Pore Alpha-toxin

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). 

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. 

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. 

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. 

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. 

Permeabilized cells allow the study of intracellular processes in situ under conditions which are believed to be close to the physiological situation in intact cells. Permeabilization by bacterial pore-forming toxins, alpha-toxin and streptolysin O (SLO) is now a widely accepted approach in the functional analysis of intracellular organelles. 

An example of pore-forming alpha helix acting via the barrel-stave models is the toxin pardaxin. This 33-residue peptide is found in the Red Sea Moses sole (a flatfish) and nsed to repel Sharks. It is found that this peptide binds to bilayer snrfaces and forms transmembrane pores consisting of 6 3 peptides. Remarkable is the efficiency... 

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