Interaction of Bordetella pertussis Adenylate Cyclase with CD11b/CD18

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the ␣ M ␤ 2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration (

Section: Discussionmentioning

confidence: 99%

Third Activity of Bordetella Adenylate Cyclase (AC) Toxin-Hemolysin

Fišer

Mašín

Basler

et al. 2007

“…We also showed recently that a region essential for CyaA binding to its cellular CD11b/CD18 (␣ M␤2 integrin) receptor encompasses a non-RTX segment between the RTX block II and block III motifs. Calcium-induced conformational rearrangements of this segment might explain the strict calcium dependence of the CyaA/CD11b association (31).…”

Section: Discussionmentioning

confidence: 99%

“…Recently, it has been demonstrated that CyaA binds specifically to target cells through the ␣ M ␤ 2 integrin receptor (CD11b/CD18) (30,31), which is expressed on a restricted subset of leukocytes including neutrophils, macrophages, and dendritic cells. Expression of this receptor most likely accounts for the high sensitivity of these cells to CyaA (13,30).…”

mentioning

confidence: 99%

Structural and Functional Characterization of an Essential RTX Subdomain of Bordetella pertussis Adenylate Cyclase Toxin

Bauche

Chenal

Knapp

et al. 2006

Self Cite

135

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells by a unique mechanism that consists in a calcium-dependent, direct translocation of the CyaA catalytic domain across the plasma membrane of the target cells. CyaA possesses a series of a glycine-and aspartate-rich nonapeptide repeats (residues 1006 -1613) of the prototype GGXG(N/D)DX(L/I/F)X (where X represents any amino acid) that are characteristic of the RTX (repeat in toxin) family of bacterial cytolysins. These repeats are arranged in a tandem fashion and may fold into a characteristic parallel ␤-helix or ␤-roll motif that constitutes a novel type of calcium binding structure, as revealed by the three-dimensional structure of the Pseudomonas aeruginosa alkaline protease. Here we have characterized the structure-function relationships of various fragments from the CyaA RTX subdomain. Our results indicate that the RTX functional unit includes both the tandem repeated nonapeptide motifs and the adjacent polypeptide segments, which are essential for the folding and calcium responsiveness of the RTX module. Upon calcium binding to the RTX repeats, a conformational rearrangement of the adjacent non-RTX sequences may act as a critical molecular switch to trigger the CyaA entry into target cells.The adenylate cyclase toxin (CyaA) 2 is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough (1-3). The 1706 residue-long CyaA is a bi-functional protein endowed with both catalytic (adenylate cyclase) and hemolytic activities (2, 4, 5). Synthesized as an inactive precursor, it is converted to the active toxin by a post translational palmitoylation of two internal lysine residues (Lys 860 and Lys 983 ) (6, 7). This active CyaA toxin is then able to deliver its catalytic domain directly across the plasma membrane of a variety of eukaryotic cells and disrupts their physiological functions by uncontrolled synthesis of cAMP (5, 8 -11), leading to the cell death by apoptosis (12)(13)(14). CyaA is constructed in a modular fashion; the calmodulinactivated catalytic domain is located in the 400-amino-proximal residues, whereas the C-terminal moiety (residues 400 -1706) is endowed with hemolytic activity (4, 5, 15, 16), which results from its ability to form cation-selective channels in membranes (17,18). It also mediates the binding and internalization of the toxin into eukaryotic cells (5,11,19). The hemolytic and the RTX domains display structural characteristics that link CyaA to the RTX (repeat in toxin) family of bacterial toxins (20, 21). Indeed, it contains a pore-forming domain (from residues 500 -700) with four hydrophobic segments (17,18,22,23), the target site for the post-translational palmitoylation (7, 24), 30 -40 copies of a characteristic glycine-and aspartate-rich nonapeptide repeats (residues 1006 -1613) of the prototype GGXG(N/ D)DX(U)X (X represents any amino acid, and U represents any large ...

“…Thus, the basolateral membranes of polarized T84 cells allow translocation even under depolarized conditions. Post-translational, covalent acylation by the acyl-transferase CyaC is required for hemolytic activity and for fully functional translocation/intoxication by ACT, although it is not required for in vitro enzymatic activity (13,25,(43)(44)(45). J774 cells express CD11b/CD18 and are intoxicated by nonacylated ACT, but with a 50-fold reduction in toxin potency.…”

Section: Rapid Induction Of I Sc By Act At the Basolateral Surface Bumentioning

confidence: 99%

Selective Translocation of the Bordetella pertussis Adenylate Cyclase Toxin across the Basolateral Membranes of Polarized Epithelial Cells

Eby

Ciesla

Hamman

et al. 2010

The catalytic domain of Bordetella pertussis adenylate cyclase toxin (ACT) translocates directly across the plasma membrane of mammalian cells to induce toxicity by the production of cAMP. Here, we use electrophysiology to examine the translocation of toxin into polarized epithelial cells that model the mucosal surfaces of the host. We find that both polarized T84 cell monolayers and human airway epithelial cultures respond to nanomolar concentrations of ACT when applied to basolateral membranes, with little or no response to toxin applied apically. The induction of toxicity is rapid and fully explained by increases in intracellular cAMP, consistent with toxin translocation directly across the basolateral membrane. Intoxication of T84 cells occurs in the absence of CD11b/CD18 or evidence of another specific membrane receptor, and it is not dependent on post-translational acylation of the toxin or on host cell membrane potential, both previously reported to be required for toxin action. Thus, elements of the basolateral membrane render epithelial cells highly sensitive to the entry of ACT in the absence of a specific receptor for toxin binding.The adenylate cyclase toxin (ACT), 3 a multifunctional, single polypeptide toxin, is expressed by six of the eight members of the genus Bordetella and was discovered by detection of adenylate cyclase enzymatic activity in a commercial pertussis vaccine (1). ACT derives its cytotoxic effects from delivery of its 400-amino acid adenylate cyclase enzymatic domain into the cell cytoplasm, resulting in the unregulated, calmodulindependent conversion of ATP into cAMP (2-4). Translocation, the process by which the catalytic domain is delivered across the cytoplasmic membrane, is unaffected by cytochalasin D or ammonium chloride and is dependent on the interaction of the ϳ1000-amino acid cell-binding domain with the cell membrane (5).This cell-binding domain is homologous to the members of the RTX (repeat-in-toxin) family of pore-forming bacterial toxins, such as Escherichia coli hemolysin, HlyA, and several leukotoxins from organisms such as Pasteurella hemolytica and Actinobacillus actinomycetemcomitans (6, 7). The RTX region of ACT oligomerizes in the cell membrane independently of translocation, forming cation-selective pores and causing hemolysis of erythrocytes and nonapoptotic death of nucleated cells (8 -14). Membrane interaction and pore formation by ACT can occur in artificial lipid bilayers and liposomes and are influenced by lipid and glycolipid composition (11,(15)(16)(17)(18). Although ACT intoxicates a broad range of cells and is able to associate with artificial lipid membranes containing no proteins, the ␤ 2 -integrin, CD11b/CD18 (Mac-1), which is expressed on phagocytic leukocytes, has been shown to increase the potency of ACT by an order of magnitude and has been considered a receptor for ACT (19). Accordingly, most of the studies on the functional, cytotoxic effects of ACT have focused on CD11b/CD18-positive cells, beginning with the initial observation that the...