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Bioengineering of Bordetella Pertussis Adenylate Cyclase Toxin for Vaccine Development and Other Biotechnological Purposes

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Bioengineering of Bordetella Pertussis Adenylate Cyclase Toxin for Vaccine Development and Other Biotechnological Purposes toxins Review Bioengineering of Bordetella pertussis Adenylate Cyclase Toxin for Vaccine Development and Other Biotechnological Purposes Daniel Ladant Biochemistry of Macromolecular Interactions Unit, Structural Biology & Chemistry Department, Institut Pasteur CNRS UMR, CEDEX 15, 3528 Paris, France; [email protected] Abstract: The adenylate cyclase toxin, CyaA, is one of the key virulent factors produced by Bordetella pertussis, the causative agent of whooping cough. This toxin primarily targets innate immunity to fa- cilitate bacterial colonization of the respiratory tract. CyaA exhibits several remarkable characteristics that have been exploited for various applications in vaccinology and other biotechnological purposes. CyaA has been engineered as a potent vaccine vehicle to deliver antigens into antigen-presenting cells, while the adenylate cyclase catalytic domain has been used to design a robust genetic assay for monitoring protein–protein interactions in bacteria. These two biotechnological applications are briefly summarized in this chapter. Keywords: Bordetella pertussis; adenylate cyclase; recombinant toxin; antigen delivery; recombinant vaccine; biological screening; protein–protein interactions; two-hybrid Key Contribution: The manuscript highlights two biotechnological applications of the adenylate cyclase toxin from Bordetella pertussis in the fields of vaccinology and biotechnology. Citation: Ladant, D. Bioengineering 1. Introduction of Bordetella pertussis Adenylate Bordetella pertussis is a strictly human pathogen that is the causative agent of whooping Cyclase Toxin for Vaccine cough, or pertussis, a respiratory infection that still kills over a 100,000 children annually, Development and Other mainly in developing countries [1]. B. pertussis is a Gram-negative bacterium that colonizes Biotechnological Purposes. Toxins the respiratory tract. It produces a number of adhesins and virulence factors, including 2021 13 , , 83. https://doi.org/ the filamentous hemagglutinin (FHA), pertussis toxin (PTX), and the adenylate cyclase 10.3390/toxins13020083 toxin (CyaA), which are all essential for pathogenesis [2,3]. CyaA plays a critical role in the early stages of bacterial respiratory tract colonization. This toxin primarily targets Received: 23 December 2020 macrophages and neutrophils, and helps bacteria to fight against the primary innate Accepted: 15 January 2021 Published: 22 January 2021 immune responses [2,3]. CyaA invades eukaryotic target cells and delivers its catalytic domain into the cell cytosol, where it is activated upon binding to endogenous calmodulin Publisher’s Note: MDPI stays neutral (CaM), which stimulates its enzymatic activity to produce a massive amount of cAMP [4–7]. with regard to jurisdictional claims in The hyperphysiological cAMP levels drastically impair the target immune cells’ bactericidal published maps and institutional affil- capacity such as chemotaxis, superoxide anion generation by peripheral blood monocytes, iations. neutrophil, and macrophage phagocytosis, and they can trigger macrophage apoptosis. CyaA is a member of a large family of bacterial cytolysins known as RTX (Repeat-in- ToXin) toxins [8,9]. CyaA is a 1706-residue long bifunctional protein (Figure1) made of a CaM-activated catalytic domain (AC) located in the ~400 amino-proximal residues and ap- pended to a 1300-residue long carboxy-terminal moiety that displays all the characteristics Copyright: © 2021 by the author. Licensee MDPI, Basel, Switzerland. of RTX cytolysins [9]. This C-terminal region is indeed endowed with hemolytic activity. This article is an open access article The RTX cytolysins are pore-forming toxins that contain characteristic tandem repetitions of distributed under the terms and nonapeptidic, “Repeat-in-ToXin” (RTX) motifs that constitute specific calcium-binding sites. conditions of the Creative Commons CyaA contains ~40 copies of RTX motifs (residues 913–1613), and calcium is essential for Attribution (CC BY) license (https:// CyaA cytotoxicity. CyaA is synthesized as an inactive precursor proCyaA that is converted creativecommons.org/licenses/by/ into an active toxin upon selective acylation of two lysine residues, K860 and K983, a 4.0/). modification that is carried out by a dedicated acyltransferase, CyaC. The acylated CyaA Toxins 2021, 13, 83. https://doi.org/10.3390/toxins13020083 https://www.mdpi.com/journal/toxins Toxins 2021, 13, x FOR PEER REVIEW 2 of 12 Toxins 2021, 13, 83 and calcium is essential for CyaA cytotoxicity. CyaA is synthesized as an inactive pre-2 of 12 cursor proCyaA that is converted into an active toxin upon selective acylation of two ly- sine residues, K860 and K983, a modification that is carried out by a dedicated acyl- polypeptidetransferase, CyaC. is then The secreted acylated across CyaA the polypeptide bacterialenvelope is then secreted by a specifc across typethe bacterial I secretion machineryenvelope by (T1SS) a specifc made type of threeI secretion components machinery encoded (T1SS) by made the cyaBof three, cyaD components, and cyaE en-genes, whichcoded form by the an cyaB operon, cyaD with, and the cyaEcyaA genes,gene which [3,9]. form Although an operon CyaA with can the intoxicate cyaA gene a variety [3,9]. of Although CyaA can intoxicate a variety of cell types by a process that is independent of cell types by a process that is independent of receptor-mediated endocytosis, it binds in a receptor-mediated endocytosis, it binds in a calcium-dependent manner to the calcium-dependent manner to the CD11b/CD18 integrin [10]. This receptor is expressed CD11b/CD18 integrin [10]. This receptor is expressed by a subset of leukocytes including by a subset of leukocytes including neutrophils, macrophages, and dendritic cells (DC), neutrophils, macrophages, and dendritic cells (DC), and consequently these cells are the and consequently these cells are the natural in vivo targets of CyaA during B. pertussis natural in vivo targets of CyaA during B. pertussis infection [4,7]. After binding to the infection [4,7]. After binding to the CD11b/CD18 receptor, the hydrophobic region (aa CD11b/CD18 receptor, the hydrophobic region (aa 500 to 750) of CyaA is inserted into the 500plasma to 750) membrane of CyaA to is firmly inserted anchor into the the toxin plasma to the membrane target cells. to Finally, firmly anchorthe catalytic the toxin do- to themain target is directly cells. delivered Finally, the across catalytic the membrane domain isinto directly the cytosol delivered, where across it binds the CaM membrane and intoproduces the cytosol, cAMP where[4,7,11,12] it binds. This CaM direct and delivery produces of th cAMPe CyaA [4 catalytic,7,11,12]. domain This direct across delivery the ofplasma the CyaA membrane catalytic of the domain target acrosscells is therather plasma unique membrane among bacterial of the toxins target that cells usually is rather uniqueexploit amongdiverse bacterialroutes of toxins endocytosis that usually to enter exploit eukaryotic diverse cells. routes One ofconsequence endocytosis of tothis enter eukaryoticoriginal entry cells. pathway One consequence is that CyaA of this can original trigger entrya very pathway rapid increase is that CyaAof intracellular can trigger a verycAMP, rapid which increase is mainly of intracellular localized incAMP, the vicinity which ofis the mainly plasma localized membrane in the [13]. vicinity This spa- of the plasmatio-temporal membrane compartmentalization [13]. This spatio-temporal of the cAMP compartmentalization signaling likely contributes of the cAMP to the signaling spe- likelycific mode contributes of action to of the CyaA specific on different mode of target action cells of CyaA [14–16]. on Currently, different target the precise cells [mo-14–16]. Currently,lecular mechanisms the precise by molecular which the mechanisms AC domain bycan which pass across the AC the domain hydrophobic can pass barrier across of the hydrophobicthe lipid bilayer barrier cells ofremain the lipid the subject bilayer of cells active remain investigations. the subject Fundamental of active investigations. aspects of Fundamentalthe biogenesis aspects of CyaA of, the includin biogenesisg the ofbiophysics CyaA, including of secretion, the biophysicscalcium and of secretion,acyla- calciumtion-dependent and acylation-dependent folding, and toxin folding, translocation and toxin across translocation membranes across have membranes been further have beendiscussed further in discussed several recent in several articles recent and articlesreviews and[4,7,12] reviews. The [remaining4,7,12]. The chapter remaining will chapterdeal willwith deal the withbiotechnological the biotechnological exploitation exploitation of the original of the characteristics original characteristics of CyaA in of vacci- CyaA in vaccinologynology and biotechnology. and biotechnology. FigureFigure 1. Schematic1. Schematic representation representation of of CyaA CyaA structural structural organization organization and and biogenesis. biogenesis. UpperUpper part:part: Structural organization organization of theof CyaA the CyaA polypeptide polypeptide with with its differentits different subdomains. subdomains. Lower Lower part part left:left: in B. pertussis, CyaACyaA is is synthesized synthesized as asan aninactive inactive precursor proCyaA that is activated by the CyaC acyltransferase and then secreted across the bacterial envelope by a dedicated type I secretion system (T1SS); Lower part right: model for CyaA invasion of eukaryotic cells. After
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