous domain essential for toxin activity, and that the region from residue 624 to 780 may be directly involved in delivery of the AC toxin into cells.

Acylation↗

PubMed1995

Cell-invasive activity of epitope-tagged adenylate cyclase of Bordetella pertussis allows in vitro presentation of a foreign epitope to CD8+ cytotoxic T cells.

The adenylate cyclase (AC) toxin (CyaA) of Bordetella pertussis has an invasive catalytic domain (AC domain) which penetrates the cytoplasmic membrane of a variety of eukaryotic cells and intoxicates them by unregulated synthesis of cyclic AMP. Previous work led to identification of five permissive sites in the AC domain at which heterologous peptides are accommodated without affecting its enzymatic properties. We have constructed a set of CyaA toxins tagged at these permissive sites by insertion of a CD8+ T-cell epitope, RPQASGVYMGNLTAQ, from the nucleoprotein of lymphocytic choriomeningitis virus. Introduction of the epitope at any of the five sites did not affect the capacity of the toxin to deliver its AC domain into target cells. Moreover, the toxin with the inserted epitope was shown to sensitize target cells for lysis by epitope-specific CD8+ cytotoxic T lymphocytes in vitro, showing that the tagged AC was processed for presentation of the lymphocytic choriomeningitis virus epitope in association with the major histocompatibility complex class I molecules. This finding indicates that by virtue of delivery of foreign epitopes into the antigen-presenting cells, purpose-designed recombinant CyaAs may be useful for induction of specific major histocompatibility complex class I-restricted cell-mediated immunity also in vivo.

Adenylyl Cyclases↗

PubMed1995

The C-terminal domain is essential for protective activity of the Bordetella pertussis adenylate cyclase-hemolysin.

The adenylate cyclase-hemolysin of Bordetella pertussis consists of a cell-invasive N-terminal adenylate cyclase domain linked to a C-terminal RTX hemolysin containing extensive glycine-rich repeats. The toxin is an essential virulence factor required in the initial stages of infection. Adenylate cyclase-hemolysin was also shown to be a potent vaccinating antigen inducing protection against B. pertussis colonization of the mouse respiratory tract. This protective activity depends on a posttranslational fatty-acylation modification. We used a set of deletion derivatives of the recombinant adenylate cyclase-hemolysin to localize the protective epitopes on the 1,706-residue toxin. We show that specific anti-adenylate cyclase-hemolysin antibodies present in the sera of B. pertussis-infected mice and humans are directed predominantly against the modification-and-repeat portion of the toxin, contained in the last 800 residues of the adenylate cyclase-hemolysin. These antibodies appear to recognize conformational epitopes present only in a structure formed by the intact C-terminal half of the toxin. There was no correlation between the capacity of the truncated adenylate cyclase-hemolysin derivatives to induce both toxin-neutralizing antibodies upon immunization of mice and protective immunity. However, only the truncated proteins which were recognized by the sera of infected mice and humans and which had their last 800 residues intact had the capacity to induce protection of mice against colonization by B. pertussis. This indicates that the structure of the modification-and-repeat region of adenylate cyclase-hemolysin is critical for its protective activity.

Adenylyl Cyclases↗

PubMed1994

Adenylate cyclase toxin (CyaA) of Bordetella pertussis. Evidence for the formation of small ion-permeable channels and comparison with HlyA of Escherichia coli.

The interaction between the adenylate cyclase toxin (CyaA) of Bordetella pertussis and lipid was studied using the lipid bilayer assay. The addition of CyaA to the aqueous phase bathing lipid bilayer membranes composed of different lipids resulted in the increase of the membrane conductance. This increase was rather small for membranes formed of pure lipids as compared with lipid mixtures such as asolectin. The toxin formed in asolectin membranes small transient ion-permeable channels with a single-channel conductance of 27 pS in 1 M KCl, which is considerably smaller than that of the alpha-hemolysin (HlyA) of Escherichia coli (1500 pS). Experiments with different salts suggested that the CyaA-induced channels were exclusively cation-selective because of negative charges localized at the channel mouth. The single-channel conductance of channels initiated by CyaA was independent of whether the toxin was purified from B. pertussis or from recombinant E. coli. However, the channel-forming activity of the CyaA expressed in B. pertussis was substantially higher than that of the recombinant toxin. Experiments with mutant forms of CyaA suggested that both the activation of CyaA by CyaC and the hemeolytic part of the toxin, but not the repeats and the cyclase activity, are required for channel formation in lipid bilayer membranes.

Adenylate Cyclase Toxin↗

PubMed1993

Bordetella pertussis adenylate cyclase: a toxin with multiple talents.

The adenylate cyclase toxin of Bordetella pertussis is a secreted multifunctional protein, endowed with calmodulin-activated catalytic, haemolytic and cytotoxic activities. Residues and domains involved in different functions have been localized and several permissive sites, able to accommodate insertion of peptides without impairing the different functions of the toxin, have been identified. A 400-bp region in the promoter upstream region of the cyaA gene, encoding the toxin, has been defined as the target of transcriptional activation.

Adenylyl Cyclases↗

PubMed1993

Repeat sequences in the Bordetella pertussis adenylate cyclase toxin can be recognized as alternative carboxy-proximal secretion signals by the Escherichia coli alpha-haemolysin translocator.

The 1706-residue adenylate cyclase toxin (CyaA) of Bordetella pertussis is an RTX protein with extensive carboxy-proximal glycine and aspartate-rich repeats. CyaA does not have a cleavable amino-terminal signal peptide and can be secreted across both bacterial membranes of the Escherichia coli cell envelope by the alpha-haemolysin (HlyA) translocator (HlyBD/TolC). We performed deletion mapping of secretion signals recognized in CyaA by this heterologous translocator. Truncated proteins with N-terminal and internal deletions were secreted at levels up to 10 times higher than intact CyaA and similar to HlyA. A secretion signal recognized by HlyBD/TolC was found within the last 74 residues of CyaA. However, secretion of CyaA was reduced but not abolished upon deletion of the last 75 or 217 residues, indicating that at least two additional secretion signals recognized by HlyBD/TolC are within CyaA. One of them was localized to the repeat sequence between residues Asp-1587 to Ile-1631. Interestingly, a conserved 'acidic' motif (Glu/Asp)-(X)11-Asp-(X)3/5-(Glu/Asp)-(X)14-Asp was found in the C-terminal sequences of HlyA, CyaA and the two secreted CyaA derivatives. We speculate that the presence and spacing of acidic residues may be an important feature of secretion signals recognized by the haemolysin translocator.

Adenylate Cyclase Toxin↗

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↗

PubMed1992

Bordetella pertussis adenylate cyclase toxin. Structural and functional independence of the catalytic and hemolytic activities.

The Bordetella pertussis calmodulin-dependent adenylate cyclase (CyaA) is a 1706-residue-long toxin, endowed with hemolytic activity. We have constructed B. pertussis mutant strains producing modified CyaAs devoid of adenylate cyclase activity. Our results show that such modified CyaAs display hemolytic activity identical to the wild-type toxin, thus demonstrating that the hemolytic activity is independent of the adenylate cyclase activity. Furthermore, B. pertussis and Escherichia coli strains producing CyaA lacking the catalytic domain (residues 1-373) were constructed. The truncated protein exhibits hemolytic activity comparable to the wild-type toxin, thus establishing that the carboxyl-terminal 1332 residues alone are endowed with hemolytic activity. Together, these findings show that adenylate cyclase and hemolytic activities are located in two distinct regions of the molecule (respectively, approximately amino acids 1-400 and 401-1706) and that the two regions of CyaA are functionally independent.

Adenylate Cyclase Toxin↗

PubMed1991

High-level synthesis of active adenylate cyclase toxin of Bordetella pertussis in a reconstructed Escherichia coli system.

The Bordetella pertussis adenylate cyclase(Cya) toxin-encoding locus (cya) is composed of five genes. The cyaA gene encodes a virulence factor (CyaA), exhibiting adenylate cyclase, hemolytic and invasive activities. The cyaB, D and E gene products are necessary for CyaA transport, and the cyaC gene product is required to activate CyaA. We reconstructed, in Escherichia coli, the cya locus of B. pertussis by cloning the different genes on appropriate vectors under the control of strong promoters and E. coli-specific translation initiation signals. We show that in the absence of additional gene products, CyaA is synthesized at high levels, is endowed with adenylate cyclase activity, but is devoid of invasive and hemolytic activities. CyaC is sufficient to confer upon the adenylate cyclase holotoxin full invasive and partial hemolytic activities. Coexpression of the cyaB, D and E genes neither stimulates nor potentiates the activation brought about by CyaC. This reconstructed system should help to elucidate both the mechanism and the structural requirements of holotoxin activation.

Adenylate Cyclase Toxin↗

PubMed1990

Delineation of the minimal portion of the Bacillus sphaericus 1593M toxin required for the expression of larvicidal activity.

The two genes of Bacillus sphaericus 1953M coding for the 51.4-kDa and 41.9-kDa proteins are both required for the expression of the active larvicidal toxin in Escherichia coli. The minimal size of the active peptide of the 41.9-kDa toxin was defined by in vitro deletion analysis of the gene and found to consist of 338 amino acids (38.3 kDa). N-terminal deletions past the Ile18 residue and C-terminal deletions past the His352 residue result in the loss of toxic activity and rapid degradation of such modified toxins by host proteases. The minimal active 38.3-kDa peptide produced in E. coli seems to mimick the stable processed form of the toxin found in larval midguts. However, it still requires the action of the synergistic 51.4-kDa protein for the larvicidal activity.

Amino Acid Sequence↗

PubMed1989

On the respective roles of the two proteins encoded by the Bacillus sphaericus 1593M toxin genes expressed in Escherichia coli and Bacillus subtilis.

The 3.6 kb HindIII DNA fragment of B. sphaericus 1593M chromosomal DNA bears two genes encoding two polypeptides of 41.9 kDa (protein "42") and 51.4 kDa (protein "51"). DNA fragments carrying only one of these two genes when expressed in E. coli yield products that are inactive towards Culex larvae. The larvicidal activity is recovered when Triton X-100 treated E. coli cells containing each one of the two genes are incubated together. In E. coli these two polypeptides are acting synergistically. The protein "51" appears to be involved in the maturation of protein "42" for expression of the larvicidal activity. In B. subtilis however the toxicity is expressed by cells carrying only the gene coding for protein "42". There is no need of the "51" gene product for the maturation of the "42" polypeptide, suggesting that the maturation is most likely accomplished by host enzymes.

Animals↗

PubMed1988

Molecular properties of elongation factor Tu from Streptomyces aureofaciens and Escherichia coli.

Some molecular properties of the elongation factor Tu of protein synthesis purified in an aggregated state from gram-positive Streptomyces aureofaciens were studied and compared with those of Tu from gram-negative Escherichia coli. Electrofocussing under reducing conditions showed that the molecule of EF-Tu from S. aureofaciens has an isoelectric point shifted more to the acidic side compared with EF-Tu from E. coli. A comparison of amino acid composition revealed minor differences in the content of several amino acids in the two factors and showed that EF-Tu from S. aureofaciens contains four half-cystines per molecule. Under denaturing conditions only two mercapto groups reacted with 5,5'-dithiobis(2-nitrobenzoic acid). Limited tryptic digestion of aggregated EF-Tu from S. aureofaciens yields six fragments: the four main fragments are of a similar size as those of the E. coli factor. All fragments detected after trypsin digestion of S. aureofaciens EF-Tu were immunologically cross-reactive with antibodies against E. coli EF-Tu. However, even after 2 h of the reaction there still remains a small part of streptomycete factor uncleaved, which documents high resistance of aggregated EF-Tu towards trypsin.

Amino Acids↗

PubMed1987

Identification of an inducible penicillinase of the lithoautotrophic hydrogen-oxidizing bacterium Alcaligenes eutrophus.

The growth of Alcaligenes eutrophus in the presence of benzylpenicillin under heterotrophic and autotrophic conditions was studied. The drug induced a penicillinase in the cells, which can be readily released and extracted from the cells after a lysozyme and EDTA treatment in the course of spheroplast formation. The isoelectric point of the enzyme is 8.1 and the molar mass was estimated to be nearly 25 kg/mol. Phenoxypenicillin is hydrolyzed in the presence of the enzyme at a higher relative rate than benzylpenicillin, ampicillin, amoxycillin and azlocillin. The cephalosporins tested, i.e. cephalosporin C, cefalexin, cefotaxime and 7-aminocephalosporanic acid, were hydrolyzed at a substantially lower relative rate than the penicillins, indicating that the enzyme is a penicillinase.

Alcaligenes↗