Sialic acid facilitates binding and cytotoxic activity of the pore-forming Clostridium perfringens NetF toxin to host cells

Abstract

NetF-producing type A Clostridium perfringens is an important cause of canine and foal necrotizing enteritis. NetF, related to the β-sheet pore-forming Leukocidin/Hemolysin superfamily, is considered a major virulence factor for this disease. The main purpose of this work is to demonstrate the pore-forming activity of NetF and characterize the chemical nature of its binding site. Electron microscopy using recombinant NetF (rNetF) confirmed that NetF is able to oligomerize and form large pores in equine ovarian (EO) cell membranes and sheep red blood cells. These oligomeric pores appear to be about 4–6 nm in diameter, and the number of oligomer subunits to vary from 6 to 9. Sodium periodate treatment rendered EO cells non-susceptible to NetF, suggesting that NetF binding requires cell surface carbohydrates. NetF cytotoxicity was also inhibited by a lectin that binds sialic acid, by sialidase, and by free sialic acid in excess, all of which clearly implicate sialic acid-containing membrane carbohydrates in NetF binding and/or toxicity for EO cells. Binding of NetF to sheep red blood cells was not inhibited by the gangliosides GM1, GM2 and GM3, nor did the latter promote membrane permeabilization in liposomes, suggesting that they do not constitute the cellular receptors. In contrast, treatment of EO cells with different proteases reduced their susceptibility to NetF, suggesting that the NetF receptor is a sialic acid-containing glycoprotein.

Sialidases From Clostridium perfringens and Their Inhibitors

“…In addition to affecting bacterial colonization, NanI can enhance the activity of α-toxin that causes gas gangrene (Flores-Díaz et al, 2005;Chiarezza et al, 2009), while also increasing the cytotoxicity of β-and ε-toxins and NetF and CPE toxin associated with gastrointestinal diseases (Li et al, 2011;Mehdizadeh Gohari et al, 2018;Theoret et al, 2018). Although many C. perfringens pathogenic strains only produce a small amount of CPB, CPE, or ETX (Collie et al, 1998;Sayeed et al, 2005;Fisher et al, 2006;Fernandez-Miyakawa et al, 2007;Ma et al, 2014), and the enhancement effect of natural levels of NanI on CPB, CPE and ETX is relatively moderate (1.5-2-fold), this activity may be important for the pathogenesis of C. perfringens during enteritis or enterotoxemia.…”

NetF-producingClostridium perfringensand its associated diseases in dogs and foals

“…The ability of NetF to form pores in biological membranes has been investigated, as well as the host cell surface receptor for NetF or some of its major characteristics; NetF is able to oligomerize and form pores with 6–8 subunits in biological cell membranes such as sheep erythrocytes and EO cells. 42 The defined size of the NetF pore was estimated to be ~4–6 nm. NetF pores are very similar in size to C. perfringens delta-toxin pores with a functional diameter of 5 nm, 33 but are larger than NetB pores (1.8 nm) and S. aureus alpha-toxin pores (2.8 nm).…”

“…16,25 In addition, a cell surface sialoprotein(s) most likely plays a crucial role in binding or cytopathic activity of NetF. 42 However, further work is required to identify the host cell surface receptor(s) for NetF because identification could not only improve understanding of the mode of action and host-specificity of this toxin but it might also lead to the development of therapeutic or preventive approaches that target the activity of NetF.…”

Pathogenicity and virulence of Clostridium perfringens

“…Recently, it was shown that NetF binds to a sialoprotein(s) on the surface of biological membranes and form pores containing 6–8 NetF monomers [ 73 ]. Osmotic protection assay results revealed that the NetF pore has a functional diameter of ~4-6 nm in RBCs and equine ovarian cell lines, which is larger than the NetB pore (1.8 nm) and S. aureus alpha-toxin pore (2.8 nm) [ 58 , 73 , 74 ].…”

“… 36 - - pCW3-like plasmid α-clostripain Cysteine Protease ? 59.6 - - Chromosome a Compiled from [ 6 , 8 , 22 , 37 , 45 , 59 , 64 , 68 , 72 , 82 , 255 , 293–296 ] b Compiled from [ 5 , 28 , 31 , 36 , 58 , 60 , 68 , 73 , 78 , 297–301 ] c Compiled from [ 12 , 62 , 109 , 112 , 115 , 120 , 123 , 136–144 , 302 , 303 ] …”