Anthrolysin O and Other Gram-positive Cytolysins Are Toll-like Receptor 4 Agonists

Exposure of bone marrow–derived macrophages (BMDMs) to low concentrations of Bacillus anthracis lethal toxin (LT), whose catalytic subunit is lethal factor (LF), results in induction of a robust apoptotic response dependent on activation of Toll-like receptor (TLR)4. A similar TLR4-dependent apoptotic response is observed when BMDMs are infected with live B. anthracis (Sterne strain). However, TLR4 is considered to be a specific signaling receptor for lipopolysaccharide (LPS), a typical product of gram-negative bacteria, whereas B. anthracis is gram-positive. To understand how B. anthracis can activate TLR4, we analyzed its culture supernatants and found them to contain a potent TLR4-stimulating activity that can also induce apoptosis in macrophages in which the antiapoptotic p38 MAP kinase (whose activation is prevented by LF) was inhibited. Purification of this activity suggested it consists of anthrolysin O (ALO), a member of the cholesterol-dependent cytolysin (CDC) family. We show that recombinant ALO can activate TLR4 in a manner independent of LPS contamination and, together with LT, can induce macrophage apoptosis. We also provide genetic evidence that ALO is required for induction of macrophage apoptosis in response to infection with live B. anthracis and that other CDC family members share the ability to activate TLR4.

[1]  Polly Matzinger,et al.  Hydrophobicity: an ancient damage-associated molecular pattern that initiates innate immune responses , 2004, Nature Reviews Immunology.

[2]  M. Caparon,et al.  Specificity of streptolysin O in cytolysin‐mediated translocation , 2004, Molecular microbiology.

[3]  C. R. Lankford,et al.  Anthrax Lethal Toxin Rapidly Activates Caspase-1/ICE and Induces Extracellular Release of Interleukin (IL)-1β and IL-18* , 2004, Journal of Biological Chemistry.

[4]  T. Merkel,et al.  Macrophages Release Tumor Necrosis Factor Alpha and Interleukin-12 in Response to Intracellular Bacillus anthracis Spores , 2004, Infection and Immunity.

[5]  M. Karin,et al.  The protein kinase PKR is required for macrophage apoptosis after activation of Toll-like receptor 4 , 2004, Nature.

[6]  Jay Wells,et al.  Systemic cytokine response in murine anthrax , 2004, Cellular microbiology.

[7]  S. Leppla,et al.  The roles of anthrax toxin in pathogenesis. , 2004, Current opinion in microbiology.

[8]  L. Hültner,et al.  The Streptococcal Exotoxin Streptolysin O Activates Mast Cells To Produce Tumor Necrosis Factor Alpha by p38 Mitogen-Activated Protein Kinase- and Protein Kinase C-Dependent Pathways , 2003, Infection and Immunity.

[9]  S. Leppla,et al.  Bacillus anthracis lethal toxin induces TNF-α–independent hypoxia-mediated toxicity in mice , 2003 .

[10]  R. Medzhitov,et al.  Recognition of microbial infection by Toll-like receptors. , 2003, Current opinion in immunology.

[11]  T. Koehler,et al.  Characterization of Anthrolysin O, the Bacillus anthracis Cholesterol-Dependent Cytolysin , 2003, Infection and Immunity.

[12]  S. Salzberg,et al.  The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria , 2003, Nature.

[13]  John A. Young,et al.  Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[14]  M. Lipsitch,et al.  Recognition of pneumolysin by Toll-like receptor 4 confers resistance to pneumococcal infection , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[15]  B. Beutler,et al.  Innate immune sensing and its roots: the story of endotoxin , 2003, Nature Reviews Immunology.

[16]  Jonathan Cohen.  The immunopathogenesis of sepsis , 2002, Nature.

[17]  P. Gilligan.  Therapeutic challenges posed by bacterial bioterrorism threats. , 2002, Current opinion in microbiology.

[18]  Michael Karin,et al.  Macrophage Apoptosis by Anthrax Lethal Factor Through p38 MAP Kinase Inhibition , 2002, Science.

[19]  John A. Young,et al.  Identification of the cellular receptor for anthrax toxin , 2001, Nature.

[20]  S. Bavari,et al.  Macrophage-Derived Cell Lines Do Not Express Proinflammatory Cytokines after Exposure to Bacillus anthracis Lethal Toxin , 2001, Infection and Immunity.

[21]  N. Ruiz,et al.  Cytolysin-Mediated Translocation (CMT) A Functional Equivalent of Type III Secretion in Gram-Positive Bacteria , 2001, Cell.

[22]  R. Bhatnagar,et al.  Anthrax Toxin , 2001, Critical reviews in microbiology.

[23]  D. Portnoy,et al.  A PEST-like sequence in listeriolysin O essential for Listeria monocytogenes pathogenicity. , 2000, Science.

[24]  H. Bouwer,et al.  Genetic immunization of mice against Listeria monocytogenes using plasmid DNA encoding listeriolysin O. , 1999, Journal of immunology.

[25]  S Falkow,et al.  The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[26]  M. Karin,et al.  JNKK1 organizes a MAP kinase module through specific and sequential interactions with upstream and downstream components mediated by its amino-terminal extension. , 1998, Genes & development.

[27]  R. Collier,et al.  Characterization of membrane translocation by anthrax protective antigen. , 1998, Biochemistry.

[28]  J. Rossjohn,et al.  Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O: an alpha-helical to beta-sheet transition identified by fluorescence spectroscopy. , 1998, Biochemistry.

[29]  M. Mock,et al.  Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages. , 1998, Biochemical and biophysical research communications.

[30]  K D Paull,et al.  Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. , 1998, Science.

[31]  M. Mock,et al.  Intracytoplasmic delivery of listeriolysin O by a vaccinal strain of Bacillus anthracis induces CD8-mediated protection against Listeria monocytogenes. , 1997, Journal of immunology.

[32]  A. Zychlinsky,et al.  A bacterial invasin induces macrophage apoptosis by binding directly to ICE. , 1996, The EMBO journal.

[33]  J C Paton,et al.  The contribution of pneumolysin to the pathogenicity of Streptococcus pneumoniae. , 1996, Trends in microbiology.

[34]  K. Klimpel,et al.  Anthrax toxin lethal factor contains a zinc metalloprotease consensus sequence which is required for lethal toxin activity , 1994, Molecular microbiology.

[35]  D. Acosta,et al.  On the role of macrophages in anthrax. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[36]  M. Bevan,et al.  CD8+ T cells specific for a single nonamer epitope of Listeria monocytogenes are protective in vivo , 1992, The Journal of experimental medicine.

[37]  H. Bouwer,et al.  Listeriolysin O is a target of the immune response to Listeria monocytogenes , 1992, The Journal of experimental medicine.

[38]  W. Goebel,et al.  Molecular determinants of Listeria monocytogenes pathogenesis , 1992, Infection and immunity.

[39]  M. Arakawa,et al.  Induction of macrophage interleukin-1 production by Listeria monocytogenes hemolysin. , 1992, Cellular immunology.

[40]  E. Pamer,et al.  Precise prediction of a dominant class I MHC-restricted epitope of Listeria monocytogenes , 1991, Nature.

[41]  C. Dempsey.  The actions of melittin on membranes. , 1990, Biochimica et biophysica acta.

[42]  S. Leppla,et al.  Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Diana Haines,et al.  Bacillus anthracis lethal toxin induces TNF-alpha-independent hypoxia-mediated toxicity in mice. , 2003, The Journal of clinical investigation.

[44]  G. Boivin,et al.  Macrophage Apoptosis by Anthrax Lethal Factor Through p 38 MAP Kinase Inhibition , 2002 .

[45]  M. Parker,et al.  The cholesterol-dependent cytolysins. , 2001, Current topics in microbiology and immunology.

[46]  J. Rood.  Virulence genes of Clostridium perfringens. , 1998, Annual review of microbiology.

[47]  R. Ulevitch,et al.  Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. , 1995, Annual review of immunology.