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

Abstract

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...

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

“…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). However, CyaA can also invade a wide variety of cells that do not express this receptor, albeit with a lower efficiency (19,(31)(32)(33)(34)(35).…”

“…How the hydrophilic CyaA catalytic domain of approximately 400 residues is able to pass across the hydrophobic barrier of the plasma membrane remains largely unknown, and whether specific eukaryotic proteins and/or cell membrane components are involved in this process is also unclear (19,32,35,38,39). To characterize the molecular mechanisms of CyaA translocation across the membrane, we performed a functional in vitro assay that exploits a recently designed biomimetic membrane assembly composed of a bilayer membrane (tBLM) tethered over an amino-grafted gold surface derivatized with CaM (40).…”

Role of CD11b/CD18 in the Process of Intoxication by the Adenylate Cyclase Toxin of Bordetella pertussis

“…The RTX component forms oligomeric pores in cell membranes and serves as the cell binding domain (55, 58). To generate cyclic AMP (cAMP), ACT binds to cells, translocates the catalytic domain across the cytoplasmic membrane without endocytosis or macropinocytosis (11,19,25), and catalyzes the conversion of intracellular ATP to cAMP.Several cytotoxic effects for ACT have been identified, starting with the discovery that toxin-generated cAMP inhibits core functions of neutrophils and macrophages, such as the generation of an oxidative burst and killing of phagocytized bacteria (8, 49). ACT elicits additional cAMP-mediated effects in a variety of eukaryotic cells; these include apoptotic cell death, dysfunction of the cell cytoskeleton, cell cycle arrest, chloride secretion from polarized epithelial cells, and dysregulation of the adaptive immune system (8,11,22,30,34,47,49,50,54).…”

“…The RTX component forms oligomeric pores in cell membranes and serves as the cell binding domain (55, 58). To generate cyclic AMP (cAMP), ACT binds to cells, translocates the catalytic domain across the cytoplasmic membrane without endocytosis or macropinocytosis (11,19,25), and catalyzes the conversion of intracellular ATP to cAMP.…”

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

“…Once secreted, CyaA binds in a calcium-dependent manner to the CD11b/CD18 integrin expressed on myeloid cells, such as macrophages, neutrophils, dendritic cells, and natural killer cells, that are the primary targets of this toxin in vivo (31). However, CyaA can also efficiently intoxicate a variety of cell types lacking this receptor (16,(32)(33)(34)(35).…”