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Tessier E, Cheutin L, Garnier A, Vigne C, Tournier JN, Rougeaux C. Early Circulating Edema Factor in Inhalational Anthrax Infection: Does It Matter? Microorganisms 2024; 12:308. [PMID: 38399712 PMCID: PMC10891819 DOI: 10.3390/microorganisms12020308] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2023] [Revised: 01/22/2024] [Accepted: 01/26/2024] [Indexed: 02/25/2024] Open
Abstract
Anthrax toxins are critical virulence factors of Bacillus anthracis and Bacillus cereus strains that cause anthrax-like disease, composed of a common binding factor, the protective antigen (PA), and two enzymatic proteins, lethal factor (LF) and edema factor (EF). While PA is required for endocytosis and activity of EF and LF, several studies showed that these enzymatic factors disseminate within the body in the absence of PA after intranasal infection. In an effort to understand the impact of EF in the absence of PA, we used a fluorescent EF chimera to facilitate the study of endocytosis in different cell lines. Unexpectedly, EF was found inside cells in the absence of PA and showed a pole-dependent endocytosis. However, looking at enzymatic activity, PA was still required for EF to induce an increase in intracellular cAMP levels. Interestingly, the sequential delivery of EF and then PA rescued the rise in cAMP levels, indicating that PA and EF may functionally associate during intracellular trafficking, as well as it did at the cell surface. Our data shed new light on EF trafficking and the potential location of PA and EF association for optimal cytosolic delivery.
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Affiliation(s)
- Emilie Tessier
- Département des Maladies Infectieuses, Institut de Recherche Biomédicale des Armées, 91220 Brétigny-sur-Orge, France (C.R.)
| | - Laurence Cheutin
- Département des Maladies Infectieuses, Institut de Recherche Biomédicale des Armées, 91220 Brétigny-sur-Orge, France (C.R.)
| | - Annabelle Garnier
- Département des Maladies Infectieuses, Institut de Recherche Biomédicale des Armées, 91220 Brétigny-sur-Orge, France (C.R.)
| | - Clarisse Vigne
- Département des Maladies Infectieuses, Institut de Recherche Biomédicale des Armées, 91220 Brétigny-sur-Orge, France (C.R.)
| | - Jean-Nicolas Tournier
- Département des Maladies Infectieuses, Institut de Recherche Biomédicale des Armées, 91220 Brétigny-sur-Orge, France (C.R.)
- Institut Pasteur, 75015 Paris, France
| | - Clémence Rougeaux
- Département des Maladies Infectieuses, Institut de Recherche Biomédicale des Armées, 91220 Brétigny-sur-Orge, France (C.R.)
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2
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Yang NJ, Isensee J, Neel DV, Quadros AU, Zhang HXB, Lauzadis J, Liu SM, Shiers S, Belu A, Palan S, Marlin S, Maignel J, Kennedy-Curran A, Tong VS, Moayeri M, Röderer P, Nitzsche A, Lu M, Pentelute BL, Brüstle O, Tripathi V, Foster KA, Price TJ, Collier RJ, Leppla SH, Puopolo M, Bean BP, Cunha TM, Hucho T, Chiu IM. Anthrax toxins regulate pain signaling and can deliver molecular cargoes into ANTXR2 + DRG sensory neurons. Nat Neurosci 2021; 25:168-179. [PMID: 34931070 DOI: 10.1038/s41593-021-00973-8] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2020] [Accepted: 11/01/2021] [Indexed: 11/09/2022]
Abstract
Bacterial products can act on neurons to alter signaling and function. In the present study, we found that dorsal root ganglion (DRG) sensory neurons are enriched for ANTXR2, the high-affinity receptor for anthrax toxins. Anthrax toxins are composed of protective antigen (PA), which binds to ANTXR2, and the protein cargoes edema factor (EF) and lethal factor (LF). Intrathecal administration of edema toxin (ET (PA + EF)) targeted DRG neurons and induced analgesia in mice. ET inhibited mechanical and thermal sensation, and pain caused by formalin, carrageenan or nerve injury. Analgesia depended on ANTXR2 expressed by Nav1.8+ or Advillin+ neurons. ET modulated protein kinase A signaling in mouse sensory and human induced pluripotent stem cell-derived sensory neurons, and attenuated spinal cord neurotransmission. We further engineered anthrax toxins to introduce exogenous protein cargoes, including botulinum toxin, into DRG neurons to silence pain. Our study highlights interactions between a bacterial toxin and nociceptors, which may lead to the development of new pain therapeutics.
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Affiliation(s)
- Nicole J Yang
- Department of Immunology, Harvard Medical School, Boston, MA, USA
| | - Jörg Isensee
- Translational Pain Research, Department of Anesthesiology and Intensive Care Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Dylan V Neel
- Department of Immunology, Harvard Medical School, Boston, MA, USA
| | - Andreza U Quadros
- Center for Research in Inflammatory Diseases, Department of Pharmacology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil
| | | | - Justas Lauzadis
- Department of Anesthesiology, Stony Brook Medicine, Stony Brook, NY, USA
| | | | - Stephanie Shiers
- Department of Neuroscience, Center for Advanced Pain Studies, University of Texas at Dallas, Richardson, TX, USA
| | - Andreea Belu
- Translational Pain Research, Department of Anesthesiology and Intensive Care Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | | | | | | | | | - Victoria S Tong
- Department of Immunology, Harvard Medical School, Boston, MA, USA
| | - Mahtab Moayeri
- Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Pascal Röderer
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Bonn, Germany.,Cellomics Unit, LIFE & BRAIN GmbH, Bonn, Germany
| | - Anja Nitzsche
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Bonn, Germany.,Cellomics Unit, LIFE & BRAIN GmbH, Bonn, Germany
| | - Mike Lu
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Bradley L Pentelute
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA.,The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.,Broad Institute of MIT and Harvard, Cambridge, MA, USA.,Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Oliver Brüstle
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Bonn, Germany
| | | | | | - Theodore J Price
- Department of Neuroscience, Center for Advanced Pain Studies, University of Texas at Dallas, Richardson, TX, USA
| | - R John Collier
- Department of Microbiology, Harvard Medical School, Boston, MA, USA
| | - Stephen H Leppla
- Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Michelino Puopolo
- Department of Anesthesiology, Stony Brook Medicine, Stony Brook, NY, USA
| | - Bruce P Bean
- Department of Neurobiology, Harvard Medical School, Boston, MA, USA
| | - Thiago M Cunha
- Center for Research in Inflammatory Diseases, Department of Pharmacology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil
| | - Tim Hucho
- Translational Pain Research, Department of Anesthesiology and Intensive Care Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Isaac M Chiu
- Department of Immunology, Harvard Medical School, Boston, MA, USA.
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3
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Bordetella Adenylate Cyclase Toxin Elicits Airway Mucin Secretion through Activation of the cAMP Response Element Binding Protein. Int J Mol Sci 2021; 22:ijms22169064. [PMID: 34445770 PMCID: PMC8396599 DOI: 10.3390/ijms22169064] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2021] [Revised: 08/14/2021] [Accepted: 08/19/2021] [Indexed: 12/30/2022] Open
Abstract
The mucus layer protects airway epithelia from damage by noxious agents. Intriguingly, Bordetella pertussis bacteria provoke massive mucus production by nasopharyngeal epithelia during the initial coryza-like catarrhal stage of human pertussis and the pathogen transmits in mucus-containing aerosol droplets expelled by sneezing and post-nasal drip-triggered cough. We investigated the role of the cAMP-elevating adenylate cyclase (CyaA) and pertussis (PT) toxins in the upregulation of mucin production in B. pertussis-infected airway epithelia. Using human pseudostratified airway epithelial cell layers cultured at air–liquid interface (ALI), we show that purified CyaA and PT toxins (100 ng/mL) can trigger production of the major airway mucins Muc5AC and Muc5B. Upregulation of mucin secretion involved activation of the cAMP response element binding protein (CREB) and was blocked by the 666-15-Calbiochem inhibitor of CREB-mediated gene transcription. Intriguingly, a B. pertussis mutant strain secreting only active PT and producing the enzymatically inactive CyaA-AC– toxoid failed to trigger any important mucus production in infected epithelial cell layers in vitro or in vivo in the tracheal epithelia of intranasally infected mice. In contrast, the PT– toxoid-producing B. pertussis mutant secreting the active CyaA toxin elicited a comparable mucin production as infection of epithelial cell layers or tracheal epithelia of infected mice by the wild-type B. pertussis secreting both PT and CyaA toxins. Hence, the cAMP-elevating activity of B. pertussis-secreted CyaA was alone sufficient for activation of mucin production through a CREB-dependent mechanism in B. pertussis-infected airway epithelia in vivo.
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4
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Hong S, Pawel GT, Pei R, Lu Y. Recent progress in developing fluorescent probes for imaging cell metabolites. Biomed Mater 2021; 16. [PMID: 33915523 DOI: 10.1088/1748-605x/abfd11] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2020] [Accepted: 04/29/2021] [Indexed: 01/12/2023]
Abstract
Cellular metabolites play a crucial role in promoting and regulating cellular activities, but it has been difficult to monitor these cellular metabolites in living cells and in real time. Over the past decades, iterative development and improvements of fluorescent probes have been made, resulting in the effective monitoring of metabolites. In this review, we highlight recent progress in the use of fluorescent probes for tracking some key metabolites, such as adenosine triphosphate, cyclic adenosine monophosphate, cyclic guanosine 5'-monophosphate, Nicotinamide adenine dinucleotide (NADH), reactive oxygen species, sugar, carbon monoxide, and nitric oxide for both whole cell and subcellular imaging.
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Affiliation(s)
- Shanni Hong
- Department of Medical Imaging Technology, School of Medical Technology and Engineering, Fujian Medical University, Fuzhou, People's Republic of China.,Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, United States of America.,CAS Key Laboratory of Nano-Bio Interfaces, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, People's Republic of China
| | - Gregory T Pawel
- Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, United States of America
| | - Renjun Pei
- CAS Key Laboratory of Nano-Bio Interfaces, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, People's Republic of China
| | - Yi Lu
- Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, United States of America
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5
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Bioengineering of Bordetella pertussis Adenylate Cyclase Toxin for Vaccine Development and Other Biotechnological Purposes. Toxins (Basel) 2021; 13:toxins13020083. [PMID: 33499260 PMCID: PMC7911819 DOI: 10.3390/toxins13020083] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2020] [Revised: 01/14/2021] [Accepted: 01/15/2021] [Indexed: 12/15/2022] Open
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 facilitate 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.
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6
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Angely C, Ladant D, Planus E, Louis B, Filoche M, Chenal A, Isabey D. Functional and structural consequences of epithelial cell invasion by Bordetella pertussis adenylate cyclase toxin. PLoS One 2020; 15:e0228606. [PMID: 32392246 PMCID: PMC7213728 DOI: 10.1371/journal.pone.0228606] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2020] [Accepted: 04/18/2020] [Indexed: 01/13/2023] Open
Abstract
Bordetella pertussis, the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at low concentrations (0.5 nM), CyaA could significantly impair the migration and wound healing capacities of the intoxicated alveolar epithelial cells. As such concentrations might be reached locally during B. pertussis infection, our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, a hallmark of pertussis.
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Affiliation(s)
- Christelle Angely
- Equipe 13, Biomécanique & Appareil Respiratoire, Inserm U955, Créteil, France
- UMR 955, UPEC, Université Paris-Est, Créteil, France
- ERL 7000, CNRS, Créteil, France
| | - Daniel Ladant
- Unité de Biochimie des Interactions Macromoléculaires (CNRS UMR 3528), Département de Biologie Structurale et Chimie, Institut Pasteur, Paris, France
| | - Emmanuelle Planus
- Institut pour l’Avancée des Biosciences (IAB), Centre de Recherche UGA/ Inserm U1209 / CNRS UMR 5309, La Tronche, France
| | - Bruno Louis
- Equipe 13, Biomécanique & Appareil Respiratoire, Inserm U955, Créteil, France
- UMR 955, UPEC, Université Paris-Est, Créteil, France
- ERL 7000, CNRS, Créteil, France
| | - Marcel Filoche
- Equipe 13, Biomécanique & Appareil Respiratoire, Inserm U955, Créteil, France
- UMR 955, UPEC, Université Paris-Est, Créteil, France
- ERL 7000, CNRS, Créteil, France
- Laboratoire de Physique de la Matière Condensée, Ecole Polytechnique, CNRS, IP Paris, Palaiseau, France
| | - Alexandre Chenal
- Unité de Biochimie des Interactions Macromoléculaires (CNRS UMR 3528), Département de Biologie Structurale et Chimie, Institut Pasteur, Paris, France
| | - Daniel Isabey
- Equipe 13, Biomécanique & Appareil Respiratoire, Inserm U955, Créteil, France
- UMR 955, UPEC, Université Paris-Est, Créteil, France
- ERL 7000, CNRS, Créteil, France
- * E-mail:
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7
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Stiles BG. Clostridial Binary Toxins: Basic Understandings that Include Cell Surface Binding and an Internal "Coup de Grâce". Curr Top Microbiol Immunol 2019; 406:135-162. [PMID: 27380267 DOI: 10.1007/82_2016_11] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Clostridium species can make a remarkable number of different protein toxins, causing many diverse diseases in humans and animals. The binary toxins of Clostridium botulinum, C. difficile, C. perfringens, and C. spiroforme are one group of enteric-acting toxins that attack the actin cytoskeleton of various cell types. These enterotoxins consist of A (enzymatic) and B (cell binding/membrane translocation) components that assemble on the targeted cell surface or in solution, forming a multimeric complex. Once translocated into the cytosol via endosomal trafficking and acidification, the A component dismantles the filamentous actin-based cytoskeleton via mono-ADP-ribosylation of globular actin. Knowledge of cell surface receptors and how these usurped, host-derived molecules facilitate intoxication can lead to novel ways of defending against these clostridial binary toxins. A molecular-based understanding of the various steps involved in toxin internalization can also unveil therapeutic intervention points that stop the intoxication process. Furthermore, using these bacterial proteins as medicinal shuttle systems into cells provides intriguing possibilities in the future. The pertinent past and state-of-the-art present, regarding clostridial binary toxins, will be evident in this chapter.
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Affiliation(s)
- Bradley G Stiles
- Biology Department, Wilson College, Chambersburg, PA, 17201, USA.
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8
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Hasan S, Rahman WU, Sebo P, Osicka R. Distinct Spatiotemporal Distribution of Bacterial Toxin-Produced Cellular cAMP Differentially Inhibits Opsonophagocytic Signaling. Toxins (Basel) 2019; 11:toxins11060362. [PMID: 31226835 PMCID: PMC6628411 DOI: 10.3390/toxins11060362] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2019] [Revised: 06/12/2019] [Accepted: 06/18/2019] [Indexed: 01/09/2023] Open
Abstract
Myeloid phagocytes have evolved to rapidly recognize invading pathogens and clear them through opsonophagocytic killing. The adenylate cyclase toxin (CyaA) of Bordetella pertussis and the edema toxin (ET) of Bacillus anthracis are both calmodulin-activated toxins with adenylyl cyclase activity that invade host cells and massively increase the cellular concentrations of a key second messenger molecule, 3',5'-cyclic adenosine monophosphate (cAMP). However, the two toxins differ in the kinetics and mode of cell entry and generate different cAMP concentration gradients within the cell. While CyaA rapidly penetrates cells directly across their plasma membrane, the cellular entry of ET depends on receptor-mediated endocytosis and translocation of the enzymatic subunit across the endosomal membrane. We show that CyaA-generated membrane-proximal cAMP gradient strongly inhibits the activation and phosphorylation of Syk, Vav, and Pyk2, thus inhibiting opsonophagocytosis. By contrast, at similar overall cellular cAMP levels, the ET-generated perinuclear cAMP gradient poorly inhibits the activation and phosphorylation of these signaling proteins. Hence, differences in spatiotemporal distribution of cAMP produced by the two adenylyl cyclase toxins differentially affect the opsonophagocytic signaling in myeloid phagocytes.
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Affiliation(s)
- Shakir Hasan
- Institute of Microbiology of the CAS, v. v. i., Videnska 1083, 142 20 Prague, Czech Republic.
| | - Waheed Ur Rahman
- Institute of Microbiology of the CAS, v. v. i., Videnska 1083, 142 20 Prague, Czech Republic.
| | - Peter Sebo
- Institute of Microbiology of the CAS, v. v. i., Videnska 1083, 142 20 Prague, Czech Republic.
| | - Radim Osicka
- Institute of Microbiology of the CAS, v. v. i., Videnska 1083, 142 20 Prague, Czech Republic.
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9
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Chenal A, Ladant D. Bioengineering of Bordetella pertussis Adenylate Cyclase Toxin for Antigen-Delivery and Immunotherapy. Toxins (Basel) 2018; 10:E302. [PMID: 30037010 PMCID: PMC6070788 DOI: 10.3390/toxins10070302] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2018] [Revised: 07/16/2018] [Accepted: 07/18/2018] [Indexed: 11/16/2022] Open
Abstract
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 where, upon activation by endogenous calmodulin, it synthesizes massive amounts of cAMP that alters cellular physiology. The CyaA toxin is a 1706 residues-long bifunctional protein: the catalytic domain is located in the 400 amino-proximal residues, whereas the carboxy-terminal 1306 residues are implicated in toxin binding to the cellular receptor, the αMβ₂ (CD11b/CD18) integrin, and subsequently in the translocation of the catalytic domain across the cytoplasmic membrane of the target cells. Indeed, this protein is endowed with the unique capability of delivering its N-terminal catalytic domain directly across the plasma membrane of eukaryotic target cells. These properties have been exploited to engineer the CyaA toxin as a potent non-replicating vector able to deliver antigens into antigen presenting cells and elicit specific cell-mediated immune responses. Antigens of interest can be inserted into the CyaA protein to yield recombinant molecules that are targeted in vivo to dendritic cells, where the antigens are processed and presented by the major class I and class II histocompatibility complexes (MHC-I and II). CyaA turned out to be a remarkably effective and versatile vaccine vector capable of inducing all the components of the immune response (T-CD4, T-CD8, and antibody). In this chapter, we summarize the basic knowledge on the adenylate cyclase toxin and then describe the application of CyaA in vaccinology, including some recent results of clinical trials of immunotherapy using a recombinant CyaA vaccine.
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Affiliation(s)
- Alexandre Chenal
- Institut Pasteur, Biochemistry of Macromolecular Interactions Unit, UMR CNRS 3528, Structural Biology and Chemistry Department, 28 rue du Docteur Roux, 75724 Paris CEDEX 15, France.
| | - Daniel Ladant
- Institut Pasteur, Biochemistry of Macromolecular Interactions Unit, UMR CNRS 3528, Structural Biology and Chemistry Department, 28 rue du Docteur Roux, 75724 Paris CEDEX 15, France.
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10
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Arumugham VB, Ulivieri C, Onnis A, Finetti F, Tonello F, Ladant D, Baldari CT. Compartmentalized Cyclic AMP Production by the Bordetella pertussis and Bacillus anthracis Adenylate Cyclase Toxins Differentially Affects the Immune Synapse in T Lymphocytes. Front Immunol 2018; 9:919. [PMID: 29765373 PMCID: PMC5938339 DOI: 10.3389/fimmu.2018.00919] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2018] [Accepted: 04/13/2018] [Indexed: 01/01/2023] Open
Abstract
A central feature of the immune synapse (IS) is the tight compartmentalization of membrane receptors and signaling mediators that is functional for its ability to coordinate T cell activation. Second messengers centrally implicated in this process, such as Ca2+ and diacyl glycerol, also undergo compartmentalization at the IS. Current evidence suggests a more complex scenario for cyclic AMP (cAMP), which acts both as positive and as negative regulator of T-cell antigen receptor (TCR) signaling and which, as such, must be subjected to a tight spatiotemporal control to allow for signaling at the IS. Here, we have used two bacterial adenylate cyclase toxins that produce cAMP at different subcellular localizations as the result of their distinct routes of cell invasion, namely Bordetella pertussis CyaA and Bacillus anthracis edema toxin (ET), to address the ability of the T cell to confine cAMP to the site of production and to address the impact of compartmentalized cAMP production on IS assembly and function. We show that CyaA, which produces cAMP close to the synaptic membrane, affects IS stability by modulating not only the distribution of LFA-1 and its partners talin and L-plastin, as previously partly reported but also by promoting the sustained synaptic accumulation of the A-kinase adaptor protein ezrin and protein kinase A while suppressing the β-arrestin-mediated recruitment of phosphodiesterase 4B. These effects are dependent on the catalytic activity of the toxin and can be reproduced by treatment with a non-hydrolyzable cAMP analog. Remarkably, none of these effects are elicited by ET, which produces cAMP at a perinuclear localization, despite its ability to suppress TCR signaling and T cell activation through its cAMP-elevating activity. These results show that the IS responds solely to local elevations of cAMP and provide evidence that potent compartmentalization mechanisms are operational in T cells to contain cAMP close to the site of production, even when produced at supraphysiological levels.
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Affiliation(s)
| | | | - Anna Onnis
- Department of Life Sciences, University of Siena, Siena, Italy
| | | | - Fiorella Tonello
- Neuroscience Institute, Italian National Research Council, Padua, Italy
| | - Daniel Ladant
- Department of Structural Biology and Chemistry, Institut Pasteur, Paris, France
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11
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Understanding the Mechanism of Translocation of Adenylate Cyclase Toxin across Biological Membranes. Toxins (Basel) 2017; 9:toxins9100295. [PMID: 28934133 PMCID: PMC5666342 DOI: 10.3390/toxins9100295] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2017] [Revised: 09/13/2017] [Accepted: 09/15/2017] [Indexed: 01/05/2023] Open
Abstract
Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal adenylate cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ≈400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called "two-step model" and the recently-proposed "toroidal pore model", will be considered.
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12
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Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain. Proc Natl Acad Sci U S A 2017; 114:E6784-E6793. [PMID: 28760979 DOI: 10.1073/pnas.1701783114] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
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.
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The Molecular Basis of Toxins' Interactions with Intracellular Signaling via Discrete Portals. Toxins (Basel) 2017; 9:toxins9030107. [PMID: 28300784 PMCID: PMC5371862 DOI: 10.3390/toxins9030107] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2017] [Revised: 03/02/2017] [Accepted: 03/04/2017] [Indexed: 12/20/2022] Open
Abstract
An understanding of the molecular mechanisms by which microbial, plant or animal-secreted toxins exert their action provides the most important element for assessment of human health risks and opens new insights into therapies addressing a plethora of pathologies, ranging from neurological disorders to cancer, using toxinomimetic agents. Recently, molecular and cellular biology dissecting tools have provided a wealth of information on the action of these diverse toxins, yet, an integrated framework to explain their selective toxicity is still lacking. In this review, specific examples of different toxins are emphasized to illustrate the fundamental mechanisms of toxicity at different biochemical, molecular and cellular- levels with particular consideration for the nervous system. The target of primary action has been highlighted and operationally classified into 13 sub-categories. Selected examples of toxins were assigned to each target category, denominated as portal, and the modulation of the different portal’s signaling was featured. The first portal encompasses the plasma membrane lipid domains, which give rise to pores when challenged for example with pardaxin, a fish toxin, or is subject to degradation when enzymes of lipid metabolism such as phospholipases A2 (PLA2) or phospholipase C (PLC) act upon it. Several major portals consist of ion channels, pumps, transporters and ligand gated ionotropic receptors which many toxins act on, disturbing the intracellular ion homeostasis. Another group of portals consists of G-protein-coupled and tyrosine kinase receptors that, upon interaction with discrete toxins, alter second messengers towards pathological levels. Lastly, subcellular organelles such as mitochondria, nucleus, protein- and RNA-synthesis machineries, cytoskeletal networks and exocytic vesicles are also portals targeted and deregulated by other diverse group of toxins. A fundamental concept can be drawn from these seemingly different toxins with respect to the site of action and the secondary messengers and signaling cascades they trigger in the host. While the interaction with the initial portal is largely determined by the chemical nature of the toxin, once inside the cell, several ubiquitous second messengers and protein kinases/ phosphatases pathways are impaired, to attain toxicity. Therefore, toxins represent one of the most promising natural molecules for developing novel therapeutics that selectively target the major cellular portals involved in human physiology and diseases.
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Interrogating cyclic AMP signaling using optical approaches. Cell Calcium 2017; 64:47-56. [PMID: 28274483 DOI: 10.1016/j.ceca.2017.02.010] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2017] [Accepted: 02/20/2017] [Indexed: 11/23/2022]
Abstract
Optical reporters for cAMP represent a fundamental advancement in our ability to investigate the dynamics of cAMP signaling. These fluorescent sensors can measure changes in cAMP in single cells or in microdomains within cells as opposed to whole populations of cells required for other methods of measuring cAMP. The first optical cAMP reporters were FRET-based sensors utilizing dissociation of purified regulatory and catalytic subunits of PKA, introduced by Roger Tsien in the early 1990s. The utility of these sensors was vastly improved by creating genetically encoded versions that could be introduced into cells with transfection, the first of which was published in the year 2000. Subsequently, improved sensors have been developed using different cAMP binding platforms, optimized fluorescent proteins, and targeting motifs that localize to specific microdomains. The most common sensors in use today are FRET-based sensors designed around an Epac backbone. These rely on the significant conformational changes in Epac when it binds cAMP, altering the signal between FRET pairs flanking Epac. Several other strategies for optically interrogating cAMP have been developed, including fluorescent translocation reporters, dimerization-dependent FP based biosensors, BRET (bioluminescence resonance energy transfer)-based sensors, non-FRET single wavelength reporters, and sensors based on bacterial cAMP-binding domains. Other newly described mammalian cAMP-binding proteins such as Popdc and CRIS may someday be exploited in sensor design. With the proliferation of engineered fluorescent proteins and the abundance of cAMP binding targets in nature, the field of optical reporters for cAMP should continue to see rapid refinement in the coming years.
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Israeli M, Rotem S, Elia U, Bar-Haim E, Cohen O, Chitlaru T. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity. Toxins (Basel) 2016; 8:E243. [PMID: 27548219 PMCID: PMC4999859 DOI: 10.3390/toxins8080243] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2016] [Revised: 08/09/2016] [Accepted: 08/15/2016] [Indexed: 01/27/2023] Open
Abstract
Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules.
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Affiliation(s)
- Ma'ayan Israeli
- Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel.
| | - Shahar Rotem
- Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel.
| | - Uri Elia
- Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel.
| | - Erez Bar-Haim
- Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel.
| | - Ofer Cohen
- Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel.
| | - Theodor Chitlaru
- Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel.
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Micropatterned macrophage analysis reveals global cytoskeleton constraints induced by Bacillus anthracis edema toxin. Infect Immun 2015; 83:3114-25. [PMID: 26015478 DOI: 10.1128/iai.00479-15] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2015] [Accepted: 05/16/2015] [Indexed: 12/20/2022] Open
Abstract
Bacillus anthracis secretes the edema toxin (ET) that disrupts the cellular physiology of endothelial and immune cells, ultimately affecting the adherens junction integrity of blood vessels that in turn leads to edema. The effects of ET on the cytoskeleton, which is critical in cell physiology, have not been described thus far on macrophages. In this study, we have developed different adhesive micropatterned surfaces (L and crossbow) to control the shape of bone marrow-derived macrophages (BMDMs) and primary peritoneal macrophages. We found that macrophage F-actin cytoskeleton adopts a specific polar organization slightly different from classical human HeLa cells on the micropatterns. Moreover, ET induced a major quantitative reorganization of F-actin within 16 h with a collapse at the nonadhesive side of BMDMs along the nucleus. There was an increase in size and deformation into a kidney-like shape, followed by a decrease in size that correlates with a global cellular collapse. The collapse of F-actin was correlated with a release of focal adhesion on the patterns and decreased cell size. Finally, the cell nucleus was affected by actin reorganization. By using this technology, we could describe many previously unknown macrophage cellular dysfunctions induced by ET. This novel tool could be used to analyze more broadly the effects of toxins and other virulence factors that target the cytoskeleton.
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Gao R, Ko J, Cha K, Jeon JH, Rhie GE, Choi J, deMello AJ, Choo J. Fast and sensitive detection of an anthrax biomarker using SERS-based solenoid microfluidic sensor. Biosens Bioelectron 2015; 72:230-6. [PMID: 25985198 DOI: 10.1016/j.bios.2015.05.005] [Citation(s) in RCA: 67] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2015] [Revised: 04/30/2015] [Accepted: 05/04/2015] [Indexed: 02/03/2023]
Abstract
We report the application of a fully automated surface-enhanced Raman scattering (SERS)-based solenoid-embedded microfluidic device to the quantitative and sensitive detection of anthrax biomarker poly-γ-D-glutamic acid (PGA) in solution. Analysis is based on the competitive reaction between PGA and PGA-conjugated gold nanoparticles with anti-PGA-immobilized magnetic beads within a microfluidic environment. Magnetic immunocomplexes are trapped by yoke-type solenoids embedded within the device, and their SERS signals were directly measured and analyzed. To improve the accuracy of measurement process, external standard values for PGA-free serum were also measured through use of a control channel. This additional measurement greatly improves the reliability of the assay by minimizing the influence of extraneous experimental variables. The limit of detection (LOD) of PGA in serum, determined by our SERS-based microfluidic sensor, is estimated to be 100 pg/mL. We believe that the defined method represents a valuable analytical tool for the detection of anthrax-related aqueous samples.
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Affiliation(s)
- Rongke Gao
- Department of Bionano Technology, Hanyang University, Ansan 426-791, South Korea
| | - Juhui Ko
- Department of Bionano Technology, Hanyang University, Ansan 426-791, South Korea
| | - Kiweon Cha
- Division of High-Risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, Cheongju 363-951, South Korea
| | - Jun Ho Jeon
- Division of High-Risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, Cheongju 363-951, South Korea
| | - Gi-eun Rhie
- Division of High-Risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, Cheongju 363-951, South Korea
| | - Jonghoon Choi
- Department of Bionano Technology, Hanyang University, Ansan 426-791, South Korea
| | - Andrew J deMello
- Department of Chemistry and Applied Biosciences, Institute of Chemical and Bioengineering, ETH Zürich, Vladimir Prelog Weg 1, 8093 Zürich, Switzerland.
| | - Jaebum Choo
- Department of Bionano Technology, Hanyang University, Ansan 426-791, South Korea.
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Goel AK. Anthrax: A disease of biowarfare and public health importance. World J Clin Cases 2015; 3:20-33. [PMID: 25610847 PMCID: PMC4295216 DOI: 10.12998/wjcc.v3.i1.20] [Citation(s) in RCA: 140] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/28/2014] [Revised: 10/23/2014] [Accepted: 10/31/2014] [Indexed: 02/05/2023] Open
Abstract
Bioterrorism has received a lot of attention in the first decade of this century. Biological agents are considered attractive weapons for bioterrorism as these are easy to obtain, comparatively inexpensive to produce and exhibit widespread fear and panic than the actual potential of physical damage. Bacillus anthracis (B. anthracis), the etiologic agent of anthrax is a Gram positive, spore forming, non-motile bacterium. This is supposed to be one of the most potent BW agents because its spores are extremely resistant to natural conditions and can survive for several decades in the environment. B. anthracis spores enter the body through skin lesion (cutaneous anthrax), lungs (pulmonary anthrax), or gastrointestinal route (gastrointestinal anthrax) and germinate, giving rise to the vegetative form. Anthrax is a concern of public health also in many countries where agriculture is the main source of income including India. Anthrax has been associated with human history for a very long time and regained its popularity after Sept 2001 incidence in United States. The present review article describes the history, biology, life cycle, pathogenicity, virulence, epidemiology and potential of B. anthracis as biological weapon.
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Abstract
INTRODUCTION Present-day rational drug design approaches are based on exploiting unique features of the target biomolecules, small- or macromolecule drug candidates and physical forces that govern their interactions. The 2013 Nobel Prize in chemistry awarded 'for the development of multiscale models for complex chemical systems' once again demonstrated the importance of the tailored drug discovery that reduces the role of the trial-and-error approach to a minimum. The intentional dissemination of Bacillus anthracis spores in 2001 via the so-called anthrax letters has led to increased efforts, politically and scientifically, to develop medical countermeasures that will protect people from the threat of anthrax bioterrorism. AREAS COVERED This article provides an overview of the recent rational drug design approaches for discovering inhibitors of anthrax toxin. The review also directs the readers to the vast literature on the recognized advances and future possibilities in the field. EXPERT OPINION Existing options to combat anthrax toxin lethality are limited. With the only anthrax toxin inhibiting therapy (protective antigen-targeting with a monoclonal antibody, raxibacumab) approved to treat inhalational anthrax, the situation, in our view, is still insecure. Further, the FDA's animal rule for drug approval, which clears compounds without validated efficacy studies on humans, creates a high level of uncertainty, especially when a well-characterized animal model does not exist. Better identification and validation of anthrax toxin therapeutic targets at the molecular level as well as elucidation of the parameters determining the corresponding therapeutic windows are still necessary for more effective therapeutic options.
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Affiliation(s)
- Ekaterina M Nestorovich
- The Catholic University of America, Department of Biology , Washington, DC , USA +1 202 319 6723 ;
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20
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Tournier JN, Ulrich RG, Quesnel-Hellmann A, Mohamadzadeh M, Stiles BG. Anthrax, toxins and vaccines: a 125-year journey targetingBacillus anthracis. Expert Rev Anti Infect Ther 2014; 7:219-36. [DOI: 10.1586/14787210.7.2.219] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
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Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer. Proc Natl Acad Sci U S A 2013; 110:20473-8. [PMID: 24297899 DOI: 10.1073/pnas.1312975110] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
Numerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model toxin, the adenylate cyclase (CyaA) from Bordetella pertussis, is able to invade eukaryotic cells by translocating its catalytic domain directly across the plasma membrane of target cells. To characterize its original translocation process, we designed an in vitro assay based on a biomimetic membrane model in which a tethered lipid bilayer (tBLM) is assembled on an amine-gold surface derivatized with calmodulin (CaM). The assembled bilayer forms a continuous and protein-impermeable boundary completely separating the underlying calmodulin (trans side) from the medium above (cis side). The binding of CyaA to the tBLM is monitored by surface plasmon resonance (SPR) spectroscopy. CyaA binding to the immobilized CaM, revealed by enzymatic activity, serves as a highly sensitive reporter of toxin translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and also on the application of a negative potential, as shown earlier in eukaryotic cells. Thus, CyaA is able to deliver its catalytic domain across a biological membrane without the need for any eukaryotic components besides CaM. This suggests that the calcium-dependent CyaA translocation may be driven in part by the electrical field across the membrane. This study's in vitro demonstration of toxin translocation across a tBLM provides an opportunity to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined experimental conditions.
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22
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Jeong SY, Martchenko M, Cohen SN. Calpain-dependent cytoskeletal rearrangement exploited for anthrax toxin endocytosis. Proc Natl Acad Sci U S A 2013; 110:E4007-15. [PMID: 24085852 PMCID: PMC3801034 DOI: 10.1073/pnas.1316852110] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
The protective antigen component of Bacillus anthracis toxins can interact with at least three distinct proteins on the host cell surface, capillary morphogenesis gene 2 (CMG2), tumor endothelial marker 8, and β1-integrin, and, with the assistance of other host proteins, enters targeted cells by receptor-mediated endocytosis. Using an antisense-based phenotypic screen, we discovered the role of calpains in this process. We show that functions of a ubiquitous Ca(2+)-dependent cysteine protease, calpain-2, and of the calpain substrate talin-1 are exploited for association of anthrax toxin and its principal receptor, CMG2, with higher-order actin filaments and consequently for toxin entry into host cells. Down-regulated expression of calpain-2 or talin-1, or pharmacological interference with calpain action, did not affect toxin binding but reduced endocytosis and increased the survival of cells exposed to anthrax lethal toxin. Adventitious expression of wild-type talin-1 promoted toxin endocytosis and lethality, whereas expression of a talin-1 mutant (L432G) that is insensitive to calpain cleavage did not. Disruption of talin-1, which links integrin-containing focal adhesion complexes to the actin cytoskeleton, facilitated association of toxin bound to its principal cell-surface receptor, CMG2, with higher-order actin filaments undergoing dynamic disassembly and reassembly during endocytosis. Our results reveal a mechanism by which a bacterial toxin uses constitutively occurring calpain-mediated cytoskeletal rearrangement for internalization.
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Affiliation(s)
| | | | - Stanley N. Cohen
- Departments of Genetics and
- Medicine, Stanford University School of Medicine, Stanford, CA 94305
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Lemichez E, Gonzalez-Rodriguez D, Bassereau P, Brochard-Wyart F. Transcellular tunnel dynamics: Control of cellular dewetting by actomyosin contractility and I-BAR proteins. Biol Cell 2013. [PMID: 23189935 DOI: 10.1111/boc.201200063] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Dewetting is the spontaneous withdrawal of a liquid film from a non-wettable surface by nucleation and growth of dry patches. Two recent reports now propose that the principles of dewetting explain the physical phenomena underpinning the opening of transendothelial cell macroaperture (TEM) tunnels, referred to as cellular dewetting. This was discovered by studying a group of bacterial toxins endowed with the property of corrupting actomyosin cytoskeleton contractility. For both liquid and cellular dewetting, the growth of holes is governed by a competition between surface forces and line tension. We also discuss how the dynamics of TEM opening and closure represent remarkable systems to investigate actin cytoskeleton regulation by sensors of plasma membrane curvature and investigate the impact on membrane tension and the role of TEM in vascular dysfunctions.
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Affiliation(s)
- Emmanuel Lemichez
- INSERM, U1065, Université de Nice-Sophia-Antipolis, Centre Méditerranéen de Médecine Moléculaire, C3M, Nice 06204, France.
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Göttle M, Dove S, Seifert R. Bacillus anthracis edema factor substrate specificity: evidence for new modes of action. Toxins (Basel) 2012; 4:505-35. [PMID: 22852066 PMCID: PMC3407890 DOI: 10.3390/toxins4070505] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2012] [Revised: 06/15/2012] [Accepted: 06/27/2012] [Indexed: 12/20/2022] Open
Abstract
Since the isolation of Bacillus anthracis exotoxins in the 1960s, the detrimental activity of edema factor (EF) was considered as adenylyl cyclase activity only. Yet the catalytic site of EF was recently shown to accomplish cyclization of cytidine 5'-triphosphate, uridine 5'-triphosphate and inosine 5'-triphosphate, in addition to adenosine 5'-triphosphate. This review discusses the broad EF substrate specificity and possible implications of intracellular accumulation of cyclic cytidine 3':5'-monophosphate, cyclic uridine 3':5'-monophosphate and cyclic inosine 3':5'-monophosphate on cellular functions vital for host defense. In particular, cAMP-independent mechanisms of action of EF on host cell signaling via protein kinase A, protein kinase G, phosphodiesterases and CNG channels are discussed.
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Affiliation(s)
- Martin Göttle
- Department of Neurology, Emory University School of Medicine, 6302 Woodruff Memorial Research Building, 101 Woodruff Circle, Atlanta, GA 30322, USA
- Author to whom correspondence should be addressed; ; Tel.: +1-404-727-1678; Fax: +1-404-727-3157
| | - Stefan Dove
- Department of Medicinal/Pharmaceutical Chemistry II, University of Regensburg, D-93040 Regensburg, Germany;
| | - Roland Seifert
- Institute of Pharmacology, Medical School of Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany;
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Lowe DE, Glomski IJ. Cellular and physiological effects of anthrax exotoxin and its relevance to disease. Front Cell Infect Microbiol 2012; 2:76. [PMID: 22919667 PMCID: PMC3417473 DOI: 10.3389/fcimb.2012.00076] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2012] [Accepted: 05/16/2012] [Indexed: 12/26/2022] Open
Abstract
Bacillus anthracis, the causative agent of anthrax, secretes a tri-partite exotoxin that exerts pleiotropic effects on the host. The purification of the exotoxin components, protective antigen, lethal factor, and edema factor allowed the rapid characterization of their physiologic effects on the host. As molecular biology matured, interest focused on the molecular mechanisms and cellular alterations induced by intoxication. Only recently have researchers begun to connect molecular and cellular knowledge back to the broader physiological effects of the exotoxin. This review focuses on the progress that has been made bridging molecular knowledge back to the exotoxin’s physiological effects on the host.
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Affiliation(s)
- David E Lowe
- Department of Microbiology, Immunology, and Cancer Biology, University of Virginia Health System, Charlottesville VA, USA
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26
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Maddugoda MP, Stefani C, Gonzalez-Rodriguez D, Saarikangas J, Torrino S, Janel S, Munro P, Doye A, Prodon F, Aurrand-Lions M, Goossens PL, Lafont F, Bassereau P, Lappalainen P, Brochard F, Lemichez E. cAMP signaling by anthrax edema toxin induces transendothelial cell tunnels, which are resealed by MIM via Arp2/3-driven actin polymerization. Cell Host Microbe 2012; 10:464-74. [PMID: 22100162 DOI: 10.1016/j.chom.2011.09.014] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2011] [Revised: 07/19/2011] [Accepted: 09/14/2011] [Indexed: 12/22/2022]
Abstract
RhoA-inhibitory bacterial toxins, such as Staphylococcus aureus EDIN toxin, induce large transendothelial cell macroaperture (TEM) tunnels that rupture the host endothelium barrier and promote bacterial dissemination. Host cells repair these tunnels by extending actin-rich membrane waves from the TEM edges. We reveal that cyclic-AMP signaling produced by Bacillus anthracis edema toxin (ET) also induces TEM formation, which correlates with increased vascular permeability. We show that ET-induced TEM formation resembles liquid dewetting, a physical process of nucleation and growth of holes within a thin liquid film. We also identify the cellular mechanisms of tunnel closure and reveal that the I-BAR domain protein Missing in Metastasis (MIM) senses de novo membrane curvature generated by the TEM, accumulates at the TEM edge, and triggers Arp2/3-dependent actin polymerization, which induces actin-rich membrane waves that close the TEM. Thus, the balance between ET-induced TEM formation and resealing likely determines the integrity of the host endothelium barrier.
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Affiliation(s)
- Madhavi P Maddugoda
- INSERM, U, Université de Nice-Sophia-Antipolis, Centre Méditerranéen de Médecine Moléculaire, Nice, France
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Trescos Y, Tournier JN. Cytoskeleton as an emerging target of anthrax toxins. Toxins (Basel) 2012; 4:83-97. [PMID: 22474568 PMCID: PMC3317109 DOI: 10.3390/toxins4020083] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2012] [Revised: 01/21/2012] [Accepted: 01/26/2012] [Indexed: 01/29/2023] Open
Abstract
Bacillus anthracis, the agent of anthrax, has gained virulence through its exotoxins produced by vegetative bacilli and is composed of three components forming lethal toxin (LT) and edema toxin (ET). So far, little is known about the effects of these toxins on the eukaryotic cytoskeleton. Here, we provide an overview on the general effects of toxin upon the cytoskeleton architecture. Thus, we shall discuss how anthrax toxins interact with their receptors and may disrupt the interface between extracellular matrix and the cytoskeleton. We then analyze what toxin molecular effects on cytoskeleton have been described, before discussing how the cytoskeleton may help the pathogen to corrupt general cell processes such as phagocytosis or vascular integrity.
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Affiliation(s)
- Yannick Trescos
- Unité Interactions Hôte-Agents pathogènes, Institut de Recherche Biomédicale des Armées, Centre de Recherche du Service de Santé des Armées, BP 87, 24 avenue des Maquis du Grésivaudan 38702 La Tronche Cedex, France;
- Ecole du Val-de-Grâce, 1 place Alphonse Lavéran, 75005 Paris, France
| | - Jean-Nicolas Tournier
- Unité Interactions Hôte-Agents pathogènes, Institut de Recherche Biomédicale des Armées, Centre de Recherche du Service de Santé des Armées, BP 87, 24 avenue des Maquis du Grésivaudan 38702 La Tronche Cedex, France;
- Ecole du Val-de-Grâce, 1 place Alphonse Lavéran, 75005 Paris, France
- Author to whom correspondence should be addressed; ; Tel.: +33-4-76636850; Fax: +33-4-76636917
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Karst JC, Barker R, Devi U, Swann MJ, Davi M, Roser SJ, Ladant D, Chenal A. Identification of a region that assists membrane insertion and translocation of the catalytic domain of Bordetella pertussis CyaA toxin. J Biol Chem 2012; 287:9200-12. [PMID: 22241477 DOI: 10.1074/jbc.m111.316166] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The adenylate cyclase (CyaA) toxin, one of the virulence factors secreted by Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, plays a critical role in the early stages of respiratory tract colonization by this bacterium. The CyaA toxin is able to invade eukaryotic cells by translocating its N-terminal catalytic domain directly across the plasma membrane of the target cells, where, activated by endogenous calmodulin, it produces supraphysiological levels of cAMP. How the catalytic domain is transferred from the hydrophilic extracellular medium into the hydrophobic environment of the membrane and then to the cell cytoplasm remains an unsolved question. In this report, we have characterized the membrane-interacting properties of the CyaA catalytic domain. We showed that a protein covering the catalytic domain (AC384, encompassing residues 1-384 of CyaA) displayed no membrane association propensity. However, a longer polypeptide (AC489), encompassing residues 1-489 of CyaA, exhibited the intrinsic property to bind to membranes and to induce lipid bilayer destabilization. We further showed that deletion of residues 375-485 within CyaA totally abrogated the toxin's ability to increase intracellular cAMP in target cells. These results indicate that, whereas the calmodulin dependent enzymatic domain is restricted to the amino-terminal residues 1-384 of CyaA, the membrane-interacting, translocation-competent domain extends up to residue 489. This thus suggests an important role of the region adjacent to the catalytic domain of CyaA in promoting its interaction with and its translocation across the plasma membrane of target cells.
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Affiliation(s)
- Johanna C Karst
- Institut Pasteur, CNRS UMR 3528, Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, Paris, France
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Nguyen C, Feng C, Zhan M, Cross AS, Goldblum SE. Bacillus anthracis-derived edema toxin (ET) counter-regulates movement of neutrophils and macromolecules through the endothelial paracellular pathway. BMC Microbiol 2012; 12:2. [PMID: 22230035 PMCID: PMC3277462 DOI: 10.1186/1471-2180-12-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2011] [Accepted: 01/09/2012] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND A common finding amongst patients with inhalational anthrax is a paucity of polymorphonuclear leukocytes (PMNs) in infected tissues in the face of abundant circulating PMNs. A major virulence determinant of anthrax is edema toxin (ET), which is formed by the combination of two proteins produced by the organism, edema factor (EF), which is an adenyl cyclase, and protective antigen (PA). Since cAMP, a product of adenyl cyclase, is known to enhance endothelial barrier integrity, we asked whether ET might decrease extravasation of PMNs into tissues through closure of the paracellular pathway through which PMNs traverse. RESULTS Pretreatment of human microvascular endothelial cell(EC)s of the lung (HMVEC-L) with ET decreased interleukin (IL)-8-driven transendothelial migration (TEM) of PMNs with a maximal reduction of nearly 60%. This effect required the presence of both EF and PA. Conversely, ET did not diminish PMN chemotaxis in an EC-free system. Pretreatment of subconfluent HMVEC-Ls decreased transendothelial 14 C-albumin flux by ~ 50% compared to medium controls. Coadministration of ET with either tumor necrosis factor-α or bacterial lipopolysaccharide, each at 100 ng/mL, attenuated the increase of transendothelial 14 C-albumin flux caused by either agent alone. The inhibitory effect of ET on TEM paralleled increases in protein kinase A (PKA) activity, but could not be blocked by inhibition of PKA with either H-89 or KT-5720. Finally, we were unable to replicate the ET effect with either forskolin or 3-isobutyl-1-methylxanthine, two agents known to increase cAMP. CONCLUSIONS We conclude that ET decreases IL-8-driven TEM of PMNs across HMVEC-L monolayers independent of cAMP/PKA activity.
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Affiliation(s)
- Chinh Nguyen
- Southern Arizona Veterans Affairs Health Care Systems, Tucson, AZ 85723, USA.
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Barochia AV, Cui X, Sun J, Li Y, Solomon SB, Migone TS, Subramanian GM, Bolmer SD, Eichacker PQ. Protective antigen antibody augments hemodynamic support in anthrax lethal toxin shock in canines. J Infect Dis 2012; 205:818-29. [PMID: 22223857 DOI: 10.1093/infdis/jir834] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
BACKGROUND Anthrax-associated shock is closely linked to lethal toxin (LT) release and is highly lethal despite conventional hemodynamic support. We investigated whether protective antigen-directed monoclonal antibody (PA-mAb) treatment further augments titrated hemodynamic support. METHODS AND RESULTS Forty sedated, mechanically ventilated, instrumented canines challenged with anthrax LT were assigned to no treatment (controls), hemodynamic support alone (protocol-titrated fluids and norepinephrine), PA-mAb alone (administered at start of LT infusion [0 hours] or 9 or 12 hours later), or both, and observed for 96 hours. Although all 8 controls died, 2 of 8 animals receiving hemodynamic support alone survived (median survival times 65 vs 85 hours, respectively; P = .03). PA-mAb alone at 0 hour improved survival (5 of 5 animals survived), but efficacy decreased progressively with delayed treatment (9 hours, 2 of 3 survived; 12 hours, 0 of 4 survived) (P = .004 comparing survival across treatment times). However, combined treatment increased survival irrespective of PA-mAb administration time (0 hours, 4 of 5 animals; 9 hours, 3 of 3 animals; and 12 hours, 4 of 5 animals survived) (P = .95 comparing treatment times). Compared to hemodynamic support alone, when combined over PA-mAb treatment times (0, 9, and 12 hours), combination therapy produced higher survival (P = .008), central venous pressures, and left ventricular ejection fractions, and lower heart rates, norepinephrine requirements and fluid retention (P ≤ .03). CONCLUSIONS PA-mAb may augment conventional hemodynamic support during anthrax LT-associated shock.
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Affiliation(s)
- Amisha V Barochia
- Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.
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Guichard A, Nizet V, Bier E. New insights into the biological effects of anthrax toxins: linking cellular to organismal responses. Microbes Infect 2011; 14:97-118. [PMID: 21930233 DOI: 10.1016/j.micinf.2011.08.016] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2011] [Revised: 08/30/2011] [Accepted: 08/30/2011] [Indexed: 12/15/2022]
Abstract
The anthrax toxins lethal toxin (LT) and edema toxin (ET) are essential virulence factors produced by Bacillus anthracis. These toxins act during two distinct phases of anthrax infection. During the first, prodromal phase, which is often asymptomatic, anthrax toxins act on cells of the immune system to help the pathogen establish infection. Then, during the rapidly progressing (or fulminant) stage of the disease bacteria disseminate via a hematological route to various target tissues and organs, which are typically highly vascularized. As bacteria proliferate in the bloodstream, LT and ET begin to accumulate rapidly reaching a critical threshold level that will cause death even when the bacterial proliferation is curtailed by antibiotics. During this final phase of infection the toxins cause an increase in vascular permeability and a decrease in function of target organs including the heart, spleen, kidney, adrenal gland, and brain. In this review, we examine the various biological effects of anthrax toxins, focusing on the fulminant stage of the disease and on mechanisms by which the two toxins may collaborate to cause cardiovascular collapse. We discuss normal mechanisms involved in maintaining vascular integrity and based on recent studies indicating that LT and ET cooperatively inhibit membrane trafficking to cell-cell junctions we explore several potential mechanisms by which the toxins may achieve their lethal effects. We also summarize the effects of other potential virulence factors secreted by B. anthracis and consider the role of toxic factors in the evolutionarily recent emergence of this devastating disease.
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Affiliation(s)
- Annabel Guichard
- Section of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0349, USA
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Kronhardt A, Rolando M, Beitzinger C, Stefani C, Leuber M, Flatau G, Popoff MR, Benz R, Lemichez E. Cross-reactivity of anthrax and C2 toxin: protective antigen promotes the uptake of botulinum C2I toxin into human endothelial cells. PLoS One 2011; 6:e23133. [PMID: 21850257 PMCID: PMC3151279 DOI: 10.1371/journal.pone.0023133] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2010] [Accepted: 07/13/2011] [Indexed: 01/03/2023] Open
Abstract
Binary toxins are among the most potent bacterial protein toxins performing a cooperative mode of translocation and exhibit fatal enzymatic activities in eukaryotic cells. Anthrax and C2 toxin are the most prominent examples for the AB7/8 type of toxins. The B subunits bind both host cell receptors and the enzymatic A polypeptides to trigger their internalization and translocation into the host cell cytosol. C2 toxin is composed of an actin ADP-ribosyltransferase (C2I) and C2II binding subunits. Anthrax toxin is composed of adenylate cyclase (EF) and MAPKK protease (LF) enzymatic components associated to protective antigen (PA) binding subunit. The binding and translocation components anthrax protective antigen (PA63) and C2II of C2 toxin share a sequence homology of about 35%, suggesting that they might substitute for each other. Here we show by conducting in vitro measurements that PA63 binds C2I and that C2II can bind both EF and LF. Anthrax edema factor (EF) and lethal factor (LF) have higher affinities to bind to channels formed by C2II than C2 toxin's C2I binds to anthrax protective antigen (PA63). Furthermore, we could demonstrate that PA in high concentration has the ability to transport the enzymatic moiety C2I into target cells, causing actin modification and cell rounding. In contrast, C2II does not show significant capacity to promote cell intoxication by EF and LF. Together, our data unveiled the remarkable flexibility of PA in promoting C2I heterologous polypeptide translocation into cells.
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Affiliation(s)
| | - Monica Rolando
- Inserm, U895, Toxines Microbiennes dans la Relation Hôte-Pathogènes, Batiment Archimed, Nice, France
- Faculté de Médecine, Institut Fédératif de Recherche 50, Université de Nice-Sophia Antipolis, Nice, France
| | | | - Caroline Stefani
- Inserm, U895, Toxines Microbiennes dans la Relation Hôte-Pathogènes, Batiment Archimed, Nice, France
| | - Michael Leuber
- Rudolf-Virchow-Center, University of Würzburg, Würzburg, Germany
| | - Gilles Flatau
- Inserm, U895, Toxines Microbiennes dans la Relation Hôte-Pathogènes, Batiment Archimed, Nice, France
| | - Michel R. Popoff
- Unité des Bactéries Anaerobies et Toxines, Institut Pasteur, Paris, France
| | - Roland Benz
- Rudolf-Virchow-Center, University of Würzburg, Würzburg, Germany
- School of Engineering and Science, Jacobs University Bremen, Bremen, Germany
- * E-mail: (EL); (RB)
| | - Emmanuel Lemichez
- Inserm, U895, Toxines Microbiennes dans la Relation Hôte-Pathogènes, Batiment Archimed, Nice, France
- Faculté de Médecine, Institut Fédératif de Recherche 50, Université de Nice-Sophia Antipolis, Nice, France
- Laboratoire central de bactériologie, Centre Hospitalier Universitaire de Nice, Nice, France
- * E-mail: (EL); (RB)
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Paccani SR, Baldari CT. T cell targeting by anthrax toxins: two faces of the same coin. Toxins (Basel) 2011; 3:660-71. [PMID: 22069732 PMCID: PMC3202842 DOI: 10.3390/toxins3060660] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2011] [Revised: 06/03/2011] [Accepted: 06/07/2011] [Indexed: 12/24/2022] Open
Abstract
Bacillus anthracis, similar to other bacterial pathogens, has evolved effective immune evasion strategies to prolong its survival in the host, thus ensuring the unchecked spread of the infection. This function is subserved by lethal (LT) and edema (ET) toxins, two exotoxins produced by vegetative anthrax bacilli following germination of the spores. The structure of these toxins and the mechanism of cell intoxication are topics covered by other reviews in this issue. Here we shall discuss how B. anthracis uses LT and ET to suppress the immune defenses of the host, focusing on T lymphocytes, the key players in adaptive immunity. We shall also summarize recent findings showing that, depending on its concentration, ET has the ability not only to suppress T cell activation but also to promote the polarization of CD4(+) T cells to the Th2 and Th17 subsets, highlighting the potential use of this toxin as an immunomodulator.
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Affiliation(s)
- Silvia Rossi Paccani
- Department of Evolutionary Biology, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy;
- Novartis Vaccines, Via Fiorentina 1, 53100 Siena, Italy
- Author to whom correspondence should be addressed; or ; Tel.: +39-0577-234396; Fax: +39-0577-234476
| | - Cosima T. Baldari
- Department of Evolutionary Biology, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy;
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Larabee JL, Shakir SM, Hightower L, Ballard JD. Adenomatous polyposis coli protein associates with C/EBP beta and increases Bacillus anthracis edema toxin-stimulated gene expression in macrophages. J Biol Chem 2011; 286:19364-72. [PMID: 21487015 DOI: 10.1074/jbc.m111.224543] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The production of cAMP from Bacillus anthracis edema toxin (ET) activates gene expression in macrophages through a complex array of signaling pathways, most of which remain poorly defined. In this study, the tumor suppressor protein adenomatous polyposis coli (APC) was found to be important for the up-regulation of previously defined ET-stimulated genes (Vegfa, Ptgs2, Arg2, Cxcl2, Sdc1, and Cebpb). A reduction in the expression of these genes after ET exposure was observed when APC was disrupted in macrophages using siRNA or in bone marrow-derived macrophages obtained from C57BL/6J-Apc(Min) mice, which are heterozygous for a truncated form of APC. In line with this observation, ET increased the expression of APC at the transcriptional level, leading to increased amounts of APC in the nucleus. The mechanism utilized by APC to increase ET-induced gene expression was determined to depend on the ability of APC to interact with C/EBP β, which is a transcription factor activated by cAMP. Coimmunoprecipitation experiments found that APC associated with C/EBP β and that levels of this complex increase after ET exposure. A further connection was uncovered when silencing APC was determined to reduce the ET-induced phosphorylation of C/EBP β at Thr-188. This ET-mediated phosphorylation of C/EBP β was blocked by glycogen synthase kinase 3 (GSK-3) inhibitors, suggesting that GSK-3 is involved in the activation of C/EBP β and supporting the idea of APC helping direct interactions between GSK-3 and C/EBP β. These results indicate that ET stimulates gene expression by promoting the formation of an inducible protein complex consisting of APC and C/EBP β.
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Affiliation(s)
- Jason L Larabee
- Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
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35
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Sayner SL. Emerging themes of cAMP regulation of the pulmonary endothelial barrier. Am J Physiol Lung Cell Mol Physiol 2011; 300:L667-78. [PMID: 21335524 DOI: 10.1152/ajplung.00433.2010] [Citation(s) in RCA: 71] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
The presence of excess fluid in the interstitium and air spaces of the lung presents severe restrictions to gas exchange. The pulmonary endothelial barrier regulates the flux of fluid and plasma proteins from the vascular space into the underlying tissue. The integrity of this endothelial barrier is dynamically regulated by transitions in cAMP (3',5'-cyclic adenosine monophosphate), which are synthesized in discrete subcellular compartments. Cyclic AMP generated in the subplasma membrane compartment acts through PKA and Epac (exchange protein directly activated by cAMP) to tighten cell adhesions, strengthen cortical actin, reduce actomyosin contraction, and decrease permeability. Confining cAMP within the subplasma membrane space is critical to its barrier-protective properties. When cAMP escapes the near membrane compartment and gains access to the cytosolic compartment, or when soluble adenylyl cyclases generate cAMP within the cytosolic compartment, this second messenger activates established cytosolic cAMP signaling cascades to perturb the endothelial barrier through PKA-mediated disruption of microtubules. Thus the concept of cAMP compartmentalization in endothelial barrier regulation is gaining momentum and new possibilities are being unveiled for cytosolic cAMP signaling with the emergence of the bicarbonate-regulated mammalian soluble adenylyl cyclase (sAC or AC10).
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Affiliation(s)
- Sarah L Sayner
- Dept. of Cell Biology and Neuroscience, Member, Center for Lung Biology, College of Medicine, Univ. of South Alabama, Mobile, AL 36688, USA.
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36
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Duverger A, Carré JM, Jee J, Leppla SH, Cormet-Boyaka E, Tang WJ, Tomé D, Boyaka PN. Contributions of edema factor and protective antigen to the induction of protective immunity by Bacillus anthracis edema toxin as an intranasal adjuvant. THE JOURNAL OF IMMUNOLOGY 2010; 185:5943-52. [PMID: 20952678 DOI: 10.4049/jimmunol.0902795] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
We have shown that intranasal coapplication of Bacillus anthracis protective Ag (PA) together with a B. anthracis edema factor (EF) mutant having reduced adenylate cyclase activity (i.e., EF-S414N) enhances anti-PA Ab responses, but also acts as a mucosal adjuvant for coadministered unrelated Ags. To elucidate the role of edema toxin (EdTx) components in its adjuvanticity, we examined how a PA mutant lacking the ability to bind EF (PA-U7) or another mutant that allows the cellular uptake of EF, but fails to efficiently mediate its translocation into the cytosol (PA-dFF), would affect EdTx-induced adaptive immunity. Native EdTx promotes costimulatory molecule expression by macrophages and B lymphocytes, and a broad spectrum of cytokine responses by cervical lymph node cells in vitro. These effects were reduced or abrogated when cells were treated with EF plus PA-dFF, or PA-U7 instead of PA. We also intranasally immunized groups of mice with a recombinant fusion protein of Yersinia pestis F1 and LcrV Ags (F1-V) together with EdTx variants consisting of wild-type or mutants PA and EF. Analysis of serum and mucosal Ab responses against F1-V or EdTx components (i.e., PA and EF) revealed no adjuvant activity in mice that received PA-U7 instead of PA. In contrast, coimmunization with PA-dFF enhanced serum Ab responses. Finally, immunization with native PA and an EF mutant lacking adenylate cyclase activity (EF-K346R) failed to enhance Ab responses. In summary, a fully functional PA and a minimum of adenylate cyclase activity are needed for EdTx to act as a mucosal adjuvant.
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Affiliation(s)
- Alexandra Duverger
- Department of Veterinary Biosciences, Ohio State University, Columbus, OH 43210, USA
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Zornetta I, Brandi L, Janowiak B, Dal Molin F, Tonello F, Collier RJ, Montecucco C. Imaging the cell entry of the anthrax oedema and lethal toxins with fluorescent protein chimeras. Cell Microbiol 2010; 12:1435-45. [PMID: 20438574 DOI: 10.1111/j.1462-5822.2010.01480.x] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
To investigate the cell entry and intracellular trafficking of anthrax oedema factor (EF) and lethal factor (LF), they were C-terminally fused to the enhanced green fluorescent protein (EGFP) and monomeric Cherry (mCherry) fluorescent proteins. Both chimeras bound to the surface of BHK cells treated with protective antigen (PA) in a patchy mode. Binding was followed by rapid internalization, and the two anthrax factors were found to traffic along the same endocytic route and with identical kinetics, indicating that their intracellular path is essentially dictated by PA. Colocalization studies indicated that anthrax toxins enter caveolin-1 containing compartments and then endosomes marked by phoshatidylinositol 3-phoshate and Rab5, but not by early endosome antigen 1 and transferrin. After 40 min, both EF and LF chimeras were observed to localize within late compartments. Eventually, LF and EF appeared in the cytosol with a time-course consistent with translocation from late endosomes. Only the EGFP derivatives reached the cytosol because they are translocated by the PA channel, while the mCherry derivatives are not. This difference is attributed to a higher resistance of mCherry to unfolding. After translocation, LF disperses in the cytosol, while EF localizes on the cytosolic face of late endosomes.
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Affiliation(s)
- Irene Zornetta
- Dipartimento di Scienze Biomediche dell'Università di Padova and Istituto di Neuroscienze del CNR, Via G. Colombo 3, 35100 Padova, Italy
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Heterodimeric integrin complexes containing beta1-integrin promote internalization and lethality of anthrax toxin. Proc Natl Acad Sci U S A 2010; 107:15583-8. [PMID: 20713715 DOI: 10.1073/pnas.1010145107] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
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 beta1, 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 integrin-activating proteins osteopontin and CD44. Further study showed that PA binds to alpha4beta1- and alpha5beta1-integrin complexes, leading to their conjoint endocytosis, and also interacts-weakly relative to CMG2 but comparably to TEM8--with purified alpha5beta1 complex in vitro. Monoclonal antibody directed against beta1-integrin or its alpha 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 beta1-integrin can both potentiate CMG2-mediated endocytosis and serve independently as a low-affinity PA receptor.
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Van Der Goot G, Young JA. Receptors of anthrax toxin and cell entry. Mol Aspects Med 2009; 30:406-12. [PMID: 19732789 PMCID: PMC2783407 DOI: 10.1016/j.mam.2009.08.007] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2009] [Accepted: 08/24/2009] [Indexed: 11/29/2022]
Abstract
Anthrax toxin-receptor interactions are critical for toxin delivery to the host cell cytoplasm. This review summarizes what is known about the molecular details of the protective antigen (PA) toxin subunit interaction with either the ANTXR1 and ANTXR2 cellular receptors, and how receptor-type can dictate the low pH threshold of PA pore formation. The roles played by cellular factors in regulating the endocytosis of toxin-receptor complexes is also discussed.
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Affiliation(s)
- Gisou Van Der Goot
- Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, SV-AI extension, Station 15, 1015 Lausanne, Switzerland,
| | - John A.T. Young
- Nomis Center for Immunobiology and Microbial Pathogenesis, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037,
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40
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Fasanella A, Galante D, Garofolo G, Jones MH. Anthrax undervalued zoonosis. Vet Microbiol 2009; 140:318-31. [PMID: 19747785 DOI: 10.1016/j.vetmic.2009.08.016] [Citation(s) in RCA: 72] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2009] [Revised: 08/03/2009] [Accepted: 08/11/2009] [Indexed: 11/19/2022]
Abstract
Anthrax is a non-contagious disease, known since ancient times. However, it became a matter of global public interest after the bioterrorist attacks in the U.S.A. during the autumn of 2001. The concern of politicians and civil authorities everywhere towards this emergency necessitated a significant research effort and the prevention of new bioterrorist acts. Anthrax is primarily a disease that affects livestock and wildlife; its distribution is worldwide; and it can represent a danger to humans but especially more so when it occurs in areas considered to be free and in atypical seasons and climatic conditions. The atypicality of the phenomenon may lead health workers to misdiagnose and, consequently, an inappropriately manage of affected carcasses with a consequent and inevitable increase in the risk of human infection. This article emphasises the importance of paying increasing attention to this zoonosis. The biggest risk is its underestimation.
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Affiliation(s)
- Antonio Fasanella
- Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Anthrax Reference Institute of Italy, Foggia, Italy.
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41
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Tonello F, Montecucco C. The anthrax lethal factor and its MAPK kinase-specific metalloprotease activity. Mol Aspects Med 2009; 30:431-8. [PMID: 19665472 DOI: 10.1016/j.mam.2009.07.006] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2009] [Accepted: 07/30/2009] [Indexed: 02/06/2023]
Abstract
The anthrax lethal factor is a multi-domain protein toxin released by Bacillus anthracis which enters cells in a process mediated by the protective antigen and specific cell receptors. In the cytosol, the lethal factor cleaves the N-terminal tail of many MAPK kinases, thus deranging a major cell signaling pathway. The structural features at the basis of these activities of LF are reviewed here with particular attention to the proteolytic activity and to the identification of specific inhibitors. A significant similarity between the metalloprotease domain of the lethal factor and of that of the clostridial neurotoxins has been noted and is discussed.
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Affiliation(s)
- Fiorella Tonello
- Dipartimento di Scienze Biomediche Sperimentali, Istituto CNR di Neuroscienze, Università di Padova, Viale G. Colombo 3, 35131 Padova, Italy
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Schwartz M. Dr. Jekyll and Mr. Hyde: a short history of anthrax. Mol Aspects Med 2009; 30:347-55. [PMID: 19577591 DOI: 10.1016/j.mam.2009.06.004] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2009] [Accepted: 06/29/2009] [Indexed: 10/20/2022]
Abstract
The anthrax letters crisis, following the discovery of a major bacterial warfare program in the USSR and the realization that Irak had been on the verge of using anthrax as a weapon during the first Gulf war, had the consequence of putting anthrax back on the agenda of scientists. Fortunately, although it was mostly unknown by the public before these events, it was far from unknown by microbiologists. Already mentioned in the bible as a disease of herbivores, it remained a major cause of death for animals all over the planet until the end of the 19th century, with occasional, sometimes extensive, contamination of human beings. The aetiological agent, Bacillus anthracis, was identified by French and German scientists in the 1860s and 1870s. This was the first time that a disease could be attributed to a specific microorganism. The discovery by Koch that this bacterium formed spores greatly contributed to the understanding of the disease epidemiology. Studies on the pathophysiology of anthrax led to the identification of two major virulence factors, the capsule, protecting the bacilli against phagocytosis, and a tripartite toxin. The latter consists of two toxins with a common component (protecting antigen, PA) that allows the binding to and penetration into cells of two enzymes, the oedema factor EF, a calmodulin dependent adenylate cyclase, and the lethal factor LF, a specific zinc metalloprotease. The primary targets of these toxins would seem to be cells of innate immunity that would otherwise impair multiplication of the bacilli. If detected early enough, B. anthracis infections can be stopped by using antibiotics such as ciprofloxacin. Infection of animals can be prevented by the administration of vaccines, the first of which was developed by Pasteur after an historical testing at Pouilly-le-Fort which marked the beginning of the science of vaccines.
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Affiliation(s)
- Maxime Schwartz
- Institut Pasteur, 25 Rue du Docteur Roux, 75015 Paris, France.
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43
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Tang WJ, Guo Q. The adenylyl cyclase activity of anthrax edema factor. Mol Aspects Med 2009; 30:423-30. [PMID: 19560485 DOI: 10.1016/j.mam.2009.06.001] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2009] [Accepted: 06/19/2009] [Indexed: 02/08/2023]
Abstract
Bacillus anthracis, the etiologic agent for anthrax, secretes edema factor (EF) to disrupt intracellular signaling pathways. Upon translocation into host cells and association with a calcium sensor, calmodulin (CaM), EF becomes a highly active adenylyl cyclase (AC) that raises the intracellular concentration of cyclic AMP (cAMP). Growing evidence shows that EF plays a key role in anthrax pathogenesis by affecting cellular functions vital for host defense. This strategy is also used by Bordetella pertussis, a bacterium that causes whooping cough. Pertussis bacteria secrete the bifunctional toxin CyaA which raises the intracellular cAMP. Here, we discuss recent advances from structural analyses that reveal the molecular basis of the conserved mechanism of activation and catalysis of EF and CyaA by CaM even though these two toxins use the completely different sequences to bind CaM. Comparison of the biochemical and structural characteristics of these two AC toxins with host ACs reveal that they have diverse strategies of catalytic activation, yet use the same two-metal-ion catalytic mechanism.
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Affiliation(s)
- Wei-Jen Tang
- Ben-May Department for Cancer Research, The University of Chicago, 929 East 57th Street, GCIS W434, Chicago, IL 60637, USA.
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Maldonado-Arocho FJ, Bradley KA. Anthrax edema toxin induces maturation of dendritic cells and enhances chemotaxis towards macrophage inflammatory protein 3beta. Infect Immun 2009; 77:2036-42. [PMID: 19273556 PMCID: PMC2681763 DOI: 10.1128/iai.01329-08] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2008] [Revised: 12/03/2008] [Accepted: 03/02/2009] [Indexed: 01/25/2023] Open
Abstract
Bacillus anthracis secretes two bipartite toxins, edema toxin (ET) and lethal toxin (LT), which impair immune responses and contribute directly to the pathology associated with the disease anthrax. Edema factor, the catalytic subunit of ET, is an adenylate cyclase that impairs host defenses by raising cellular cyclic AMP (cAMP) levels. Synthetic cAMP analogues and compounds that raise intracellular cAMP levels lead to phenotypic and functional changes in dendritic cells (DCs). Here, we demonstrate that ET induces a maturation state in human monocyte-derived DCs (MDDCs) similar to that induced by lipopolysaccharide (LPS). ET treatment results in downregulation of DC-SIGN, a marker of immature DCs, and upregulation of DC maturation markers CD83 and CD86. Maturation of DCs by ET is accompanied by an increased ability to migrate toward the lymph node-homing chemokine macrophage inflammatory protein 3beta, like LPS-matured DCs. Interestingly, cotreating with LT differentially affects the ET-induced maturation of MDDCs while not inhibiting ET-induced migration. These findings reveal a mechanism by which ET impairs normal innate immune function and may explain the reported adjuvant effect of ET.
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Affiliation(s)
- Francisco J Maldonado-Arocho
- Department of Microbiology, Immunology, & Molecular Genetics, University of California at Los Angeles, 609 Charles E. Young Dr. East, Los Angeles, CA 90095, USA
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Kaiser E, Pust S, Kroll C, Barth H. Cyclophilin A facilitates translocation of theClostridium botulinumC2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells. Cell Microbiol 2009; 11:780-95. [DOI: 10.1111/j.1462-5822.2009.01291.x] [Citation(s) in RCA: 66] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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Abstract
Inhalation anthrax results in high-grade bacteremia and is accompanied by a delay in the rise of the peripheral polymorphonuclear neutrophil (PMN) count and a paucity of PMNs in the infected pleural fluid and mediastinum. Edema toxin (ET) is one of the major Bacillus anthracis virulence factors and consists of the adenylate cyclase edema factor (EF) and protective antigen (PA). Relatively low concentrations of ET (100 to 500 ng/ml of PA and EF) significantly impair human PMN chemokinesis, chemotaxis, and ability to polarize. These changes are accompanied by a reduction in chemoattractant-stimulated PMN actin assembly. ET also causes a significant decrease in Listeria monocytogenes intracellular actin-based motility within HeLa cells. These defects in actin assembly are accompanied by a >50-fold increase in intracellular cyclic AMP and a >4-fold increase in the phosphorylation of protein kinase A. We have previously shown that anthrax lethal toxin (LT) also impairs neutrophil actin-based motility (R. L. During, W. Li, B. Hao, J. M. Koenig, D. S. Stephens, C. P. Quinn, and F. S. Southwick, J. Infect. Dis. 192:837-845, 2005), and we now find that LT combined with ET causes an additive inhibition of PMN chemokinesis, polarization, chemotaxis, and FMLP (N-formyl-met-leu-phe)-induced actin assembly. We conclude that ET alone or combined with LT impairs PMN actin assembly, resulting in paralysis of PMN chemotaxis.
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Rossi Paccani S, Benagiano M, Capitani N, Zornetta I, Ladant D, Montecucco C, D'Elios MM, Baldari CT. The adenylate cyclase toxins of Bacillus anthracis and Bordetella pertussis promote Th2 cell development by shaping T cell antigen receptor signaling. PLoS Pathog 2009; 5:e1000325. [PMID: 19266022 PMCID: PMC2643477 DOI: 10.1371/journal.ppat.1000325] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2008] [Accepted: 02/03/2009] [Indexed: 01/28/2023] Open
Abstract
The adjuvanticity of bacterial adenylate cyclase toxins has been ascribed to their capacity, largely mediated by cAMP, to modulate APC activation, resulting in the expression of Th2–driving cytokines. On the other hand, cAMP has been demonstrated to induce a Th2 bias when present during T cell priming, suggesting that bacterial cAMP elevating toxins may directly affect the Th1/Th2 balance. Here we have investigated the effects on human CD4+ T cell differentiation of two adenylate cyclase toxins, Bacillus anthracis edema toxin (ET) and Bordetella pertussis CyaA, which differ in structure, mode of cell entry, and subcellular localization. We show that low concentrations of ET and CyaA, but not of their genetically detoxified adenylate cyclase defective counterparts, potently promote Th2 cell differentiation by inducing expression of the master Th2 transcription factors, c-maf and GATA-3. We also present evidence that the Th2–polarizing concentrations of ET and CyaA selectively inhibit TCR–dependent activation of Akt1, which is required for Th1 cell differentiation, while enhancing the activation of two TCR–signaling mediators, Vav1 and p38, implicated in Th2 cell differentiation. This is at variance from the immunosuppressive toxin concentrations, which interfere with the earliest step in TCR signaling, activation of the tyrosine kinase Lck, resulting in impaired CD3ζ phosphorylation and inhibition of TCR coupling to ZAP-70 and Erk activation. These results demonstrate that, notwithstanding their differences in their intracellular localization, which result in focalized cAMP production, both toxins directly affect the Th1/Th2 balance by interfering with the same steps in TCR signaling, and suggest that their adjuvanticity is likely to result from their combined effects on APC and CD4+ T cells. Furthermore, our results strongly support the key role of cAMP in the adjuvanticity of these toxins. Colonization by pathogens requires keeping at bay the host immune defenses, at least at the onset of infection. The adenylate cyclase (AC) toxins produced by many pathogenic bacteria assist in this crucial function by catalyzing the production of cAMP, which acts as a potent immunosuppressant. Nevertheless, at low concentrations, these toxins act as adjuvants, enhancing antibody responses to vaccination. We have investigated the molecular basis of the immunomodulatory activities of two AC toxins, Bacillus anthracis edema toxin and Bordetella pertussis CyaA. We show that high toxin concentrations inhibit activation of T lymphocytes, which orchestrate the adaptive immune response against pathogens, whereas low toxin concentrations promote differentiation of helper T lymphocytes to Th2 effectors, which are required for development of antibody-producing cells. Both the immunosuppressant and Th2–driving activities of the toxins are dependent on cAMP. The results demonstrate that, dependent on their concentration, the AC toxins of B. anthracis and B. pertussis evoke distinct responses on target T lymphocytes by differentially modulating antigen receptor signaling, resulting either in suppression of T cell activation or Th2 cell differentiation. These results are of relevance to the evolution of disease in infected individuals and provide novel mechanistic insight into the adjuvanticity of these toxins.
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Affiliation(s)
| | - Marisa Benagiano
- Department of Internal Medicine and Immunoallergology, University of Florence, Florence, Italy
| | - Nagaja Capitani
- Department of Evolutionary Biology, University of Siena, Siena, Italy
| | - Irene Zornetta
- Department of Biomedical Sciences, University of Padua, Padua, Italy
| | - Daniel Ladant
- Unité de Biochimie des Interactions Macromoléculaires, CNRS URA 2185, Institut Pasteur, Paris, France
| | - Cesare Montecucco
- Department of Biomedical Sciences, University of Padua, Padua, Italy
| | - Mario M. D'Elios
- Department of Internal Medicine and Immunoallergology, University of Florence, Florence, Italy
| | - Cosima T. Baldari
- Department of Evolutionary Biology, University of Siena, Siena, Italy
- * E-mail:
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Ratio of lethal and edema factors in rabbit systemic anthrax. Toxicon 2008; 52:824-8. [DOI: 10.1016/j.toxicon.2008.08.011] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2008] [Revised: 08/13/2008] [Accepted: 08/15/2008] [Indexed: 01/07/2023]
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Anthrax edema toxin modulates PKA- and CREB-dependent signaling in two phases. PLoS One 2008; 3:e3564. [PMID: 18958164 PMCID: PMC2569206 DOI: 10.1371/journal.pone.0003564] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2008] [Accepted: 09/22/2008] [Indexed: 01/03/2023] Open
Abstract
Background Anthrax edema toxin (EdTx) is an adenylate cyclase which operates in the perinuclear region of host cells. However, the action of EdTx is poorly understood, especially at molecular level. The ability of EdTx to modulate cAMP-dependent signaling was studied in Jurkat T cells and was compared with that of other cAMP-rising agents: Bordetella pertussis adenylate cyclase toxin, cholera toxin and forskolin. Methodology/Principal Findings EdTx caused a prolonged increase of the intracellular cAMP concentration. This led to nuclear translocation of the cAMP-dependent protein kinase (PKA) catalytic subunit, phosphorylation of cAMP response element binding protein (CREB) and expression of a reporter gene under control of the cAMP response element. Neither p90 ribosomal S6 kinase nor mitogen- and stress-activated kinase, which mediate CREB phosphorylation during T cell activation, were involved. The duration of phospho-CREB binding to chromatin correlated with the spatio-temporal rise of cAMP levels. Strikingly, EdTx pre-treated T cells were unresponsive to other stimuli involving CREB phosphorylation such as addition of forskolin or T cell receptor cross-linking. Conclusions/Significance We concluded that, in a first intoxication phase, EdTx induces PKA-dependent signaling, which culminates in CREB phosphorylation and activation of gene transcription. Subsequently CREB phosphorylation is impaired and therefore T cells are not able to respond to cues involving CREB. The present data functionally link the perinuclear localization of EdTx to its intoxication mechanism, indicating that this is a specific feature of its intoxication mechanism.
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Dal Molin F, Zornetta I, Puhar A, Tonello F, Zaccolo M, Montecucco C. cAMP imaging of cells treated with pertussis toxin, cholera toxin, and anthrax edema toxin. Biochem Biophys Res Commun 2008; 376:429-33. [PMID: 18793614 DOI: 10.1016/j.bbrc.2008.09.012] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2008] [Accepted: 09/03/2008] [Indexed: 12/12/2022]
Abstract
The enzymatic activity of the three most studied bacterial toxins that increase the cytosolic cAMP level: pertussis toxin (PT), cholera toxin (CT), and anthrax edema toxin (ET), was imaged by fluorescence videomicroscopy. Three different cell lines were transfected with a fluorescence resonance energy transfer biosensor based on the PKA regulatory and catalytic subunits fused to CFP and YFP, respectively. Real-time imaging of cells expressing this cAMP biosensor provided time and space resolved pictures of the toxins action. The time course of the PT-induced cAMP increase suggests that its active subunit enters the cytosol more rapidly than that deduced by biochemical experiments. ET generated cAMP concentration gradients decreasing from the nucleus to the cell periphery. On the contrary, CT, which acts on the plasma membrane adenylate cyclase, did not. The potential of imaging methods in studying the mode of entry and the intracellular action of bacterial toxins is discussed.
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Affiliation(s)
- Federica Dal Molin
- Dipartimento di Scienze Biomediche and Istituto C.N.R. Neuroscienze, Università di Padova, Viale G. Colombo n. 3, 35121 Padova, Italy
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