THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 258, No. 9, Issue of May 10, pp. 5899-5904, 1983 Printed in U. S. A.

Binding and Partial Inactivation of Staphylococcus aureus a-Toxin by Human Plasma Low Density Lipoprotein*

(Received for publication, November 3, 1982)

Sucharit BhakdiSg, Jqrgen Tranum-Jensenn, Gerd Utermannll, and Roswitha FiissleS From the $Institute of Medical Microbiology, University of Giessen, Schubertstrde 1, 0-6300 Giessen, West Germany, the IAnatomy Institute C, Uniuersity of Copenhagen, Slegdamsvej 3 C, DK-22Do Copenhagen N, Denmark, and the ((Department of Human Genetics, University of Marburg, 0-3550 Marburg, West Germany

Human low density lipoprotein (LDL) in isolated form or in unfractionated serum binds and partially inacti- vates Staphylococcus aureus a-toxin. Under conditions of LDL excess, up to 90% inactivation occurs as esti- mated by hemolytic titration. Inactivation is accompa- nied by, and probably due to, oligomerization of 3 S native toxin molecules into 11 S hexamers on the LDL molecules. This process is believed to be mediated by the lipids contained in the lipoprotein. The toxin hex- amers are visualized as ring structures and stubs bound to the spherical LDL molecules by negative staining electron microscopy. These structures appear identical with those formed on target erythrocyte membranes during toxin-dependent lysis. The toxin hexamers are trypsin-resistant and do not spontaneously dissociate from the lipoprotein. The binding process appears to be highly selective, and no similar interaction of toxin with any other serum protein, including high density lipoprotein, has been observed. LDL/a-toxin complexes exhibit some residual hemolytic activity which possibly derives from the presence of lipoprotein-bound but nonoligomerized 3 S toxin. This fraction of bound a- toxin appears to have the capacity of dissociating from the lipoprotein molecules and attack cells. The collec- tive results imply a dual role of LDL relative to S. aureus a-toxin in the organism. The lipoprotein may exert a beneficial effect through nonimmune inactiva- tion of a-toxin on the one hand, but may also serve as a carrier for a small fraction of potentially cytotoxic native toxin within the host.

Staphylococcus aureus a-toxin is a water-soluble, 3.3 S polypeptide of M, = 34,000 that is produced by most strains of pathogenic staphylococci (1, 2). Its biological significance lies in its capacity to damage a wide variety of cells. In the erythrocyte model, we have recently presented data that hemolysis ensues from an oligomerization of a-toxin molecules in the target lipid bilayer (3). This process leads to formation of amphiphilic, detergent- and lipid-binding 11 S toxin hex- amers that possess the structure of hollow rings or short cylinders (3,4). The insertion of the 11 S toxin complexes into the bilayer apparently creates functional pores of -3-nm effective diameter. Ion leakage is thought to occur through these channels, leading to colloid-osmotic swelling and, ulti- mately, rupture of the cells.

* This work was supported by the Deutsche Forschungsgemein- schaft (SFB 47). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed.

The oligomerization process leading to formation of amphi- philic toxin hexamers seems to be triggered quite unspecifi- cally when the native toxin comes into contact with a variety of lipid substrates. Thus, earlier studies showed that the toxin can bind to and damage liposomes of widely differing lipid composition (1, 2, 5, 6). More recently, hexamerization was induced simply through toxin contact with deoxycholate de- tergent micells (4). In the present study, we sought to deter- mine whether toxin oligomerization might also occur upon contact with lipid-containing serum proteins, e.g. the lipopro- teins. The affirmative results reported here demonstrate se- lective binding of a-toxin to human serum LDL.’ They suggest a role for nonimmune inactivation, but also possibly for trans- port of a small fraction of the active toxin by this lipoprotein in the human organism.

MATERIALS AND METHODS

Native 3 S toxin was obtained by chromatography of an a-toxin preparation from the Behringwerke (Marburg) as described previ- ously (3). Human serum LDL and HDL were isolated from human serum by density gradient ultracentrifugation as described (7). Anti- sera to apoprotein A and apoprotein B were purchased from the Behringwerke, Marburg. Antibodies to human serum proteins were obtained from Dakopatts Immunoglobulins, Copenhagen. Antisera to the I1 S form of a-toxin were raised in rabbits as described (3). The antiserum was found to precipitate the 11 S, but not the native 3 S, form of a-toxin in electroimmunoassays.

Electroimmunoassays-Crossed and fused rocket immunoelectro- phoresis with and without intermediate gels was performed in 1% agarose gels as described in Ref. 8, using an 0.1 M glycine, 0.038 M Tris buffer, pH 8.7.

SDS-PAGE4556 gel columns (5-mm diameter) were prepared and used as described by Fairbanks et al. (9). Samples were made 2% in SDS and incubated either at room temperature or at 100 “C for 30 s prior to application to the gels.

Sephacryl S-300 Gel Chromatography-A gel column (1 X 60 cm) equilibrated with 50 nm Tris, 50 rn NaC1,15 nm NaNR, pH 8.2, was used. Thirty-min fractions were collected at a flow rate of 3.6 ml/h in a cold room (5 “C). Sample volumes were 1-1.2 ml.

Hemolytic Assays-50-pl aliquots of individual column chromatog- raphy fractions were serially diluted with 0.9% NaCl solution in microtiter wells. An equal volume (50 pl) of a 2.5% rabbit erythrocyte suspension (in saline) was added to each well and hemolysis read visually after 60 min at 37 “C. The titer was defined as the last dilution giving total hemolysis.

Protein Determination-Protein was determined according to Lowry et al. (IO) in the presence of 0.1% SDS (final concentration) using bovine serum albumin as a standard. The protein content of LDL was assumed to be 25% of the lipoprotein mass (11).

Electron Microscopy-Negative staining with sodium silicotung- state or phosphotungstate was performed as described (4). Specimens were examined at 80 kV in a JEOL 100 CX electron microscope with apertures of 200 and 60 pm in the condenser and objective, respec-

’ The abbreviations used are: LDL, low density lipoprotein; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; HDL, high density lipoprotein.

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Nonimmune Toxin Inactivation by Plasma Lipoproteins

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FIG. 1. Crossed immunoelectrophoresis of human serum after incubation with a-toxin. S. aureus a-toxin was incubated with human serum and the toxin/serum sample was analyzed by crossed immunoelectrophoresis with an intermediate gel. Each upper gel contained rabbit antibodies to human serum proteins (10 pl/cm'). A, control plate with blank intermediate gel; B, intermediate gel containing rabbit antiserum against a-toxin (3 pl/cm2). First dimen- sion electrophoresis, 45 min at 10 V/cm (right to left); second dimen- sion immunoelectrophoresis, 2 V/cm overnight. Note the precipita- tion of a-toxin present in the unfractionated serum by the specific antibodies in the intermediate gel of B and the simultaneous disap- pearance of one human serum protein from the upper gel (marked with arrows in the reference pattern, A).

tively. Images were recorded on Agfa 23056 emulsion using a low dose exposure technique.

RESULTS

Selective Binding of a-Toxin to LDL: Demonstration by Crossed Immunoelectrophoresis

30 pg of a-toxin contained in 60 pl of buffer were incubated with 60 pl of fresh human serum for 10 min at 22 "C. There- after, the serum/a-toxin sample was analyzed by crossed immunoelectrophoresis. Fig. lA depicts an immunoelectro- phoresis developed with polyspecific antibodies to human serum proteins. The intermediate gel was blank. Approxi- mately 25 immunoprecipitates were discernible. In a plate run in parallel, specific antiserum to a-toxin was incorporated into the intermediate gel, and an immunoprecipitate representing a-toxin was then discerned in the lower gel (Fig. 1B). A comparison between plate A and B revealed that one serum protein Fig. L4, arrows) had selectively disappeared from the reference gel pattern. The suspicion thus arose that this protein might have co-precipitated with a-toxin in the inter- mediate gel of plate B. The /3-electrophoretic mobility of this putative a-toxin/serum protein complex suggested LDL as a possible candidate for the toxin-binding component. There- fore, further immunoelectrophoretical analyses were per- formed using specific antiserum to apolipoproteins A-I and B.

Fig. 2 depicts a crossed immunoelectrophoresis of the same serum/a-toxin sample, developed with anti-HDL' in the left gels. The intermediate gel in the control plate A was blank; plate B contained anti-a-toxin antiserum. The immunoprecip- itation of a-toxin in plate B left the immunoprecipitation of HDL entirely unaffected. Thus, an interaction between this lipoprotein and a-toxin could not be discerned by this method. A parallel experiment performed with anti-LDL is depicted in

For the sake of simplicity, anti-apoprotein A and B will be referred to as anti-HDL and anti-LDL, respectively.

anti (1-Tox

A '

LDL

I

LDL

FIG. 2. Crossed immunoelectrophoresis reveals specific binding of a-toxin to LDL. A similar toxin/serum sample was analyzed as in Fig. 1 using antisera specific for apoprotein A (A and B ) and apoprotein B (C and D) to develop the immunoprecipitates in the upper gels. A and C, the control plates with blank intermediate gels; E and D, intermediate gels containing antiserum to a-toxin. The antisera were used in the following concentrations: anti-apoprotein A, 10 pl/cm2; anti-apoprotein B, 6 pl/cm'; anti-a-toxin, 3 pl/cm'. First dimension electrophoresis (right to left) was at 10 V/cm for 65 min; second dimension immunoelectrophoresis was at 2 V/cm overnight. Note the co-precipitation of LDL, but not HDL with a-toxin, in the intermediate gels of E and D.

Fig. 2, C and D. Fig. 2C is the control plate with a blank intermediate gel. Incorporation of anti-a-toxin in the inter- mediate gel (plate D) led to precipitation of the toxin here, as in Fig. 2B. Concomitantly, however, the precipitate corre- sponding to LDL almost entirely disappeared from the upper gel. The conclusion that this reflected co-precipitation of LDL through binding to a-toxin was confirmed in the following experiments using purified LDL.

Sephacryl Gel Chromatography Distribution ofHemolytic Activity-In control experiments,

LDL and a-toxin were separately chromatographed over Sephacryl S-300. The LDL eluted immediately after the col- umn void volume (not shown), whereas native 3 S toxin eluted as a sharp, symmetrical peak with K., - 0.6 as previously reported (4). Hemolytic titrations of individual column frac- tions showed that hemolytic toxin activity faithfully followed the toxin protein peak (Fig. 3). In agreement with previous reports, the critical toxin concentration eliciting total hemol- ysis of an equal volume of 2.5% rabbit erythrocyte suspensions was in the order of 0.2 pg/ml.

In order to obtain an estimate of the maximal binding capacity for a-toxin by LDL, aliquots of 600 pg of toxin contained in 0.4 ml of buffer were incubated with 375, 1000, and 2000 pg of LDL (total lipoprotein). Each sample was chromatographed over Sephacryl. The results of hemolytic titrations are depicted in Fig. 3. As the amount of LDL offered to a-toxin increased, the total hemolytic activity of the native 3 S toxin peak decreased. When 1000 and 2000 pg of LDL were given to 600 pg of toxin, less than 10% of the original 3 S

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FIG. 3. Distribution of hemolytic activity of a-toxin upon Sephacryl gel chromatography in the absence and presence of LDL. A, hemolytic titers of fractions recovered after Sephacryl chro- matography of a-toxin alone (0) and the same amount of a-toxin (600 pg) after preincubation with increasing amounts of LDL (I, 375 pg; II , 1000 pg; and III, 2000 pg). Native a-toxin eluted as a sharp, symmet- rical peak. Preincubation with LDL led to progressive decreases in the hemolytic titers. Concomitantly, small peaks of hemolytic activity appeared just after the void volume, corresponding to LDL-bound toxin. B, a similar experiment was performed wherein a large amount of LDL (2000 pg) was incubated with less a-toxin (60 pg). Despite the marked increase in the LDL/toxin ratio, the overall hemolytic activity in the native a-toxin fractions (0) could not be depressed beyond a plateau inhibition of approximately 90%, as compared to the control column (A). Vo and V, indicate the column void and bed volumes, respectively.

hemolytic activity was recovered. An additional, small peak of hemolytic activity was discerned in early column fractions containing LDL/a-toxin complexes (discussed below).

To determine whether the entire disappearance of the 3 S toxin peak might be obtained by a large excess of LDL, 2000 pg of LDL were incubated with 60 pg of toxin (Fig. 3B). However, the overall degree of toxin inactivation was not significantly higher (approximately 90%). This appeared to be a plateau value of inactivation found in several other inde- pendent experiments as well.

In order to ascertain that the observed toxin inactivation would not only occur with isolated LDL, similar chromatog- raphy experiments were performed with whole human serum incubated with a-toxin. Essentially the same patterns of toxin inactivation were observed as found for isolated LDL. Exper- iments were also performed with purified HDL. In these cases, no binding and/or inactivation of a-toxin could be discerned (results not shown).

Protein Distribution-The distribution of protein in the Sephacryl chromatography fractions is shown in Fig. 4. The 3 S toxin control peak shown coincided exactly with the peak of hemolysis of Fig. 3. Incubation of a-toxin with 375 and 1000 pg of LDL caused reduction of the 3 S protein peak to comparable extents as the reduction of hemolytic activity. The protein peaks eluting just after the column void volume concomitantly increased. That these peaks represented LDL plus bound a-toxin was c o n f i e d by electroimmunoassays, SDS-PAGE, and electron microscopy.

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20

15

10

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FIG. 4. Protein content of fractions recovered from chro- matographies of Fig. 3. U, control a-toxin chromatography (600 pg of toxin applied, corresponding to the control curve of Fig. 3 A ) . Curues I and II (0 and A, respectively) correspond to curves I and II of Fig. 3A. Note the reduction of protein in the native 3 S toxin peak upon preincubation of the toxin with LDL and the concomitant increase in protein eluting after the void volume, corresponding to LDL/toxin complexes.

Immunochemical Analyses-Fig. 5 depicts a fused rocket immunoelectrophoresis of column fractions obtained from an experiment performed as described for Fig. 3 (2000 pg of LDL incubated with 600 pg of toxin). Anti-LDL was incorporated into the upper gels. In 5A, the intermediate gel was blank, and LDL was seen eluting immediately after the void volume. When anti-a-toxin was incorporated into the intermediate gel (Fig. 5B), precipitable toxin became detectable in the same fractions as LDL. As observed earlier (Fig. 2, C and D ) , precipitation of the toxin in the lower gel markedly diminished the area of the LDL precipitate in the upper gel. The eluting position of native 3 S toxin on the same column is indicated by the arrow. The antiserum did not precipitate the residual, unbound 3 S toxin (compare with Fig. 3A) which was, how- ever, detectable by hemolytic assay and also by SDS-PAGE.

Fig. 6 shows SDS-PAGE analyses. LDL (get a) appeared as a major polypeptide of apparent M, = 250,000, representing apoprotein B. Minor bands probably represented degradation products of the apoprotein. Gel b is the native toxin, which gave rise to a single major polypeptide band of M, = 34,000. Gel c is the pattern obtained through electrophoresis of an LDL/toxin column fraction (denoted x in Fig. 5) . Additional to the LDL and native a-toxin bands, there was a Mr = 200,000 band that corresponded to a-toxin hexamer. Upon boiling in SDS, this band virtually disappeared, whereas the M, = 34,000 a-toxin band became strengthened (gel d). The incomplete dissociation of toxin hexamers with SDS at room temperature

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FIG. 5. Fused rocket immunoelectrophoresis of fractions re- covered from ar~ experiment in which 600 pg of a-toxin were preincubated with 2000 pg of LDL and chromatographed over Sephacryl S-300.5~1 aliquots of each fraction were applied and the plates developed by incorporation of anti-apoprotein B (6 pl/cm') in the upper gels. A, blank intermediate gel (control); B, intermediate gel containing 2 pl/cm2 of anti-a-toxin antiserum. The column void (VO) and bed volumes (V,) and the eluting position of native a-toxin on the same column are depicted. Note the elution of precipitable a- toxin in the front column fractions and the co-precipitation of LDL with a-toxin in the intermediate gel of B.

apo-B-,

34 K+

b 4-200K

4-34 K

0

a b c d e f FIG. 6. SDS-PAGE of LDL (gel a), native a-toxin (gel b),

aliquot of fraction 14 (marked x in Fig. 5) from the Sephacryl chromatography experiment of Fig. 5 applied in SDS without heating (gel c), or after boiling for 30 s in SDS (ge ld) . Note the appearance of the M, = 200,000 ( 2 0 toxin hexamer band in gel c and the heat sensitivity of this band, which dissociates to yield the M, = 34,000 ( 3 4 K ) toxin band (gel d). Gel e shows the M, = 34,000 toxin band obtained by electrophoresing an unheated, rear fraction (frac- tion 22) from the column that exhibited hemolytic activity; this band represented non-LDL-bound boxin. Gel f is the SDS-PAGE pattern obtained after extensive trypsin-treatment of the same LDL/toxin sample as applied to gel c (unheated, sample volume doubled com- pared to gel c ) . note the persistence of the protease-resistant M, = 200,000 toxin hexamer band and the persistence of some M, = 34,000 material that probably stemmed from dissociation of a small fraction of the hexamers through the action of SDS. Minor bands in gel 6 represent contaminating degradation products of a-toxin.

and their augmented dissociation a t 100 "C have been de- scribed earlier (3, 4, 12).

SDS-PAGE of an unheated, rear peak fraction containing hemolytic activity gave rise to a weak M, = 34,000 band corresponding to native toxin (gel e). By densitometric ap- proximation, it was found that a maximum of 10-15% of residual native toxin eluted in this peak. This estimate was in

good agreement with that obtained from the hemolytic titra- tions and the protein determinations.

Molecular Nature of LDL-bound a-Toxin The appearance of the heat-sensitive M, = 200,000 band in

SDS-PAGE, as well as the presence of immunoprecipitable a-toxin in the LDL/a-toxin fractions by electroimmunoassay, indicated the presence of toxin hexamers in these fractions. This contention was directly confirmed by electron micros- copy. Negative stainings of LDL control preparations ex- hibited a uniform population of round, smoothly contoured particles, approximately 25 nm in diameter (Fig. 7A). Fig. 7, B and C, depicts the same preparation after incubation with a-toxin. Seen attached to the LDL particles are numerous ring and stub structures, indistinguishable from those ob- served on target erythrocyte membranes or liposomes carrying the 11 S toxin hexamer (Ref. 3). The particles observed in such preparations of toxin-treated LDL fell into four distinct categories. 1) One substantial fraction of the 25-nm LDL particles was devoid of recognizable toxin structures; 2) an- other large fraction of 25-nm particles carried one to four clearly identifiable ring or stub structures (Fig. 7, B and 0; 3) a small fraction (-1%) of the 25-nm particles carried markedly more (8-12) toxin structures (Fig. 7B); and 4) another small fraction (51%) of large 100-150-nm particles carried large numbers of toxin structures (Fig. 70). Particles of this size were not present in control LDL preparations, and they dif- fered further from the 25-nm LDL control particles by an apparently smaller mass density of protein/lipid material as indicated by their higher electron density relative to the 25- nm particles in negative staining.

All toxin-treated LDL preparations examined contained some free toxin hexamer structures. Because these were also observed in preparations that had been rechromatographed with no biochemically detectable loss of 11 S toxin, it is likely that detachment of some toxin from the LDL particles occurs as an artifact during the process of negative staining. Due to this, and because toxin intercalated between the grid surface and the LDL particles probably escapes detection, attempts at numerical correlation on the average number of toxin hexamers/LDL particle between biochemical and microscop- ical quantitations seemed fruitless.

Further support for the presence of toxin oligomers on the LDL particles derived from their trypsin insensitivity. Earlier studies have shown that whereas native 3 S toxin is destroyed by trypsin (13), the toxin hexamers are not (3, 4). The LDL/ a-toxin complexes were therefore trypsin-treated at an en- zyme/protein ratio of 1:20 (w/w) overnight at 37 "C. In the electron microscope, there was no alteration of the appearance of the LDL/toxin complexes (not shown). In SDS-PAGE, apoprotein B was extensively degraded, whereas the MI = 200,000 band and a fainter M, = 34,000 band persisted (Fig. 6, gel f ) . We have confirmed the observation (13) that high trypsin concentrations totally abolish the appearance of the MI = 34,000 polypeptide representing native toxin. The per- sistence of a M, = 34,000 band in the present experiments (Fig. 6, gel f ) therefore indicates its origin from the protease- resistant hexamers, a small fraction of which are dissociated to monomers by treatment with SDS at room temperature (see also Refs. 3 and 4).

Hemolytic Activity of LDL/a-Toxin Complexes LDL/a-toxin complexes recovered from the Sephacryl col-

umns exhibited low titered hemolytic activity (Figs. 3 and 4) . Assuming that the toxin disappearing from the original 3 S peak position on the Sephacryl column had all bound to LDL, we estimated that this hemolytic activity was in the order of

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FIG. 7. Ultrastructural appearance of native and a-toxin- treated LDL. A, control preparation of human LDL showing a uniform population of smooth and round, approximately 25-nm par- ticles; B and C, a-toxin-treated LDL. Typical ring and stub profiles of 11 S a-toxin hexamers are seen attached to the LDL particles (arrow- heads). "Free" 11 S a-toxin complexes are indicated by small ar~ows. A minor fraction of the 25-nm LDL particles carry markedly more toxin structures than others (asterisk). 0, a small fraction of large 100-150-nm particles, possibly of a composition different from the 25- nm particles, are heavily loaded with 11 S toxin structures (sodium silicotungstate negative staining). Scale bars indicate 50 nm.

2-5% of that displayed by equivalent concentrations of native toxin. Since preformed toxin hexamers (e.g. as recovered after their isolation from membranes or after their generation through contact with detergent; Refs. 3 and 4) are incapable of binding to cell membranes, we attributed the marked reduction in hemolytic activity of LDL-bound a-toxin to the formation of these oligomeric structures on the lipoprotein molecules.

Nevertheless, LDL/a-toxin complexes did display some re- sidual, albeit very weak, hemolytic activity (Fig. 3). In order to obtain an indication on the nature of this toxin fraction, LDL/a-toxin complexes were trypsin-treated and subse- quently tested. It was found that all residual hemolytic activity was destroyed by this treatment, indicating that the hemolytic activity had derived from small amounts of LDL-bound, but nonhexamerized toxin.

Stability of LDL/a-Toxin Complexes LDL/a-toxin preparations were trypsin-treated to destroy

residual, hemolytically active toxin. Thereafter, the LDL/a- toxin complexes were rechromatographed repeatedly over Sephacryl S-300, and fractions were analyzed by SDS-PAGE and tested for hemolytic activity. No dissociation of the 11 S toxin from LDL and no reappearance of any hemolytic activity whatsoever could be discerned. Thus, once transformed to the 11 S form, toxin remained tenaciously attached in a hemolyt- ically inactive state to the LDL molecules.

DISCUSSION

The results of the present investigation lead us to conclude that human plasma LDL binds and partially inactivates S. aureus a-toxin. Binding to LDL is highly selective. No similar interaction with any other nonlipoprotein plasma protein has been observed. With regard to other lipoproteins, we have also not been able to detect any interaction between the toxin and HDL. Preliminary studies do, however, indicate binding and inactivation by very low density lipoprotein.:' Binding by intermediate density lipoprotein and by chylomicrons has not yet been studied. The cause of the differential binding of a- toxin to LDL with respect to HDL is unknown. However, it is known that there is only a quantitative difference in lipid composition between LDL and HDL, and that LDL molecules (20-nm diameter) are much larger than HDL (7-10-nm di- ameter) molecules. Since there is more free cholesterol and less protein in LDL than in HDL, the surface area of exposed lipid is probably much higher in the former. Thus, it is possible that the binding and/or oligomerization of a-toxin requires a critical exposed lipid surface area which HDL fails to provide.

When a-toxin is incubated with LDL, three toxin popula- tions are distinguishable. First, there is residual, native 3 S toxin, which is hemolytic and can be separated from LDL by gel chromatography. Why quantitative binding of a-toxin to LDL cannot be achieved through increasing the LDL/toxin ratio is unclear. In experiments not depicted here, the free toxin recovered by gel chromatography was reincubated with a fresh sample of LDL, and binding with inactivation was again observed.

The second toxin population is represented by LDL-bound toxin in its hexamerized, nonlytic form. These toxin molecules can be directly visualized as LDL-bound structures in the electron microscope. It appears probable that, analogous to the mode of toxin binding to erythrocytes (3), the 11 S rings are primarily attached to the lipid moiety of LDL. In this connection, it is of interest to note that membranes and

Recent experiments indicate that a-toxin also binds to very low density lipoprotein (unpublished observations).

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liposomes with which a-toxin has previously been shown to interact contain lipid in bilayer form, whereas LDL-exposed lipid may be present as a monolayer. The binding of a-toxin to lipid monolayers has not been studied to date, and it will be of interest to conduct such experiments in the future.

A third, minor fraction of a-toxin co-elutes with LDL upon Sephacryl chromatography and is hemolytic. The trypsin sensitivity of this toxin population indicates its presence in native 3 S form. Additional experiments (not depicted) have, indeed, shown that when high molecular weight fractions of LDL/a-toxin complexes recovered from Sephacryl columns are rechromatographed, a small amount of hemolytic toxin appears eluting as 3 S monomer. By contrast, the 11 S toxin rings appear not to dissociate from LDL. The factors govern- ing formation of the various toxin subpopulations have not been delineated. The hexamers are thought to form simply upon contact with lipid contained in the lipoprotein. Why a population of toxin molecules should bind to LDL without being converted to the hexamer form is unknown.

Estimates of molar binding ratios of a-toxin to LDL under conditions of toxin excess indicate that 1 g of LDL can bind approximately 0.6-0.8 g of toxin. Assuming a molecular weight of LDL of 2,500,000 and a toxin monomer molecular weight of 34,000, this corresponds to a mean binding ratio of 40-50 toxin molecules or seven to eight hexamers/LDL molecule. Electron microscopy shows that large variations in the binding ratio actually exist, such that some LDL particles carry large num- bers of toxin rings, whereas others carry few or none. It is presently unclear which factors are responsible for this appar- ently differential binding. Possibly, they relate to variations in the lipid composition of the individual LDL molecules. The numerically small fraction of large, 100-150-nm particles found only in toxin-treated LDL preparations and carrying large numbers of toxin hexamers may result from confluesc- ence of several LDL particles. Alternatively, they may repre- sent particles of an altogether different nature, e.g. lipid vesicles formed through toxin-mediated detachment of lipid from LDL molecules.

Notwithstanding these uncertainties, it is apparent that plasma LDL is an effective, hitherto unrecognized nonimmune inactivator of a-toxin. Under conditions of LDL excess, such as is probably the case in the human circulation, overall inactivation approaches 90% as estimated by hemolytic titra- tions. Since binding and similar inactivation were found upon the use of whole human serum, we infer that this defense mechanism may be of physiological importance, e.g. in cases of septicemia or generalized S. aureus infections. A small amount of cytolytic toxin will, however, be transported by LDL within the organism. If active toxin thus finds a way to reach target tissues remote from the site of actual toxin

production in sufficiently high concentration, the ensuing damage would partially counteract the beneficial effects of LDL.

In view of the rapidly increasing knowledge on the physi- ology and metabolism of LDL (14), it would seem of interest to determine the influence of bound toxin on the fate of the lipoprotein particles. It will also be of interest to determine whether a-toxin might be taken up into cells together with LDL to exert some as yet unrecognized intracellular effects. The interaction of a-toxin with LDL appears not to be an exclusive property of this toxin. LDL has been reported to bind and partially inactivate staphylococcal 8-toxin (15), strep- tolysin 0 (16), and streptolysin S (17), although these inter- actions have not been characterized at a molecular level. Plasma LDL may emerge as a biologically significant factor responsible for nonimmune inactivation of these and of several other lipid-binding toxins in the host organism.

Acknowledgment-We thank Marion Muhly for expert technical assistance.

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