Quantitation of monomeric and oligomeric forms of membrane-bound staphylococcal alpha-toxin by enzyme-linked immunosorbent assay with a neutralizing monoclonal antibody

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

doi:10.1128/iai.55.12.2933-2939.1987

Cited by 18 publications

(14 citation statements)

References 27 publications

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“…Monoclonal antibodies (MAb) of murine origin against alpha-toxin were produced as described previously (25). MAb a4C1 is a neutralizing antibody, whereas MAb clones a4D6 and a4D9 exhibit no neutralizing capacity.…”

Section: Methodsmentioning

confidence: 99%

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Release of interleukin-1 beta associated with potent cytocidal action of staphylococcal alpha-toxin on human monocytes

et al. 1989

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The pathogenetic relevance of Staphylococcus aureus alpha-toxin in humans has been debated because human cells have been thought to display a natural resistance toward the cytotoxic action of this cytolysin. Following our previous demonstration that human platelets represent sensitive targets for toxin attack, we have now identified monocytes as a second, highly vulnerable human cell species that succumb to attack by low doses (20 ng/ml) of alpha-toxin. The cytotoxic action of alpha-toxin is reflected in a rapid depletion of cellular ATP that is essentially complete within 30 min. The presence of human plasma proteins affords some protection of monocytes against the action of the toxin. In 10% autologous serum, ATP depletion commences at 80 to 300 ng of toxin per ml. Subcytolytic doses stimulate the release of tumor necrosis factor alpha, a process that is slightly accentuated in the presence of 50% serum. Cytocidal toxin doses unfailingly cause the release of large amounts of interleukin-1 beta from cultured cells, with levels of this monokine generally exceeding 10 ng/ml in the cell supernatants 60 min after application of toxin. Initial evidence suggests that this is due to processing of intracellular interleukin-1 rather than to de novo synthesis of the cytokine. All noted effects are abrogated in the presence of a neutralizing monoclonal antibody against alpha-toxin. Through its capacity to provoke cytokine release from monocytes and its attack on platelets, alpha-toxin may initiate cellular events that are relevant to the pathogenesis of staphylococcal infection.

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Section: Methodsmentioning

confidence: 99%

“…When indicated, cycloheximide or actinomycin D was added to the medium at a final concentration of 2 or 10 ,ug/ml, respectively. Cell numbers were obtained by counting 25 fields per well through a calibrated microscopic objective. Each culture well typically contained approximately 105 monocytes.…”

Section: Methodsmentioning

confidence: 99%

Release of interleukin-1 beta associated with potent cytocidal action of staphylococcal alpha-toxin on human monocytes

et al. 1989

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“…Fibronogen was from Kabi Vitrum (Munich, FRG) . mAb a4C1 against a toxin has been described (20). Another mAb, 4G3, was produced that does not inhibit toxin binding but inhibits lateral aggregation and oligomer formation in the cell membrane (Hugo, F, B. Eberspacher, and S .…”

Section: Methodsmentioning

confidence: 99%

“…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

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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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“…The method described by Hugo et al [10] was followed with few modifications. Briefly, the toxin-treated human erythrocyte membranes were prepared by incubating 3 X 108 washed erythrocytes with 20 /xg of purified CTox in a final volume of 1 ml of Krebs Ringer phosphate buffer (KRP; 120 mM NaCl, 4.9 mM KCI, 0.33 mM NaHEPO4, 16.47 mM NaEHPO4, pH 7.4), for 10 min at 4°C.…”

Section: Extraction and Determination Of Ctox Inserted Into Erythrocymentioning

confidence: 99%

Physical characterization of the pore forming cytolysine from Gardnerella vaginalis

Morán

1992

FEMS Microbiology Letters

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Quantitation of monomeric and oligomeric forms of membrane-bound staphylococcal alpha-toxin by enzyme-linked immunosorbent assay with a neutralizing monoclonal antibody

Cited by 18 publications

References 27 publications

Release of interleukin-1 beta associated with potent cytocidal action of staphylococcal alpha-toxin on human monocytes

Release of interleukin-1 beta associated with potent cytocidal action of staphylococcal alpha-toxin on human monocytes

Staphylococcal α toxin promotes blood coagulation via attack on human platelets

Physical characterization of the pore forming cytolysine from Gardnerella vaginalis

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