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

Marietta Flores-Díaz,Alberto Alape-Girón,Graeme Clark,Bruno Catimel,Yoshio Hirabayashi,Ed Nice,José-María Gutiérrez,Richard Titball,Monica Thelestam

doi:10.1074/jbc.m500278200

Abstract

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-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.

Highlights

Bacterial phospholipases C (PLCs)1 (EC 3.1.4.3) are secreted proteins that display a variable preference for different phos
Inhibition of Proteoglycan Synthesis Does Not Affect Cellular Sensitivity to Clostridium perfringens phospholipase C (Cp-PLC)—To evaluate the role of proteoglycans in the cell sensitivity to Cp-PLC, the following two cell lines having extreme differences in their proteoglycan content due to a genetic reason were used: the proteoglycan-deficient mutant cell line, pgsG-110, which has an impaired activity of the enzyme GlcUAT-I, that catalyzes the first step in the synthesis of the tetrasaccharide primer of glycosaminoglycans [20], and the clone pgs-T, which is a stable transfectant having a wild type GlcUAT-I gene
Pretreatment of Don wt with tunicamycin, an inhibitor of the N-acetylglucosamine phosphotransferase, which blocks the synthesis of all N-linked glycoproteins [21], was found to increase the cellular sensitivity to both Cp-PLC and wheat germ lectin, in comparison with control cells (Fig. 3, A and B, bars 8)

Summary

Introduction

Bacterial phospholipases C (PLCs) (EC 3.1.4.3) are secreted proteins that display a variable preference for different phos-. Cp-PLC is called ␣-toxin and has been associated with enteritis in domestic animals, Crohn disease, and gas gangrene in humans [6]. The plc gene, encoded in the C. perfringens chromosome, shows minimal inter-strain sequence variations and is highly expressed in all strains associated with gas gangrene [6, 9]. Several lines of evidence indicate that Cp-PLC is the major virulence factor in C. perfringens-induced gas gangrene. When injected intramuscularly in mice, recombinant Cp-PLC causes myonecrosis and reproduces the histopathological features of gas gangrene [12]. A C. perfringens mutant strain, in which the plc gene has been inactivated by homologous recombination, is unable to produce gas gangrene [14]. Ganglioside Reduction Causes ␣-Toxin Hypersensitivity considerable progress has been made in recent years in the knowledge of the structure of Cp-PLC, our understanding of the cellular and molecular basis of its toxic effects is still incomplete [15]

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