Cell entry and cAMP imaging of anthrax edema toxin

To kill macrophages, the lethal factor component of Bacillus anthracis toxin binds to a carrier protein (PA), which then interacts with the CMG2 receptor protein on the cell surface and is endocytosed into the cytoplasm. CMG2, as well as TEM8, a second PA receptor not present on macrophages, contain a von Willebrand A domain that is crucial for toxin binding. Here we report that integrin β1, another cell surface von Willebrand A domain protein, can mediate and potentiate anthrax toxin endocytosis. By using microarray-based analysis to globally correlate gene expression profiles with toxin sensitivity, we associated toxin effects with the integrinactivating proteins osteopontin and CD44. Further study showed that PA binds to α4β1-and α5β1-integrin complexes, leading to their conjoint endocytosis, and also interacts-weakly relative to CMG2 but comparably to TEM8-with purified α5β1 complex in vitro. Monoclonal antibody directed against β1-integrin or its α integrin partners reduced PA/integrin endocytosis and anthrax toxin lethality, and hyaluronic acid-which interferes with CD44-mediated integrin activation-had similar effects. Remarkably, whereas deficiency of CMG2 protected macrophages from rapid killing by large toxin doses (>50 ng/mL), by 24 h the toxin-treated cells were dead. Such late killing of CMG2-deficient cells by high dose toxin as well as the late death observed during exposure of CMG2-producing macrophages to low-dose toxin (<1 ng/mL), was dependent on integrin function. Effects of inactivating both CMG2 and integrin were synergistic. Collectively, our findings argue strongly that β1-integrin can both potentiate CMG2-mediated endocytosis and serve independently as a low-affinity PA receptor.CD44 | microarray | osteopontin | CMG2 | lethal factor

Section: Resultsmentioning

confidence: 99%

Heterodimeric integrin complexes containing β1-integrin promote internalization and lethality of anthrax toxin

Martchenko¹,

Jeong²,

Cohen

2010

“…ACT also has hemolytic properties conferred by its C-terminal domain, which may form pores that permeabilize the cell membrane for monovalent cations, thus contributing to overall cytoxicity of ACT toward phagocytes (8)(9)(10). Bordetella ACT toxin, together with the anthrax edema factor, are the two prototypes of bacterial protein toxin that attack the immune response by overboosting the major signaling pathway in the immune response (cAMP, together with the MAPK pathway) (11).…”

mentioning

confidence: 99%

Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain

González-Bullón

Uribe

Martı́n

et al. 2017

Adenylate cyclase toxin (ACT or CyaA) plays a crucial role in respiratory tract colonization and virulence of the whooping cough causative bacterium Bordetella pertussis. Secreted as soluble protein, it targets myeloid cells expressing the CD11b/CD18 integrin and on delivery of its N-terminal adenylate cyclase catalytic domain (AC domain) into the cytosol, generates uncontrolled toxic levels of cAMP that ablates bactericidal capacities of phagocytes. Our study deciphers the fundamentals of the heretofore poorly understood molecular mechanism by which the ACT enzyme domain directly crosses the host cell membrane. By combining molecular biology, biochemistry, and biophysics techniques, we discover that ACT has intrinsic phospholipase A (PLA) activity, and that such activity determines AC translocation. Moreover, we show that elimination of the ACT-PLA activity abrogates ACT toxicity in macrophages, particularly at toxin concentrations close to biological reality of bacterial infection. Our data support a molecular mechanism in which in situ generation of nonlamellar lysophospholipids by ACT-PLA activity into the cell membrane would form, likely in combination with membrane-interacting ACT segments, a proteolipidic toroidal pore through which AC domain transfer could directly take place. Regulation of ACT-PLA activity thus emerges as novel target for therapeutic control of the disease.bacterial toxins | RTX toxin family | protein translocation | biological membranes | membrane remodeling

“…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).…”

mentioning

confidence: 99%

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

Veneziano

Rossi

Chenal

et al. 2013

Self Cite

Significance Many bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, although how they pass through a lipid bilayer remains largely unknown. Bordetella pertussis adenylate cyclase (CyaA) toxin delivers its catalytic domain directly across the cell membrane. To characterize this unique translocation process, we designed an in vitro assay based on a tethered lipid bilayer assembled over a biosensor surface derivatized with calmodulin, a natural activator of the toxin. CyaA activation by calmodulin provided a highly sensitive readout for toxin translocation across the bilayer. CyaA translocation was calcium- and membrane potential-dependent but independent of any additional eukaryotic protein. This biomimetic membrane will permit in vitro studies of protein translocation in precisely defined conditions.