Alpha-toxin

This bacterium can produce numerous types of toxins that cause a range of medical problems. The most potent toxin of C. perfringens is the alpha toxin — the one most likely to be used by terrorists. It would be lethal by aerosol delivery. 

Y Ji, A Marra, M Rosenberg, G Woodnutt. Regulated antisense RNA eliminates alpha-toxin virulence in Staphylococcus aureus infection. J Bacteriol 181 6585— 6590, 1999. 

Sine, S.M. Taylor, P. The relationship between agonist occupation and the permeability response of the chohnergic receptor revealed by bound cobra alpha-toxin. J. Biol. Chem. 1980, 225, 10144-10156. 

Bette P, Frevert J, Mauler F, etal. (1989) Pharmacological and biochemical, studies of cytotoxicity of Clostridium novyi type A alpha-toxin. In Infect. Immun. 57 2507-2513. 

Bette P, Oksche A, Mauler F, et al. (1991) A comparative biochemical, pharmacological and immunological study of Clostridium novyi alpha-toxin, C.difficile toxin B and C.sordellii lethal toxin. In Toxicon, 29 877-887. 

Hofmann F, Herrmann A, Habermann E, etal. (1995) Sequencing and analysis of the gene encoding the alpha-toxin of Clostridium novyi proves its homology to toxins A and B of Clostridium difficile. In A4o/ Gen Genet, 247 670—679. 

The concept of membrane damage as an effector mechanism of bacterial exotoxins arose during the 1970s, mainly through the work on alpha-toxin by Freer, Arbuthnott and Bernheimer (Bernheimer, 1974 Freer et al., 1968) and, subsequently, Thelestam and Mollby (1975). These studies gave rise to the idea that staphylococcal alpha-toxin produced permeability defects in membranes. However, it was... 

Binding studies have only been undertaken with alpha-toxin (Cassidy and Harshman, 1976 Hildebrand etai, 1991), aerolysin (Howard and Buckley, 1982), streptolysin O (Palmer etal., 1993) and perfringolysin... 

Certain cellular processes are specifically influenced by gradients of monovalent ions across the plasma membrane. It appears that stimulation of some such processes are actually counteracted by concomitant flux. Ca -dependent processes in turn may be abrogated when pores are large enough to permit rapid egress of cytoplasmic proteins. Therefore, it is useful to differentiate between three types of pores (a) those that are selectively permissive for monovalent ions (e.g. staphylococcal alpha-toxin) (b) those that are permissive for Ca and small molecules, but not for proteins (e.g. E. coli hemolysin) and (c) large pores that allow passage of macromolecules (e.g. streptolysin O). 

Two possibly related phenomena have been found to be dependent on the flux of monovalent ions. The hypothetical common link is represented by a newly discovered family of intracellular proteases whose activity may be influenced by concentrations. Interleukin converting enzyme (ICE) is the best studied member of this family. Efflux of from monocytes leads to activation of ICE, so that the cells rapidly process and export IL-16 (Walev etal., 1995). An ICE-related protease is involved in regulating programmed cell death, which may be the reason why formation of K -permissive pores by alpha-toxin in human T-lymphoctes causes apoptosis (Jonas et a/., 1994). Both apoptosis and ICE-activation are inhibited when alpha-toxin treated cells are suspended in K" -rich medium. It is of interest that simultaneous flooding of cells with Ca , such as occurs when larger pores are formed in lymphocytes (e.g. at high alpha-toxin concentrations or with . coli hemolysin) counteracts the apoptosis-promoting effect of K -efflux (Jonas et a/., 1994). 

Secretion. Exocytotic liberation of granular constituents has been demonstrated in leukocytes, platelets and neurological cells. Platelets attacked by alpha-toxin secrete platelet factor 4 and factor 5 (Arvand et a/., 1990 Bhakdi ef al., 1988). Release of the latter leads to assembly of platelet-bound prothrombinase complexes that generate thrombin. Alpha-toxin can thus promote coagulation via its perme-abilizing action on platelets. Release of granule constituents from leukocytes has been observed after permeabilization of these cells by HlyA. For example, large amounts of elastase are secreted into the extracellular medium (Bhakdi eta/., 1989). 

Stimulation of constitutive NO-synthase. Both S. aureus alpha-toxin and HlyA increase synthesis of NO by the constitutive NO synthase in endothelial cells (Suttorp et al., 1993). NO in turn can provoke an array of further reactions in bystander cells. 

In the following, we will describe our current protocols for isolating staphylococcal alpha-toxin and streptolysin-O from bacterial culture supernatants. The protocol for isolating recombinant streptolysin-O from call has been published (Weller et al., 1996) and will not be... 

The strain most widely used tor toxin production is S. aureus Wood 46. Note that the presence of proteases may give rise to proteolytic cleavage of alpha-toxin, which results in altered pore-forming activity (Palmer et al., 1993). Moreover, proteolytic activity contaminating the final toxin preparation may interfere with cell-biological experiments. Per liter of culture, the following protocol typically yields 20 mg of alpha-toxin suitable for cell-biological applications with a strain available from this laboratory. 

Since high concentration promotes membrane-independent oligomerization of alpha-toxin, the following steps should be carried out at 4°C. The culture supernatant is cycled through a suitable membrane concentration device until the volume has been reduced by about 90 %. 

The advantages of PFTs are manifold. The effects of digitonin or saponin are normally difficult to predict, control or monitor. In contrast, the actions of PFTs are well characterized and easy to control. The toxins are easy to handle. The two agents currently used by most groups, alpha-toxin and streptolysin O, are stable and can be stored in lyophilized form tor years. No special procedures are required to... 

When very small pores that are permissive for monovalent but not for divalent ions are required, alpha-toxin should be used in a concentration range of 0.5-5 ng/ml. Most cells will become perme-abilized.Note, however, that certain cells exhibit a natural resistance towards alpha-toxin and pore formation will not occur. A simple means to discern whether permeabilization has taken place is to observe whether the cells swell increases in cell volume are the consequence of an uncontrolled flux of monovalent ions and water, and can be observed microscopically or by flow cytometry. Another simple method is measurement of cellular ATP. One hour after toxin application, cells are lysed with Triton X-100, and ATP is quantified using the luciferase assay (method described in (Bhakdi ef al., 1989)). ATP depletion will always be found in cells that have been permeabilized. 

Arvand M, Bhakdi S, Dahiback B et al. (1990) Staphylococcus aureus alpha-toxin attack on human platelets promotes assembly of the prothrombinase complex. In J Biol Chem 265 14377-14381... 

Bhakdi S, Bayley H, Valeva A etal. (1996) Staphylococcal alpha-toxin, streptolysin O, and Escherichia coli hemolysin prototypes of pore forming bacterial cytoly-sins. In Arch Microbiol 165 73-79. 

Bhakdi S, Tranum-Jensen J (1991) Alpha-toxin of Staphylococcus aureus. In A4/cro-biol Rev 55 733-751. 

Bhakdi S, Muhly M, Mannhardt U et al. (1988) Staphylococcal alpha toxin promotes blood coagulation via attack on human platelets. In J Exp Med 168 527-542. 

Bramley AJ, Patel AH, O Reilly M etal. (1989) Roles of alpha-toxin and beta-toxin in virulence of Staphylococcus aureus tor the mouse mammary gland. In Infect Immun 57 2489-2494. 

Cassidi P, Harshman S. (1976) Biochemical studies on the binding of staphylococcal l-labeled alpha-toxin to rabbit erythrocytes. In Biochemistry 15 2348-2355. 

Freer JH, Arbuthnott JP, Bernheimer AW (1968) Interaction of staphylococcal alpha toxin with artificial and natural membranes. In J Bacterial 95 1153-1168. 

FOssle R, Bhakdi S, Sziegoleit A et ol. (1981) On the mechanism of membrane damage by S. aureus alpha-toxin. In J Cell Biol 91 83-94. 

Jonas D, Walev I, Berger T et al. (1994) Small transmembrane pores created by staphylococcal alpha-toxin in T lymphocytes evoke internucleosomal DNA-degradation. In Infect Immun 62 1304-1312. 

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