Effect of Bacillus anthracis lethal toxin on human peripheral blood mononuclear cells

Lethal toxin (LeTx) plays a central role in anthrax pathogenesis, however a cytotoxicity of LeTx has been difficult to demonstrate in vitro. No cytolytic effect has been reported for human cells, in contrast to murine cell lines, indicating that cell lysis can not be considered as a marker of LeTx activity. We have recently shown that murine macrophage‐like RAW 264.7 cells treated with LeTx or infected with anthrax spores underwent changes typical of apoptotic death. Here we demonstrate that cells from human peripheral blood display a proapoptotic behavior similar to murine cells. TUNEL assay detected a nucleosomal degradation typical of apoptosis in peripheral blood mononuclear cells (PBMC) treated with LeTx. Membrane staining with apoptotic dyes was detected in macrophages derived from monocytes in presence of LeTx. The toxin inhibited production of proinflammatory cytokines in PBMC stimulated with a preparation of Bacillus anthracis cell wall. Infection of PBMC with anthrax spores led to the appearance of a large population of cells stained positively for apoptosis, with a reduced capacity to eliminate spores and vegetative bacteria. The aminopeptidase inhibitor, bestatin, capable of protecting cells from LeTx, restored a bactericidal activity of infected cells. These findings may be explained by LeTx expression within phagocytes and support an important role of LeTx as an early intracellular virulence factor contributing to bacterial dissemination and disease progression.

[1]  P. Gounon,et al.  OlpB, a new outer layer protein of Clostridium thermocellum, and binding of its S-layer-like domains to components of the cell envelope , 1995, Journal of bacteriology.

[2]  Charles L. Bailey,et al.  Lethal toxin of Bacillus anthracis causes apoptosis of macrophages. , 2002, Biochemical and biophysical research communications.

[3]  C. Kuo,et al.  Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases , 1997, Molecular and cellular biology.

[4]  G. Woude,et al.  Anthrax lethal factor causes proteolytic inactivation of mitogen‐activated protein kinase kinase , 1999, Journal of applied microbiology.

[5]  D. Green,et al.  The c-Jun N-terminal kinase cascade plays a role in stress-induced apoptosis in Jurkat cells by up-regulating Fas ligand expression. , 1998, Journal of immunology.

[6]  S. Leppla,et al.  Optimized production and purification of Bacillus anthracis lethal factor. , 2000, Protein expression and purification.

[7]  S. Leppla,et al.  Internalization and processing of Bacillus anthracis lethal toxin by toxin-sensitive and -resistant cells. , 1989, The Journal of biological chemistry.

[8]  A. Friedlander,et al.  Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process. , 1986, The Journal of biological chemistry.

[9]  P. M. Davis,et al.  Differential Induction of Apoptosis by Fas–Fas Ligand Interactions in Human Monocytes and Macrophages , 1997, The Journal of experimental medicine.

[10]  M. Kubbutat,et al.  Regulation of p53 Function and Stability by Phosphorylation , 1999, Molecular and Cellular Biology.

[11]  B. Zhivotovsky,et al.  Apoptosis in human disease: a new skin for the old ceremony? , 1999, Biochemical and biophysical research communications.

[12]  S. Bhakdi,et al.  Novel path to apoptosis: small transmembrane pores created by staphylococcal alpha-toxin in T lymphocytes evoke internucleosomal DNA degradation , 1994, Infection and immunity.

[13]  B. Bloom,et al.  Role of Macrophage Oxidative Burst in the Action of Anthrax Lethal Toxin , 1994, Molecular medicine.

[14]  M. Mock,et al.  Anthrax lethal factor cleaves the N‐terminus of MAPKKS and induces tyrosine/threonine phosphorylation of MAPKS in cultured macrophages , 1999, Journal of applied microbiology.

[15]  D. Munn,et al.  Activation-induced apoptosis in human macrophages: developmental regulation of a novel cell death pathway by macrophage colony- stimulating factor and interferon gamma , 1995, The Journal of experimental medicine.

[16]  M. Mock,et al.  The cytotoxic activity of Bacillus anthracis lethal factor is inhibited by leukotriene A4 hydrolase and metallopeptidase inhibitors. , 1996, The Biochemical journal.

[17]  Cesare Montecucco,et al.  Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/IFNγ‐induced release of NO and TNFα , 1999 .

[18]  D. Green,et al.  Withdrawal of Survival Factors Results in Activation of the JNK Pathway in Neuronal Cells Leading to Fas Ligand Induction and Cell Death , 1999, Molecular and Cellular Biology.

[19]  F. Klein,et al.  DUAL NATURE OF RESISTANCE MECHANISMS AS REVEALED BY STUDIES OF ANTHRAX SEPTICEMIA. , 1963, Journal of bacteriology.

[20]  T. Koehler,et al.  Early Bacillus anthracis–macrophage interactions: intracellular survival and escape , 2000 .

[21]  S. Orrenius,et al.  Suppression of Fas/APO-1-mediated apoptosis by mitogen-activated kinase signaling. , 1998, Journal of immunology.

[22]  P. Hanna.  Anthrax pathogenesis and host response. , 1998, Current topics in microbiology and immunology.

[23]  M. Weller,et al.  Apoptosis in human alveolar macrophages is induced by endotoxin and is modulated by cytokines. , 1996, American journal of respiratory cell and molecular biology.

[24]  S. Wahl,et al.  Apoptosis in human monocytes: possible role in chronic inflammatory diseases. , 1993, Journal of periodontology.

[25]  S. Akira,et al.  Toll-like receptors: critical proteins linking innate and acquired immunity , 2001, Nature Immunology.

[26]  S. Welkos,et al.  Differences in susceptibility of inbred mice to Bacillus anthracis , 1986, Infection and immunity.

[27]  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.

[28]  M. Mock,et al.  Lethal factor of Bacillus anthracis cleaves the N-terminus of MAPKKs: analysis of the intracellular consequences in macrophages. , 2000, International journal of medical microbiology : IJMM.

[29]  N. Kaplowitz,et al.  Mechanism of staurosporine-induced apoptosis in murine hepatocytes. , 2002, American journal of physiology. Gastrointestinal and liver physiology.

[30]  S. Chauvaux,et al.  Distinct Affinity of Binding Sites for S-Layer Homologous Domains in Clostridium thermocellum and Bacillus anthracis Cell Envelopes , 1999, Journal of bacteriology.

[31]  M. Mock,et al.  Anthrax lethal toxin-induced mitogenic response of human T-cells. , 1997, FEMS microbiology letters.

[32]  S. Leppla.  Production and purification of anthrax toxin. , 1988, Methods in enzymology.

[33]  J. Austin,et al.  Antibacterial effect of protamine in combination with EDTA and refrigeration. , 2001, International journal of food microbiology.

[34]  B. Gee,et al.  Colorimetric method for determining MICs of antimicrobial agents for Mycobacterium tuberculosis , 1995, Journal of clinical microbiology.

[35]  Sun-Mi Park,et al.  Non-apoptotic Signaling Pathways Activated by Soluble Fas Ligand in Serum-starved Human Fibroblasts , 2001, The Journal of Biological Chemistry.

[36]  M. Mock,et al.  Germination of Bacillus anthracis spores within alveolar macrophages , 1999, Molecular microbiology.