Trends in Microbiology
ReviewBordetella pertussis adenylate cyclase: a toxin with multiple talents
Section snippets
Structure–function relationships
The CyaA toxin is encoded by the cyaA gene, whose expression, like that of the other virulence genes of B. pertussis, is regulated by environmental signals10. The 1706-residue CyaA is a bifunctional protein with both adenylate cyclase and haemolytic activities1, 11. Synthesized as an inactive precursor, it is converted to the active toxin by palmitoylation of Lys983, a process that is dependent on the product of the accessory gene cyaC (Ref. 12). CyaA is constructed in a modular fashion (Fig. 1
Mechanisms of entry into eukaryotic cells
CyaA can interact with a variety of cell types and deliver its catalytic domain into the host cell cytoplasm, where, upon binding by CaM, it produces high levels of cAMP. The novelty of the CyaA toxin, with respect to other bacterial or plant toxins, stems from its unique mechanism of penetration into eukaryotic cells. The catalytic domain appears to be translocated directly across the plasma membrane of the target cells into the cytosol by a process known as internalization or intoxication.
Delivery of CD8+ T-cell epitopes
Recently, we have shown that exogenous peptides can be inserted into defined permissive sites within the catalytic domain of CyaA without altering the biological activities of the molecule, suggesting that the toxin could be used as a vehicle to deliver polypeptides into eukaryotic cells21. In particular, we have attempted to use CyaA to target major histocompatibility complex (MHC) class I-associated T-cell epitopes within antigen-presenting cells (APCs) (Fig. 3), as it is well established
Reporter for protein targeting
The CaM dependence of the AC activity is a key feature of CyaA, as it allows a tight coupling between cAMP generation by the toxin, which is responsible for toxicity, and its internalization into eukaryotic cells (a constitutive enzymatic activity would be lethal for the bacterial host). As bacteria do not generally express CaM, the AC activity constitutes a selective reporter of the eukaryotic environment. This property has been exploited recently to study the mechanisms by which pathogenic
Reporter of protein association
We have taken advantage of the modular structure of the catalytic domain of CyaA to design a genetic system that allows easy screening and selection of functional interactions between two proteins in vivo. This bacterial two-hybrid system is based on functional complementation between the T25 and T18 fragments of the catalytic AC domain14 (Fig. 1). This complementation can be easily tested in an E. coli AC-deficient (cya−) strain. In E. coli, cAMP is a global regulator that, upon binding to the
Conclusions
The CyaA toxin of B. pertussis exhibits several novel features, the most significant being its activation by CaM, a protein absent in bacteria. It shares this property with another adenylate cyclase toxin, the edema factor (EF), produced by a taxonomically unrelated pathogen, Bacillus anthracis. In spite of differences in their size and overall sequence, these toxins share three regions of high sequence homology; however, their mechanisms of secretion and penetration into target cells are
Questions for future research
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How is adenylate cyclase (CyaA) activated by calmodulin?
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Does the haemolytic activity of CyaA have a role in vivo?
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How does the catalytic domain of CyaA cross the plasma membrane of eukaryotic cells?
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Does CyaA have some biological effect on the immune system that could underlie the cellular immune response against recombinant toxins carrying foreign epitopes?
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Can the detoxified recombinant CyaA toxins be used as vaccines?
Acknowledgements
We are grateful to Gouzel Karimova for helpful collaboration and discussions, Dara Frank for sharing results before publication, and David Grausz and Claude Leclerc for critical reading of this manuscript. Financial support came from the Institut Pasteur, the Centre National de la Recherche Scientifique (URA 1129) and Association pour la Recherche contre le Cancer (ARC).
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