Mechanism of induction of complement susceptibility of erythrocytes by spider and bacterial sphingomyelinases

Abstract

SUMMARYWe have recently shown that the sphingomyelinase toxins P1 and P2 from the venom of the spider Loxosceles intermedia induce complement (C)-dependent lysis of autologous erythrocytes by induction of the cleavage of cell surface glycophorins through activation of an endogenous metalloproteinase facilitating the activation of the alternative pathway of C. Phospholipase D (PLD) from Corynebacterium pseudotuberculosis shows some degree of homology with the spider sphingomyelinases and can induce similar clinical symptoms to those observed after spider envenomation. The aim of this study was to investigate if the bacterial PLD-induced haemolysis of human erythrocytes was C dependent and if cleavage of glycophorins occurred. We show here that haemolysis of both PLD-and P1-treated human erythrocytes was C dependent, but while PLD-mediated haemolysis was dependent on activation of the classical pathway of C, P1 induced lysis via both the classical and alternative pathways. P1, but not PLD, induced cleavage of glycophorins and no change in expression of complement regulators was induced by either of the toxins. In both cases, annexin V binding sites were exposed, suggesting that the membrane asymmetry had been disturbed causing exposure of phosphatidylserine to the cell surface. Our results suggest that C susceptibility induced by L. intermedia and C. pseudotuberculosis PLD is a result of exposure of phosphatidylserine, and the higher potency of P1 toxin can be explained by its additional effect of cleavage of glycophorins.

Spider and Bacterial Sphingomyelinases D Target Cellular Lysophosphatidic Acid Receptors by Hydrolyzing Lysophosphatidylcholine

“…It is also of note that LPA stimulates Ca 2ϩ entry into human erythrocytes (43) with subsequent exposure of phosphatidylserine (PS) on the cell surface. 2 SMaseD likewise induces PS exposure in erythrocytes and this apparent loss of plasma membrane asymmetry is thought to contribute to complement-dependent hemolysis (16). The potential role of lyso-PLD activity in the hemolytic action of SMaseD obviously warrants further study.…”

Corynebacterium pseudotuberculosis: microbiology, biochemical properties, pathogenesis and molecular studies of virulence

“…This exotoxin is a permeability factor that promotes the hydrolysis of ester bonds in sphingomyelin in mammalian cell membranes, possibly contributing to the spread of the bacteria from the initial site of infection to secondary sites within the host [19,30,65,69,89,106,108]. Moreover, it provokes dermonecrotic lesions, and at higher doses it is lethal to a number of different species of laboratory and domestic animals [34,102].…”

“…Several of the biological activities of C. pseudotuberculosis PLD, as well as its molecular structure, have also been found in sphingomyelinases in the venom of the medically important spider genus Loxosceles [7,10,30,102,108,112].…”

A description of genes of Corynebacterium pseudotuberculosis useful in diagnostics and vaccine applications

“…Phospholipase D (PLD) has for some years been implicated as the major virulence factor of C. pseudotuberculosis (Hodgson et al, 1999). The pld gene encodes an exotoxin (Burrel, 1983;Hodgson et al, 1994) that probably promotes bacterial dissemination, increasing vascular peret al, 1994) that probably promotes bacterial dissemination, increasing vascular per-1994) that probably promotes bacterial dissemination, increasing vascular permeability (Egen et al, 1989;Cardenas and Clements, 1992;Hodgson et al, 1994) through the hydroet al, 1989;Cardenas and Clements, 1992;Hodgson et al, 1994) through the hydro-1989; Cardenas and Clements, 1992;Hodgson et al, 1994) through the hydroet al, 1994) through the hydro-1994) through the hydrolysis of ester bonds in sphingomyelin in mammalian cell membranes following infection (Carne and Onon, 1978;Lipsky et al, 1982;Coyle and Lipsky, 1990;�ached�ian et al, 1995;Navas, 1996;�am-1978;Lipsky et al, 1982;Coyle and Lipsky, 1990;�ached�ian et al, 1995;Navas, 1996;�am-et al, 1982;Coyle and Lipsky, 1990;�ached�ian et al, 1995;Navas, 1996;�am-1982;Coyle and Lipsky, 1990;�ached�ian et al, 1995;Navas, 1996;�am-et al, 1995;Navas, 1996;Tam-1995;Navas, 1996;Tambourgi et al, 2002;Dorella et al, 2006a), be...…”

“…Several studies have been carried out involving the biological activities of C. pseudotuberculosis PLD, as well as its molecular structures, and results have shown similarities with sphingomyelinases present in the venom of the medically important spider genus Loxosceles (Bernheimer et al, 1985;Songer, 1997;Tambourgi et al, 2002;van Meeet al, 1985;Songer, 1997;Tambourgi et al, 2002;van Mee-1985;Songer, 1997;Tambourgi et al, 2002;van Meeet al, 2002;van Mee-2002;van Meeteren et al, 2004;Binford et al, 2005). Unlike diphtheria toxin, which occurs in about half of the isolates of C. diphtheria (Saragea et al, 1966;Toshach et al, 1977), PLD probably is characteristic of most or almost all strains of C. pseudotuberculosis and C. ulcerans and have not been found in any other Corynebacteria (Barksdale et al, 1981).…”