Intermedilysin, a novel cytotoxin specific for human cells secreted by Streptococcus intermedius UNS46 isolated from a human liver abscess

Abstract

A novel cytotoxin (intermedilysin) specific for human cells was identified as a cytolytic factor of Streptococcus intermedius UNS46 isolated from a human liver abscess. Intermedilysin caused human cell death with membrane blebs. Intermedilysin was purified from UNS46 culture medium by means of gel filtration and hydrophobic chromatography. The purified toxin was resolved into major and minor bands of 54 and 53 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These proteins reacted with an antibody against intermedilysin. Five internal peptide fragments of intermedilysin were sequenced and found to have 42 to 71% homology with the thiol-activated cytotoxin pneumolysin. However, the action of intermedilysin differed from that of thiol-activated cytotoxins, especially in terms of a lack of activation by dithiothreitol and resistance to treatments with N-ethylmaleimide and 5,5-dithio-bis-(2-nitrobenzoic acid), although cholesterol inhibited the toxin activity. Intermedilysin was potently hemolytic on human erythrocytes but was 100-fold less effective on chimpanzee and cynomolgus monkey erythrocytes. Intermedilysin was not hemolytic in nine other animal species tested. Since human erythrocytes treated with trypsin were far less sensitive to intermedilysin than were the intact cells, a cell membrane protein(s) may participate in the intermedilysin action. These data demonstrated that intermedilysin is distinguishable from all known bacterial cytolysins.

Intermedilysin Is Essential for the Invasion of Hepatoma HepG2 Cells by Streptococcus intermedius

“…PLY (the closest CDC relative of ILY) was shown to facilitate pneumococcal invasion/ penetration and proliferation in alveolar cells and, following bacteremia (31), damage endothelial cells of the blood brain barrier (44) and cause meningitis in the mouse (36). The cytolytic characteristics and steric molecular structure of ILY are similar to those of other CDCs (24,25,29), although ILY shows an unusual and perhaps significant species specificity for human cells and recognizes an alternative cell membrane receptor to cholesterol, the receptor molecule for typical CDCs (24,25). It has recently been reported that the ILY receptor is CD59, a glycosylphosphatidylinositol (GPI)-anchored cell surface protein (10) production levels and deep-seated infection has been shown (27) together with modulation of the PMN functions by ILY (22), the role of ILY in infection by S. intermedius was evaluated using an ily knockout mutant and complementation strain in this study.…”

“…Previously we reported a human-specific pore-forming cytolysin, intermedilysin (ILY) secreted from a strain of S. intermedius originally isolated from the human liver (25,26). ILY is a member of the cholesterol-dependent cytolysins (CDC) which include streptolysin O (SLO) (4,19), and it has been shown to directly cause cell membrane damage in human erythrocytes and in cell lines derived from the major organs involved in deep-seated infections by S. intermedius (25). Therefore, ILY has been suggested as a potentially important virulence factor of this species (17,25).…”

“…ILY is a member of the cholesterol-dependent cytolysins (CDC) which include streptolysin O (SLO) (4,19), and it has been shown to directly cause cell membrane damage in human erythrocytes and in cell lines derived from the major organs involved in deep-seated infections by S. intermedius (25). Therefore, ILY has been suggested as a potentially important virulence factor of this species (17,25). It has also been shown that the production level of ILY in isolates from deep-seated abscesses is 6.2-10.2-fold higher than in strains from normal habitats such as dental plaque, although no differences in production levels of other potential virulence factors such as hyaluronidase and sialidase were found (27).…”

Streptococcal toxins: role in pathogenesis and disease

“…AGS are opportunist human pathogens capable of causing liver and brain abscesses, dentoalveolar infections and endocarditis (Jacobs et al, 1995). The secreted intermedilysin (ILY) of S. intermedius binds complement receptor CD59 on human cells and forms cytotoxic pores, but only in the presence of sufficient levels of cholesterol (Nagamune et al, 1996;Farrand et al, 2008;Heuck et al, 2010;Johnson et al, 2013). ILY damages host tissues and immune cells to promote bacterial survival and dissemination (Nagamune et al, 1996(Nagamune et al, , 2000.…”

“…The secreted intermedilysin (ILY) of S. intermedius binds complement receptor CD59 on human cells and forms cytotoxic pores, but only in the presence of sufficient levels of cholesterol (Nagamune et al, 1996;Farrand et al, 2008;Heuck et al, 2010;Johnson et al, 2013). ILY damages host tissues and immune cells to promote bacterial survival and dissemination (Nagamune et al, 1996(Nagamune et al, , 2000. ILY is considered to be a major virulence factor required for adherence to and invasion of human liver cells (Sukeno et al, 2005).…”