Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation

Barry, Eileen M.; Weiss, Alison A.; Ehrmann, Ingrid; Gray, Mary C.; Hewlett, Erik L.; Goodwin, M.

doi:10.1128/jb.173.2.720-726.1991

Cited by 150 publications

(95 citation statements)

References 34 publications

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“…Because the CyaA polypeptide expressed in E. coli also lacks the ability to induce cAMP accumulation in target cells, they postulated that a posttranslational modification should occur in B. pertussis but not in E. coli, to confer cytotoxic capabilities to CyaA. The CyaCmodifying acyltransferase and the specific acylation of CyaA on lysine residues 860 and 983 were later described by Hewlett and colleagues (10). How this modification could convert the noncytotoxic pro-CyaA into an invasive and hemolytic protein has remained elusive until now (14,15).…”

Section: Discussionmentioning

confidence: 99%

“…The acylation region, spanning residues 800 -1000, contains two post-translational modification sites that are essential for the cytotoxic activities of CyaA (10 -12). The toxin is indeed synthesized as an inactive precursor, pro-CyaA that is converted into the active CyaA toxin upon specific acylation of Lys-860 and Lys-983 by a dedicated acyltransferase, CyaC (10,11,13). The C-terminal part of CyaA is the cell receptor-binding domain (RD; residues 1000 -1706).…”

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confidence: 99%

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Calcium, Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

Karst

Enguéné

Cannella

et al. 2014

Journal of Biological Chemistry

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The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Calcium, Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

Karst

Enguéné

Cannella

et al. 2014

Journal of Biological Chemistry

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“…It is well known that CyaA's invasive capacity depends on its acylation on the lysine residues K860 and K983 (26,27). Thus, we tested nonacylated proCyaA in our in vitro tBLM/CaM translocation assay.…”

Section: Resultsmentioning

confidence: 99%

“…The CyaA toxin is synthesized as an inactive precursor, proCyaA, which is converted into the active toxin form (CyaA) on specific acylation of two lysine residues (26,27). Then CyaA is secreted across the bacterial envelope by a dedicated type I secretion machinery and binds to the CD11b/CD18 integrin expressed by a subset of leukocytes including neutrophils, macrophages, and dendritic cells (22,(28)(29)(30).…”

mentioning

confidence: 99%

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

Veneziano

Rossi

Chenal

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Significance Many bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, although how they pass through a lipid bilayer remains largely unknown. Bordetella pertussis adenylate cyclase (CyaA) toxin delivers its catalytic domain directly across the cell membrane. To characterize this unique translocation process, we designed an in vitro assay based on a tethered lipid bilayer assembled over a biosensor surface derivatized with calmodulin, a natural activator of the toxin. CyaA activation by calmodulin provided a highly sensitive readout for toxin translocation across the bilayer. CyaA translocation was calcium- and membrane potential-dependent but independent of any additional eukaryotic protein. This biomimetic membrane will permit in vitro studies of protein translocation in precisely defined conditions.

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“…The Bordetella gene is encoded on the opposite strand from its E. coli counterpart and the sequence contains at least one error that separates two regions of clear similarity. This interpretation is now known to be correct, the error has been found, and the gene product has been characterized (20). Fig.…”

Section: Resultsmentioning

confidence: 98%

Finding errors in DNA sequences.

Roberts

1992

Proc. Natl. Acad. Sci. U.S.A.

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An algorithm is described that can detect certain errors within coding regions of DNA sequences. The algorithm is based on the idea that an insertion or deletion error within a coding sequence would interrupt the reading frame and cause the correct translation of a DNA sequence to require one or more frameshifts. If the coding sequence shows similarity to a known protein sequence then such errors can be detected by comparing the conceptual tntions of DNA sequences in all six reading frames with every sequence in a protein sequence data base. We have incorporated these ideas into a computer program, called DETECT, that can serve as an aid to the experimentalist who is determining new DNA sequences so that obvious errors may be located and corrected. The program has been tested using raw experimental data and against sequences from the European Molecular Biology Laboratory data base, annotated as co ining fr ss. We have also tested it using unidentified open reading frames that flank known, annotated genes in the GenBank data base. Many potential errors are apparent and in some caes functions can be suggested for the "corrected" versions of these reading frames leading to the identification of new genes. As more sequences are determined the power of this method will increase substantially.During the determination of a DNA sequence there are many opportunities for errors. These include simple human errors, such as incorrectly recording a sequence from one medium to another or misinterpreting experimental data, as might occur at a compression in a sequencing gel. Such errors can lead to changes in protein coding sequences and obscure the interpretation of the final sequence. Often they are detected serendipitously because a particular sequence is known to encode a product and the absence of a continuous open reading frame leads to a suspicion of error. Finding the location of such errors can be time-consuming, although programs such as BLAST (1) can greatly assist in the endeavor. Nevertheless, the use of manual methods to locate potential frameshifts has several disadvantages. They are rarely systematic and often require considerable interpretation of the results before their significance can be assessed.Already computational aids are available to avoid many of the pitfalls of error accumulation during DNA sequence determination. Semi-automated gel readers can help to ensure the correct recording of a sequence from a radioautogram to a computer (2-6), while many programs can manipulate raw sequence data and assemble it into a final sequence (7-9). More recently, new DNA sequencing machines have been developed that reduce the labor required for the accumulation of sequence data and further reduce the possibility for manual introduction of errors. However data interpretation remains a problem and the initial sequence is still likely to contain errors. In this paper we introduce a program, called DETECT, that can scan newly determined DNA sequences for the presence of errors in regions that code for proteins. ...

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Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation

Cited by 150 publications

References 34 publications

Calcium, Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

Calcium, Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

Finding errors in DNA sequences.

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