The calmodulin‐sensitive adenylate cyclase of <i>Bordetella pertussis</i>: cloning and expression in <i>Escherichia col</i>

Glaser, Philippe; Ladant, Daniel; Sezer, Odile; Pichot, FrÃ©dÃ©rique; Ullmann, Agnès; Danchin, Antoine

doi:10.1111/j.1365-2958.1988.tb00003.x

Cited by 306 publications

(179 citation statements)

References 47 publications

(45 reference statements)

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“…The cyaA and bapC genes were also examined for variation by dideoxy sequencing. Oligonucleotide primers were designed from available sequences for cyaA (Y00545) (Glaser et al, 1988) and bapC (AF081494). For cyaA, five primer pairs were used to amplify approx.…”

Section: Methodsmentioning

confidence: 99%

Sequence variation and conservation in virulence-related genes of Bordetella pertussis isolates from the UK

Packard

Parton

Coote

et al. 2004

Journal of Medical Microbiology

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To determine the value of gene markers for surveillance and to assess the genetic stability of potential acellular pertussis vaccine components, the sequence variation in ten virulence-related genes of Bordetella pertussis was investigated in strains isolated in the UK between 1920 and 2002. These genes encode: pertactin (prnA); pertussis toxin subunits S1 (ptxA) and S3 (ptxC); tracheal colonization factor (tcfA); bordetella autotransporter protein C (bapC); bordetella resistance to killing protein (brkA); fimbrial antigen 2 (fim2); outer-membrane protein Q (ompQ); virulence-activated gene 8 (vag8) and adenylate cyclase toxin (cyaA). The encoded proteins are either components of current acellular vaccines (ACVs), or potential virulence markers for B. pertussis. Three strains used in the pertussis UK whole-cell vaccine (WCV), strain Tohama-I used for production of ACV components and the type strain of B. pertussis (18323 T ) were also analysed. Several novel alleles were found. The UK isolates were assigned multi-locus sequence types (MLSTs) according to a previously described scheme for B. pertussis based on three of these genes (ptxA, ptxC and tcfA). Compared with isolates from other countries, the UK clinical strains showed a distinct distribution of MLSTs. Apart from one strain that was MLST-3, all other recent isolates (2000)(2001)(2002) were identified as MLST-5. These isolates differed from the three WCV strains, which were MLST-2 or MLST-3, the Tohama-I strain (MLST-2) and the type strain of B. pertussis (MLST-9). MLST-3 and MLST-5 differ only by a single synonymous mutation, but this method does indicate that currently circulating strains of B. pertussis are not identical to the vaccine types, and they may differ in other important characteristics. Two new MLSTs were identified amongst historical UK isolates. Sequence-based typing offers a convenient method of analysing and comparing populations of B. pertussis from different time periods and from different countries. The variation exhibited by prnA and fim2 suggests that they could be useful, additional epidemiological markers in such a typing scheme.

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Section: Methodsmentioning

confidence: 99%

Sequence variation and conservation in virulence-related genes of Bordetella pertussis isolates from the UK

Packard

Parton

Coote

et al. 2004

Journal of Medical Microbiology

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“…Host factor activation of YpkA may be a strategy by which Yersinia prevents YpkA from being active within the bacterial cell. This strategy is utilized by pathogens such as Bordetella pertussis and Pseudomonas aeruginosa which express a calmodulin-activated adenylate cyclase (Cya) and a 14-3-3-activated Ras modifying toxin (ExoS), respectively (42)(43)(44). In these latter examples it is thought that the host cell activator is simply an abundant cytosolic protein that serves as an "on" switch for the toxin.…”

Section: Fig 6 Yeast Two-hybrid Assaysmentioning

confidence: 99%

The Yersinia Protein Kinase A Is a Host Factor Inducible RhoA/Rac-binding Virulence Factor

Dukuzumuremyi

Rosqvist

Hållberg

et al. 2000

Journal of Biological Chemistry

103

112

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The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpkA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino terminus, which contains the kinase domain, is sufficient to localize YpkA to the plasma membrane while the carboxyl terminus of YpkA is required for YpkAs morphological effects. YpkAs carboxyl-terminal region was found to affect the levels of actin-containing stress fibers as well as block the activation of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl-terminal region of YpkA, which contains sequences that bear similarity to the RhoAbinding domains of several eukaryotic RhoA-binding kinases, directly interacts with RhoA as well as Rac (but not Cdc42) and displays a slight but measurable binding preference for the GDP-bound form of RhoA. Surprisingly, YpkA binding to RhoA GDP affected neither the intrinsic nor guanine nucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting that YpkA controls activated RhoA levels by a mechanism other than by simply blocking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interaction with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that YpkA possesses both similarities and differences with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersiniae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.

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“…CyaA plays an important role in the early stages of respiratory tract colonization by B. pertussis. CyaA is able to invade eukaryotic target cells, where it is activated by an endogenous protein, calmodulin (CaM), 4 and produces high levels of cAMP that, in turn, alters the cellular physiology.…”

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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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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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The calmodulin‐sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia col

Cited by 306 publications

References 47 publications

Sequence variation and conservation in virulence-related genes of Bordetella pertussis isolates from the UK

Sequence variation and conservation in virulence-related genes of Bordetella pertussis isolates from the UK

The Yersinia Protein Kinase A Is a Host Factor Inducible RhoA/Rac-binding Virulence Factor

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

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