The vacuolar ATPase proton pump is required for the cytotoxicity of <i>Bacillus anthracis</i> lethal toxin

Ménard, Armelle; Altendorf, Karlheinz; Breves, Daniel; Mock, Michèle; Montecucco, Cesare

doi:10.1016/0014-5793(96)00422-x

Cited by 45 publications

(42 citation statements)

References 30 publications

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“…These data indicate that pore formation can occur at near-neutral pH when the toxin is bound to TEM8 but not when it is bound to CMG2. Furthermore, these results revealed an unexpected difference between three cell types (HeLa, CHO-K1, and RAW 264.7 cells) that were previously used to derive a unified model for anthrax toxin entry (9,11,15).…”

Section: Resultsmentioning

confidence: 85%

“…These complexes are then exposed to increasingly acidic environments as they are trafficked from early͞sorting endosomes to late endosomes. The current model of anthrax toxin entry invokes (PA 63 ) 7 prepore conversion to a 14-stranded ␤-barrel-containing pore on the membranes of intraluminal vesicles that are formed within mildly acidic early͞sorting endosomes (7,(11)(12)(13)(14)(15)(16) (Fig. 1a).…”

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confidence: 99%

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Receptor-specific requirements for anthrax toxin delivery into cells

Rainey

Wigelsworth

Ryan

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

119

157

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The three proteins that constitute anthrax toxin self-assemble into toxic complexes after one of these proteins, protective antigen (PA), binds to tumor endothelial marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2) cellular receptors. The toxin receptor complexes are internalized, and acidic endosomal pH triggers pore formation by PA and translocation of the catalytic subunits into the cytosol. In this study we show that the pH threshold for conversion of the PA prepore to the pore and for translocation differs by approximately a pH unit, depending on whether the TEM8 or CMG2 receptor is used. For TEM8-associated toxin, these events can occur at close to neutral pH values, and they show relatively low sensitivity to ammonium chloride treatment in cells. In contrast, with CMG2-associated toxin, these events require more acidic conditions and are highly sensitive to ammonium chloride. We show, furthermore, that PA dissociates from TEM8 and CMG2 upon pore formation. Our results are consistent with a model in which translocation depends on pore formation and pore formation, in turn, depends on release of PA from its receptor. We propose that because PA binds to CMG2 with much higher affinity than it does to TEM8, a lower pH is needed to attenuate CMG2 binding to allow pore formation. Our results suggest that toxin can form pores at different points in the endocytic pathway, depending on which receptor is used for entry.capillary morphogenesis protein 2 ͉ tumor endothelial marker 8 ͉ toxin entry B acillus anthracis, the causative agent of anthrax, secretes a toxin that is believed to be instrumental in causing anthrax disease symptoms leading to death. Anthrax toxin consists of three proteins, protective antigen (PA), which is a receptorbinding and pore-forming subunit; lethal factor (LF), which is a protease that cleaves mitogen-activated protein kinase kinase family members; and edema factor (EF), which is an adenylate cyclase that raises cAMP levels in cells (1). PA is synthesized as an 83-kDa protein (PA 83 ) for which two cell surface receptors have been identified: tumor endothelial marker 8 (TEM8) (2, 3) and capillary morphogenesis protein 2 (CMG2) (4). Two splice variant mRNAs derived from the TEM8 gene (sv1 and sv2) encode functional anthrax toxin receptors (2, 5). TEM8 expression has been documented in epithelium of the lung, intestine, and skin, the three routes of entry in anthrax infection (6). The CMG2 gene, which has been shown to be broadly expressed in different tissues (4), encodes three protein isoforms, two of which, CMG2 488 and CMG2 489 , are anthrax toxin receptors (4) (H.M.S., unpublished data).Receptor-bound PA 83 is cleaved by a cellular protease to generate a 20-kDa PA 20 subunit and a 63-kDa subunit (PA 63 ). The larger subunit assembles into the heptameric (PA 63 ) 7 prepore in lipid rafts (7-9). EF and LF bind to the prepore, and the toxin-receptor complexes are internalized by clathrindependent endocytosis and by other endocytic mechanisms (9, 10). These complexes are then exp...

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Section: Resultsmentioning

confidence: 85%

mentioning

confidence: 99%

Receptor-specific requirements for anthrax toxin delivery into cells

Rainey

Wigelsworth

Ryan

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

119

157

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“…To investigate a possible role of this environment in the sporicidal activity of MQs, the effect of inhibitors of phagolysosomal acidification on viability of the phagocytosed spore was studied. Bafilomycins are specific inhibitors of the vacuolar ATPase proton pump, which is responsible for the acidic pH of endosomes and lysosomes [20]; the lysosomotropic amine chloroquine also dissipates intracellular proton gradients and raises the pH of intracellular vesicles [16]. MQs treated with either bafilomycin A1 or chloroquine were compromised in their sporicidal activity and the phagocytosed spores were able to germinate, outgrow and escape from the MQ (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Compounds tested included bafilomycin A1 and the lysosomotropic amine chloroquine [16][17][18][19][20]. MQs treated with bafilomycin A1 exhibited larger numbers of germinated spores microscopically after the 1-h phagocytosis period than untreated cultures and the treated MQs permitted the outgrowth and replication of the organisms (Figs 1b and 3a).…”

Section: Viability Of B Anthracis Strain Ames After Phagocytosismentioning

confidence: 99%

In-vitro characterisation of the phagocytosis and fate of anthrax spores in macrophages and the effects of anti-PA antibody

Welkos¹,

Friedlander

Weeks³

et al. 2002

Journal of Medical Microbiology

131

157

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“…4) (32, 49, 469). A decreased pH probably induces conformational changes and membrane insertion of the heptameric "B" component, followed by translocation of the "A" component(s) across the endosomal membrane, which is also mimicked on the cell surface by simply lowering the pH of the medium (32,49,50,131,157,247,267,272,392,400). However, there are unique pH requirements for binary toxins such as C2 and , since the latter requires a lower pH (Յ5.0) than the former (Յ5.5) for direct cytosolic entry through the cell membrane.…”

Section: Cell Entry and Intoxication "B" Binding To The Cellmentioning

confidence: 99%

Binary Bacterial Toxins: Biochemistry, Biology, and Applications of CommonClostridiumandBacillusProteins

Barth

Aktories

Popoff

et al. 2004

Microbiol Mol Biol Rev

375

388

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Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic “A-B” paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The “B” components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated “B” components form homoheptameric rings that subsequently dock with an “A” component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, “A” molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria

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The vacuolar ATPase proton pump is required for the cytotoxicity of Bacillus anthracis lethal toxin

Cited by 45 publications

References 30 publications

Receptor-specific requirements for anthrax toxin delivery into cells

Receptor-specific requirements for anthrax toxin delivery into cells

In-vitro characterisation of the phagocytosis and fate of anthrax spores in macrophages and the effects of anti-PA antibody

Binary Bacterial Toxins: Biochemistry, Biology, and Applications of CommonClostridiumandBacillusProteins

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