A Cellular Deficiency of Gangliosides Causes Hypersensitivity to Clostridium perfringens Phospholipase C*

Clostridium perfringens phospholipase C (Cp-PLC), also called α-toxin, is the major virulence factor in the pathogenesis of gas gangrene. Previously, a cellular UDP-Glc deficiency was related with a hypersensitivity to the cytotoxic effect of Cp-PLC. Because UDP-Glc is required in the synthesis of proteoglycans, N-linked glycoproteins, and glycosphingolipids, the role of these gly-coconjugates in the cellular sensitivity to Cp-PLC was studied. The cellular sensitivity to Cp-PLC was significantly enhanced by glycosphingolipid synthesis inhibitors, and a mutant cell line deficient in gangliosides was found to be hypersensitive to Cp-PLC. Gangliosides protected hypersensitive cells from the cytotoxic effect of Cp-PLC and prevented its membrane-disrupting effect on artificial membranes. Removal of sialic acids by C. perfringens sialidase increases the sensitivity of cultured cells to Cp-PLC and intramuscular co-injection of C. perfringens sialidase, and Cp-PLC in mice potentiates the myotoxic effect of the latter. This work demonstrated that a reduction in gangliosides renders cells more susceptible to the membrane damage caused by Cp-PLC and revealed a previously unrecognized synergism between Cp-PLC and C. perfringens sialidase, providing new insights toward understanding the pathogenesis of clostridial myonecrosis.

[1]  W. Liu,et al.  Wheat germ lectin induces G2/M arrest in mouse L929 fibroblasts , 2004, Journal of cellular biochemistry.

[2]  G. Schwarzmann,et al.  Uptake and metabolism of exogenous glycosphingolipids by cultured cells. , 2001, Seminars in cell & developmental biology.

[3]  B. Maggio,et al.  The surface behavior of glycosphingolipids in biomembranes: a new frontier of molecular ecology. , 1994, Progress in biophysics and molecular biology.

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

[5]  B. Persson,et al.  Cellular UDP-Glucose Deficiency Caused by a Single Point Mutation in the UDP-Glucose Pyrophosphorylase Gene* , 1997, The Journal of Biological Chemistry.

[6]  C. Kent,et al.  Regulation of phosphatidylcholine biosynthesis in mammalian cells. I. Effects of phospholipase C treatment on phosphatidylcholine metabolism in Chinese hamster ovary cells and LM mouse fibroblasts. , 1983, The Journal of biological chemistry.

[7]  I. Bianco,et al.  Effect of sulfatide and gangliosides on phospholipase C and phospholipase A2 activity. A monolayer study. , 1990, Biochimica et biophysica acta.

[8]  B. Lomonte,et al.  Broad cytolytic specificity of myotoxin II, a lysine-49 phospholipase A2 of Bothrops asper snake venom. , 1994, Toxicon : official journal of the International Society on Toxinology.

[9]  Claudio Cobelli,et al.  Roles of Glucose Transport and Glucose Phosphorylation in Muscle Insulin Resistance of NIDDM , 1996, Diabetes.

[10]  D. Stevens,et al.  The pathogenesis of clostridial myonecrosis. , 2000, International journal of medical microbiology : IJMM.

[11]  K. Prydz,et al.  Synthesis and sorting of proteoglycans. , 2000, Journal of cell science.

[12]  P. Roggentin,et al.  Diversity in the properties of two sialidase isoenzymes produced by Clostridium perfringens spp. , 1995, Biological chemistry Hoppe-Seyler.

[13]  J. Vázquez-Boland,et al.  The Comprehensive Sourcebook of Bacterial Protein Toxins , 2006 .

[14]  M. Burke,et al.  Nontraumatic clostridial myonecrosis. , 1999, The American journal of forensic medicine and pathology.

[15]  S. Sprang,et al.  Structure of the protein kinase Cbeta phospholipid-binding C2 domain complexed with Ca2+. , 1998, Structure.

[16]  P. Roggentin,et al.  The sialidase gene from Clostridium septicum: cloning, sequencing, expression in Escherichia coli and identification of conserved sequences in sialidases and other proteins , 1991, Molecular and General Genetics MGG.

[17]  J. M. Gutierrez,et al.  Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangrene. , 2000, European journal of biochemistry.

[18]  H. Rasmussen,et al.  Regulation of protein kinase C activity by gangliosides. , 1987, The Journal of biological chemistry.

[19]  R. Titball,et al.  Differences in the Carboxy-Terminal (Putative Phospholipid Binding) Domains of Clostridium perfringens andClostridium bifermentans Phospholipases C Influence the Hemolytic and Lethal Properties of These Enzymes , 1999, Infection and Immunity.

[20]  I. Florin.  Isolation of a fibroblast mutant resistant to Clostridium difficile toxins A and B. , 1991, Microbial pathogenesis.

[21]  K. Sandhoff,et al.  Tunicamycin inhibits ganglioside biosynthesis in rat liver Golgi apparatus by blocking sugar nucleotide transport across the membrane vesicles. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[22]  D. Moss,et al.  Identification of residues in the carboxy-terminal domain of Clostridium perfringens alpha-toxin (phospholipase C) which are required for its biological activities. , 2000, Archives of biochemistry and biophysics.

[23]  David S Moss,et al.  Crystal structure of the C. perfringens alpha-toxin with the active site closed by a flexible loop region. , 2002, Journal of molecular biology.

[24]  E. Heinz,et al.  In vitro characterization of anti-glucosylceramide rabbit antisera. , 2000, Glycobiology.

[25]  E. Berger,et al.  Golgi-disturbing agents , 1998, Histochemistry and Cell Biology.

[26]  Shinichi Nakamura,et al.  Clostridium absonum alpha-toxin: new insights into clostridial phospholipase C substrate binding and specificity. , 2003, Journal of molecular biology.

[27]  A. Elbein.  Glycosidase inhibitors: inhibitors of N‐linked oligosaccharide processing , 1991, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[28]  Y. Hirabayashi,et al.  Glucosylceramide synthase and glycosphingolipid synthesis. , 1998, Trends in cell biology.

[29]  G. Zimmerman,et al.  Alpha toxin from Clostridium perfringens induces proinflammatory changes in endothelial cells. , 1997, The Journal of clinical investigation.

[30]  Hui Zhao,et al.  Anoxia and reoxygenation of human endothelial cells decreases ceramide glucosyltransferase expression and activates caspases , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[31]  M. Nagahama,et al.  Membrane-damaging action of Clostridium perfringens alpha-toxin on phospholipid liposomes. , 1996, Biochimica et biophysica acta.

[32]  M. Čačić,et al.  Glycosphingolipid expression in human skeletal and heart muscle assessed by immunostaining thin-layer chromatography , 2004, Glycoconjugate Journal.

[33]  R. Titball,et al.  The Clostridium perfringensα-toxin , 1999 .

[34]  M. Flores-Díaz,et al.  Tyrosine 331 and phenylalanine 334 in Clostridium perfringens α‐toxin are essential for cytotoxic activity , 2001, FEBS letters.

[35]  R. Schnaar,et al.  High‐Affinity Anti‐Ganglioside IgG Antibodies Raised in Complex Ganglioside Knockout Mice , 2000, Journal of neurochemistry.

[36]  E C Nice,et al.  Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization. , 1998, Glycobiology.

[37]  U. Neumann,et al.  Glycosphingolipids of skeletal muscle: I. Subcellular distribution of neutral glycosphingolipids and gangliosides in rabbit skeletal muscle. , 1998, Carbohydrate research.

[38]  P. Roggentin,et al.  Sialidase activity in the sera of patients and rabbits with clostridial myonecrosis. , 1985, Clinica chimica acta; international journal of clinical chemistry.

[39]  F. Goñi,et al.  Dual inhibitory effect of gangliosides on phospholipase C-promoted fusion of lipidic vesicles. , 1996, Biochemistry.

[40]  M. Jensen,et al.  Effects of type 2 diabetes on the ability of insulin and glucose to regulate splanchnic and muscle glucose metabolism: evidence for a defect in hepatic glucokinase activity. , 2000, Diabetes.

[41]  D. Moss,et al.  Structure of the key toxin in gas gangrene , 1998, Nature Structural &Molecular Biology.

[42]  F. Goñi,et al.  Inhibition by gangliosides of Bacillus cereus phospholipase C activity against monolayers, micelles and bilayer vesicles. , 1996, European journal of biochemistry.

[43]  Thilo Stehle,et al.  Gangliosides are receptors for murine polyoma virus and SV40 , 2003, The EMBO journal.

[44]  Wesley E. Martin,et al.  A novel class of microbial phosphocholine‐specific phospholipases C , 2002, Molecular microbiology.

[45]  Alfred H. Merrill,et al.  De Novo Sphingolipid Biosynthesis: A Necessary, but Dangerous, Pathway* , 2002, The Journal of Biological Chemistry.

[46]  S. Cole,et al.  UDP-glucose Deficiency Causes Hypersensitivity to the Cytotoxic Effect of Clostridium perfringens Phospholipase C* , 1998, Journal of Biological Chemistry.

[47]  Y. Hirabayashi,et al.  Complete Removal of Sphingolipids from the Plasma Membrane Disrupts Cell to Substratum Adhesion of Mouse Melanoma Cells* , 1996, The Journal of Biological Chemistry.

[48]  G. Tettamanti.  Ganglioside/glycosphingolipid turnover: New concepts , 2003, Glycoconjugate Journal.

[49]  P. Roggentin,et al.  Gene structure of the ‘large’ sialidase isoenzyme fromClostridium perfringens A99 and its relationship with other clostridialnanH proteins , 1994, Glycoconjugate Journal.

[50]  M. Hattori,et al.  Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[51]  R. Titball,et al.  A genetically engineered vaccine against the alpha-toxin of Clostridium perfringens protects mice against experimental gas gangrene. , 1993, Vaccine.

[52]  W. Goebel,et al.  Listeria Pathogenesis and Molecular Virulence Determinants , 2001, Clinical Microbiology Reviews.

[53]  J. Esko,et al.  Chinese Hamster Ovary Cell Mutants Defective in Glycosaminoglycan Assembly and Glucuronosyltransferase I* , 1999, The Journal of Biological Chemistry.

[54]  D. Court,et al.  Transcription antitermination: the λ paradigm updated , 1995 .

[55]  M. Flores-Díaz,et al.  Effects of Clostridium perfringens phospholipase C in mammalian cells. , 2004, Anaerobe.

[56]  S. Hakomori,et al.  An IgG3 monoclonal antibody established after immunization with GM3 lactone: immunochemical specificity and inhibition of melanoma cell growth in vitro and in vivo. , 1988, Cancer research.

[57]  A. Helenius,et al.  Intracellular functions of N-linked glycans. , 2001, Science.

[58]  R. Schnaar,et al.  Tunicamycin inhibits ganglioside biosynthesis in neuronal cells. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[59]  T. Karasawa,et al.  Clostridium sordellii Phospholipase C: Gene Cloning and Comparison of Enzymatic and Biological Activities with Those of Clostridium perfringens and Clostridium bifermentans Phospholipase C , 2003, Infection and Immunity.

[60]  J. Songer,et al.  Bacterial phospholipases and their role in virulence. , 1997, Trends in microbiology.