Quantitative analysis of the binding and oligomerization of staphylococcal alpha-toxin in target erythrocyte membranes

Reichwein, J; Hugo, F; Roth, Markus; Sinner, Axel; Bhakdi, Sucharit

doi:10.1128/iai.55.12.2940-2944.1987

Cited by 32 publications

(21 citation statements)

References 27 publications

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“…The binding exhibited no recognizable saturation and displayed no characteristics of a receptor-ligand interaction ; the total net binding was calculated to be -10% at all toxin concentrations between 0.5 and 12 .5 Pg/ml. A similar binding behavior was previously noted with rabbit erythrocytes (21). The ELISA permitted quantitative differentiation between toxin monomers and oligomers.…”

Section: Resultssupporting

confidence: 77%

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Staphylococcal α toxin promotes blood coagulation via attack on human platelets

Bhakdi¹,

Muhly²,

Mannhardt³

et al. 1988

The Journal of Experimental Medicine

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125

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Staphylococcus aureus plays a major role as a bacterial pathogen in human medicine, causing diseases that range from superficial skin and wound to systemic nosocomial infections . The majority of S. aureus strains produces a toxin, a proteinaceous exotoxin whose hemolytic, dermonecrotic, and lethal properties have long been known (1-6). The toxin is secreted as a single- chained, nonglycosylated polypeptide with a M(r) of 3.4 x 10(4) (7, 8). The protein spontaneously binds to lipid monolayers and bilayers (9-14), producing functional transmembrane pores that have been sized to 1.5-2.0-nm diameters (15-18). The majority of pores formed at high toxin concentrations (20 μg/ml) is visible in the electron microscope as circularized rings with central pores of approximately 2 nm in diameter. The rings have been isolated, and molecular weight determinations indicate that they represent hexamers of the native toxin (7). We have proposed that transmembrane leakiness is due to embedment of these ring structures in the bilayer, with molecular flux occurring through the central channels (15, 19). Pore formation is dissectable into two steps (20, 21). Toxin monomers first bind to the bilayer without invoking bilayer leakiness . Membrane-bound monomers then laterally diffuse and associate to form non-covalently bonded oligomers that generate the pores. When toxin pores form in membranes of nucleated cells, they may elicit detrimental secondary effects by serving as nonphysiologic calcium channels, influx of this cation triggering diverse reactions, including release of potent lipid mediators originating from the arachidonate cascade (22-24). That α toxin represents an important factor of staphylococcal pathogenicity has been clearly established in several models of animal infections through the use of genetically engineered bacterial strains deleted of an active α toxin gene (25-27). Whether the toxin is pathogenetically relevant in human disease, however, is a matter of continuing debate. Doubts surrounding this issue originate from two main findings. First, whereas 60 percent hemolysis of washed rabbit erythrocytes is effected by approximately 75 ng/ml α toxin, approximately 100-fold concentrations are required to effect similar lysis of human cells (4-6, 13). The general consensus is that human cells display a natural resistance towards toxin attack. The reason for the wide inter-species variations in susceptibility towards α toxin is unknown but does not seem to be due to the presence or absence of high-affinity binding sites on the respective target cells (20, 21). Second, low-density lipoprotein (28) and neutralizing antibodies present in plasma of all healthy human individuals inactivate a substantial fraction of α toxin in vitro. These inactivating mechanisms presumably further raise the concentration threshold required for effective toxin attack, and it is most unlikely that such high toxin levels will ever be encountered during infections in the human organism. The aforegoing arguments rest on the validity of two general assumptions. First, the noted natural resistance of human erythrocytes to α toxin must be exhibited by other human cells. Second, toxin neutralization by plasma components, usually tested and quantified after their preincubation with toxin in vitro, must be similarly effective under natural conditions, and protection afforded by these components must not be restricted to specific cell species.

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Section: Resultssupporting

confidence: 77%

“…We have proposed that transmembrane leakiness is due to embedment of these ring structures in the bilayer, with molecular flux occurring through the central channels (15,19) . Pore formation is dissectable into two steps (20,21) . Toxin monomers first bind to the bilayer without invoking bilayer leakiness .…”

mentioning

confidence: 99%

Staphylococcal α toxin promotes blood coagulation via attack on human platelets

Bhakdi¹,

Muhly²,

Mannhardt³

et al. 1988

The Journal of Experimental Medicine

Self Cite

125

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“…Aerolysin, the only toxin known to be related to C. septicum alpha toxin (Ballard et al, 1995), has also been found to oligomerize at low temperature and does not appear to be cytolytic at that temperature (Garland and Buckley, 1988) although pore formation has not been directly measured at low temperature for aerolysin. Similar results have not, as yet, been conclusively demonstrated for Staphylococcus aureus alpha toxin (Bhakdi and Tranum-Jensen, 1991;Fussle et al, 1981;Harshman et al, 1989;Reichwein et al, 1987;Valeva et al, 1995;Walker et al, 1992), but a similar mechanism is probably utilized by this toxin. The current findings with C. septicum alpha toxin suggest a pathway for the transition of alpha toxin from a soluble monomer to a membrane-bound complex (Fig.…”

Section: Discussionsupporting

confidence: 58%

**Generation of a membrane‐bound, oligomerized pre‐pore complex is necessary for pore formation by Clostridium septicum alpha toxin**

Sellman

Kagan

Tweten

1997

Molecular Microbiology

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SummaryLow-temperature inhibition of the cytolytic activity of alpha toxin has facilitated the identification of an important step in the cytolytic mechanism of this toxin. When alpha toxin-dependent haemolysis was measured on erythrocytes at various temperatures it was clear that at temperatures Յ15ЊC the haemolysis rate was significantly inhibited with little or no haemolysis occurring at 4ЊC. Alpha toxin appeared to bind to and oligomerize on erythrocyte membranes with similar kinetics at 4ЊC and 37ЊC. The slight differences in these two processes at 4ЊC and 37ЊC could not account for the loss of cytolytic activity at low temperature. At 4ЊC alpha toxin neither stimulated potassium release from erythrocytes nor formed pores in planar membranes. In contrast, at temperatures Ն 25ЊC both processes proceeded rapidly. Pores that were opened in osmotically stabilized erythrocytes could not be closed by low temperature. Therefore, low temperature appeared to prevent the oligomerized complex from forming a pore in the membrane. These data support the hypothesis that alpha toxin oligomerizes into a membrane-bound, pre-pore complex prior to formation of a pore in a lipid bilayer.

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“…Pore formation by a-toxin requires the association of membrane-bound monomers into hexameric ring structures. This process is drastically retarded in cold membranes [33]. Accordingly, we found hemolysis by a-toxin to be almost abolished on ice (data not shown).…”

Section: Discussionmentioning

confidence: 65%

Interaction of Serratia marcescens hemolysin (ShlA) with artificial and erythrocyte membranes

Schönherr

Hilger

Bröer

et al. 1994

European Journal of Biochemistry

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Pore formation by hemolysin (ShlA) of Serratia marcescens was studied in erythrocytes and in artificial lipid bilayer membranes. The results with erythrocytes demonstrated that hemolysin pores varied in size. In erythrocyte membranes with reduced fluidity (0°C), the toxin formed small pores with diameter 1–1.5 nm. In fluid membranes (above 20°C), hemolysin pores with larger diameters (approximately 2.5–3.0 nm) were observed, which may be caused by association of ShlA monomers into oligomers. Comparison of the channels formed by Staphylococcus aureusα‐toxin with channels formed by ShlA indicated a slightly smaller pore diameter of ShlA pores. Analysis of ShlA in artificial lipid bilayers showed the formation of pores with a broad distribution of single channel conductances, suggesting variable sizes of the ShlA pore. The lower limit for the pore diameter was approximately 1.0 nm. The ShlA pores did not exhibit pronounced ion selectivity nor voltage dependence, supporting the presence of a large water‐filled pore.

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Quantitative analysis of the binding and oligomerization of staphylococcal alpha-toxin in target erythrocyte membranes

Cited by 32 publications

References 27 publications

Staphylococcal α toxin promotes blood coagulation via attack on human platelets

Staphylococcal α toxin promotes blood coagulation via attack on human platelets

**Generation of a membrane‐bound, oligomerized pre‐pore complex is necessary for pore formation by Clostridium septicum alpha toxin**

Interaction of Serratia marcescens hemolysin (ShlA) with artificial and erythrocyte membranes

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