Hydrophobic Interaction Chromatography of Staphylococcus aureus Delta-Toxin

Transcription

1 INFECTION AND IMMUNITY, Mar. 1981, p /81/ $02.00/0 Vol. 31, No.3 Hydrophobic Interaction Chromatography of Staphylococcus aureus Delta-Toxin FREDERICK S. NOLTEt AND FRANK A. KAPRAL* Department ofmedical Microbiology and Immunology, The Ohio State University, Columbus, Ohio Staphylococcus aureus delta-toxin bound avidly to agarose gels containing phenyl, octyl, or decyl ligands, but less so to agarose with hexyl groups. Agarose with ethyl or butyl moieties did not bind any more toxin than did agarose without attached ligands. About 10% of the applied toxin preparation did not bind to gels and eluted with the starting buffer. The nonadsorbed material was not hemolytic, did not react with anti-delta-toxin immunoglobulin G, and did not appear to be a peptide. Toxin bound to phenyl-sepharose was not eluted with water, solutions containing chaotropic ions or ethylene glycol, or by increasing the ph, but was eluted with 50% ethanol. The ethanol-eluted delta-toxin (EEDT) was soluble in water, ethanol, 10% sucrose, or 6 M urea, but was poorly soluble in aqueous salt solutions at neutral ph. Regardless of whether the soluble or insoluble form of delta-toxin was applied to the gel, the resultant EEDT fraction was water soluble. The hemolytic activity of EEDT was markedly reduced when assayed in saline, but was the same as that of the original toxin preparation when assayed in isotonic sucrose. A significant portion of EEDT, when rechromatographed on phenyl-sepharose, did not bind to the gel. This unbound fraction may represent toxin aggregates in which the hydrophobic regions of the toxin monomers are interiorized within the aggregates. Several investigators have shown that Staphylococcus aureus delta-toxin contains an unusually high percentage of amino acids with nonpolar side chains, especially leucine, isoleucine, and valine (5, 8, 13). This polypeptide is also unusual in that it is soluble in aqueous solutions, 80% ethanol, or chloroform-methanol (2:1) and has the ability to form high-pressure films at airwater interfaces (3). In the presence of phospholipids or fatty acids, the toxin's hemolytic activity is either inhibited or augmented, depending on the lipid involved (9, 10). Purified delta-toxin exists in two forms; one is water soluble, and the other is relatively water insoluble. The specific activity of the latter is generally twice that of the former. Hydrophobic interaction chromatography is a technique that exploits the interaction between nonpolar groups chemically linked to an uncharged gel matrix and nonpolar regions of a solute (6, 7). Because of its amino acid composition, the hydrophobic interaction of purified delta-toxin with substituted agarose gels was investigated. MATERIALS AND METHODS Hemolysins. Delta-toxin was prepared and purified as previously described (11) and exhibited a single t Present address: Microbiology Laboratories, School of Medicine and Dentistry, University of Rochester, Rochester, NY band when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (14). In this study, both the water-soluble and -insoluble forms of delta-toxin were used. Tyrocidine was obtained from the Nutritional Biochemical Corp. and was prepared as a stock solution in 95% ethanol. Gels. Phenyl-Sepharose CIA-B, octyl-sepharose CL-4B, and Sepharose 2B were obtained from Pharmacia Fine Chemicals. Ethyl-, butyl-, hexyl-, and decyl-agarose gels were purchased from Miles Laboratories. Gels were washed with distilled water and absolute ethanol before use. Analytical chromatography. Columns containing 1 ml of either unsubstituted Sepharose, ethylagarose, butyl-agarose, hexyl-agarose, phenyl-sepharose, octyl-sepharose, or decyl-agarose were equilibrated with 0.15 M NaCl. Soluble delta-toxin, dissolved in saline at a concentration of 1 mg/ml (absorbancy at 280 nm = units), was applied to the gel until the absorbancy of the effluent at 280 nm was equal to that of the applied solution, or until a total of 20 mg of toxin had been loaded on the gel. Successive 0.5-ml fractions of the eluate were collected, and the absorbancy at 280 nm of each fraction was determined. Preparative chromatography on phenyl- and octyl-sepharose. Chromatography was accomplished using a Pharmacia solvent-resistant column (10 mm ID) with upward flow adaptors. The effluent was monitored continuously for absorbance at 280 nm. Delta-toxin was loaded into the gel in starting buffer, and any unbound material washed through with this same buffer. Bound toxin was eluted by either a continuous or discontinuous ethanol gradient. Fractions representing elution peaks were pooled, dialyzed 094

2 VOL. 31, % of the sample did not adsorb, but was pres- ent in the first bed volume through the column. No other material was recovered until the adsorbed toxin was eluted with 40 to 50% ethanol.,r from the Increasing the ethanol concentration to 100% or RESULTS Analytical chromatography. Six different hydrophobic ligands were exi igels with their ability to bind purified delta-toxininied n a low- for ionic-strength environment. It was fsound ethyl- and butyl-agarose did not diffe: that Sepharose control in their ability to Ibind delta- toxin (Fig. 1). Phenyl-Sepharose, oc rose, and decyl-agarose, however, we're able to O. 0 f40.5 { DELTA-TOXIN HYDROPHOBIC INTERACTIONS 1095 against distilled water, and lyophilized. An3y remaining bind large amounts of toxin. Hexyl-agarose was bound material was eluted from the gel Mvith several intermediate in its affinity for delta-toxin. With bed volumes of n-butanol. the phenyl-, octyl-, and decyl-substituted gels, Assay of hemolytic activity. WashE cytes from freshly drawn human blood wereae erythro- even 20 mg of delta-toxin was not sufficient to in the appropriate diluent to give a final co] ncuesnptreationd saturate the 1-ml amounts of gel u.ed. Although of 1% (vol/vol). Serial twofold dilutions of the sample these gels bound significant amounts of toxin, it (delta-toxin or tyrocidine) were preparedi in a final was also apparent that the toxin preparation volume of 0.6 ml, and 0.4 ml of the 1% Eerythrocyte contained matprial that had little or no affinity suspension was added. The mixtures weree incubated for the hydrophobic ligands in 0.15 M NaCl. at 370C for 20 min and centrifuged, and thie hemoglo- Preparative chromatography on phenyl- spec- and octyl-sepharose. The above results indi- bin content of the supernatants was deterimined trophotometrically at 430 rm. One 50% hennolytic dose cated that either phenyl-, octyl, or decyl-seph- releas- arose could be used to bind delta-toxin. In initial (HD!5,1) was defined as that amount of material ing 50% of the hemoglobin from the test st In experiments where the effect of varicausesalitson experiments using octyl-sepharose, delta-toxin the specific activity of delta-toxin was irduestigated was applied to the gel in 2 M NaCl. Only small either the salts were added to toxin dillutions just amounts of the applied toxin preparation did not before addition of the erythrocyte suslpension, or adsorb or were subsequently eluted by decreasthe diluent ing the ionic strength, lowering the polarity by erythrocytes were allowed to equilibrate in with salts for specified periods before beirng added to inclusion of ethylene glycol in the eluant, increasing the ph, or by including chaotropic ions toxin dilutions. Immunodiffusion. Ouchterlony tests vvere carried (Ba2+) in the eluant. Most of the toxin was out on microscope slides coated with 0. 5% Ionagar desorbed by using increasing ethanol concentra- (Oxoid) in phosphate-buffered saline (pf1 7.2). The results were read after 18 h of incubation Xat 230C. tions When in water. delta-toxin was applied to phenyl-sepharose columns in a low-ionic-strength solution or in distilled water, regardless of the ph, about washing the column with n-butanol did not retyl-sepha- lease any more 280-nm-absorbing material. The unbound material recovered from fractions passing through the column with starting buffer was not hemolytic. Although the material did absorb at 280 nm, there was no absorption maximum in this region, and the absorbance spectrum in the ultraviolet range (240 to 320 nm) was totally different from that of delta-toxin (13). Furthermore, the material did not react with anti-delta-toxin immunoglobulin G in agar gels (Fig. 2). No other attempts were made to characterize this contaminant. On phenyl-sepharose, the water-soluble and -insoluble forms of delta-toxin exhibited similar elution profiles and eluted in unison when chro- FYL A matographed together (Fig. 3). In these experiments, both forms were applied in 0.05 N NH40H to bring the insoluble form into solution. -,----. The columns were eluted with distilled water , 7 DELTA TOXIN(mg) * *'10 followed by a continuous ethanol gradient. In each case, most of the toxin eluted as a single FIG. 1. Analytical hydrophobic intera matography. Delta-toxin (1 mg/ml) was c otiinoursoy peak at a concentration of approximately 50% applied to a 1.0-ml column of either etlhyl-, butyl-, ethanol. hexyl, phenyl-, octyl-, or decyl-substitut'ed agarose The 50% ethanol-eluted delta toxin (EEDT) gels, and 0.5-ml fractions of the effluent were col- had the same characteristic ultraviolet absorb- as a con- ance spectrum as unfractionated toxin (13). lected. Unsubstituted agarose was includeed trol. These preparations were soluble in distilled wa-

3 1096 NOLTE AND KAPRAL FIG. 2. Immunodiffusion analysis ofphenyl-sepharose fractions. Delta-toxin was chromatographed and separated into unadsorbed and EEDT fractions. WellA, Unadsorbed material; well B, unfractionated delta-toxin; well C, EEDT; wells D and E, immunoglobulin isolated from rabbit antiserum directed against the unfractionated toxin. FIG. 3. Phenyl-Sepharose chromatography of delta-toxin. Either 25 mtg of soluble delta-toxin, 25 mg of insoluble delta-toxin, or a mixture containing 12.5 mg of each form was applied to 25 ml of gel in 0.05 N NH40H. The absorbancy at 280 nm of the effluent was monitored continuously. Elution was with water followed by a water-ethanol gradient. ter, ethanol, 10% sucrose, and 6 M urea, but were poorly soluble in salt solutions at a neutral ph. Although the EEDT obtained upon fractionation of the water-insoluble forn of the toxin was freely soluble in water, its specific activity was still twice that of the EEDT obtained by fractionation of the soluble forn. However, regardless of the fonn of delta-toxin applied to the gel, the EEDT consistently had a specific activity one-third to one-fourth that of the starting material. Attempts to restore the initial hemolytic activity by recombining all eluted fractions proved unsuccessful. When EEDT was rechromatographed on phenyl-sepharose, it again eluted as two distinct peaks (Fig. 4). The first peak eluted with water and represented unadsorbed material; the second peak eluted with 50% ethanol. The ultraviolet absorbance spectra of both peaks were identical and characteristic of delta-toxin. Whereas the specific activity of the applied material was 132 HD,,O/mg, that of the first peak (A) was <5 HDso/mg and that of the second peak (B) was 32 HDs,o/mg when assayed with erythrocytes suspended in saline. Assay of hemolytic activity. Because of the poor solubility of EEDT in salt solutions, the hemolytic activity of such preparations was also determined by assaying the toxin in isotonic sucrose. Both EEDT and unfractionated deltatoxin displayed increased hemolytic activity when assayed in isotonic sucrose as compared to assays done in saline (Table 1). The degree to which hemolytic activity increased depended upon the length of time erythrocytes had been stored in the sucrose diluent before use in the IL INFECT. IMMUN. jr\~~~~~~ AI-.O%e FRACTM NO. r-hz0 q 5H -qp IWO%KTOHI1 j,o^ FIG. 4. Elution profile of EEDT rechromatographed on phenyl-sepharose. EEDT (50 mg) was applied to the gel in water and separated into unadsorbed (A) and adsorbed (B) peaks. The absorbancy at 280 nm of the effluent was monitored continuously. TABLE 1. Specific activity of delta-toxin preparations and tyrocidine when assayed in saline or isotonic sucrose Sp act' Diluent storage Tyrocistorage" DTEEDT dine 0.9% NaCl % sucrose ,640 2, "Days erythrocytes were stored in diluent. ' Average of duplicate assays (HDr,o per milligram). DT, Delta-toxin.

4 VOL. 31, 1981 assay. An increase in susceptibility was noted when cells were stored in the diluent for as little as 2 h and was maximal after 3 days. Cells held in isotonic sucrose for longer periods (up to 7 days) did not display any further change in susceptibility to the toxin. Erythrocytes held in isotonic sucrose did not exhibit an increase in spontaneous lysis and did not change significantly in their susceptibility to the cyclic peptide tyrocidine. This increased susceptibility to delta-toxin appeared to result from a lack of ions in the suspending medium since inclusion of either NaCl, KCI, or NaNO3 in the diluent abrogated any change in erythrocyte susceptibility (Table 2). However, this occurred only when erythrocytes were permitted to equilibrate with the sucrosesalt diluent. Salts in the same final concentration, added to the diluent at the time of the assay, did not alter erythrocyte susceptibility. Immunodiffusion studies. Delta-toxin chromatographed on phenyl-sepharose was separated into the unadsorbed and EEDT fractions. The unbound material, when reacted with rabbit anti-delta-toxin immunoglobulin G in agar gels, did not fonn a line of precipitation. EEDT did precipitate with antibody and gave a line of identity with the unfractionated delta-toxin (Fig. 2). DISCUSSION This study demonstrated that delta-toxin was bound to hydrophobic ligands and that phenyl, octyl, and decyl groups were equally effective in this binding. The fact that toxin binds to these ligands in low-ionic-strength buffers probably is the result of the high concentration of hydrophobic amino acids in the molecule. Inability to elute toxin by lowering polarity of the eluant, decreasing the ionic strength, increasing the ph, TABLE 2. Effect of salts on the specific activity of delta-toxin Sp act' of cells stored in: Diluent Days of storage " Diluent 9.25% with Susalt crose' 9.25% sucrose , % sucrose + 0.5% NaCI , % sucrose % KCI , % sucrose % NaNO:, ,520 " Days erythrocytes were stored in diluent. b Average of duplicate assays (HDro per milligram). 'Salts were added at the time of assay. DELTA-TOXIN HYDROPHOBIC INTERACTIONS 1097 or using chaotropic ions suggests that this binding is of high avidity. The avidity of this interaction might result from simultaneous interactions between the same toxin molecule and several ligand groups, but present knowledge does not reveal whether the toxin monomer has single or multiple hydrophobic domains. An alternate explanation for the strong binding is that deltatoxin aggregates interact with many ligand groups. There is ample evidence that delta-toxin in aqueous solutions exists in a polymeric or aggregated form (8, 13). Preparative chromatography of purified deltatoxin on phenyl-sepharose revealed the presence of a contaminant. This material was not bound by the gel and was not hemolytic. Its ultraviolet absorbance spectrum was distinct from that of delta-toxin, and the lack of an adsorption peak at about 280 nm suggested the material was not a peptide. This material did not appear to be immunogenic, since antisera raised against the unfractionated toxin did not precipitate the contaminant in agar gels. Recombining the contaminant with EEDT failed to restore the original hemolytic activity of fractionated toxin when assayed in saline. Bernheimer (1) has suggested that the aggregation of delta-toxin in aqueous solution involves its association with a carrier molecule. If this hypothesis is true, the unbound contaminant could function in this capacity. A significant portion of EEDT fails to bind to phenyl-sepharose when it is rechromatographed. Although it is possible that initial chromatography might result in denaturation of the toxin, this seems unlikely. An altemate explanation is that small polar compounds nonnally associate with the toxin molecule, partially covering its hydrophobic domain(s). During binding of toxin to phenyl-sepharose, the compounds are displaced, elute from the column, and are recognized as the contaminant. The resultant EEDT, in the absence of ethanol, then forms aggregates with the hydrophobic domains interiorized. These aggregates, now exposing a hydrophilic surface, are subsequently unable to bind to hydrophobic ligands on the gel. Such a process could also account for the apparent loss in hemolytic activity of EEDT. This hypothesis is supported by recent data obtained by studying spectral changes in certain dyes when bound to delta-toxin, and which suggest that the toxin, above a critical concentration, forms micellar structures in aqueous solutions (unpublished data). Delta-toxin has been shown to be active against a wide variety of membranes, including bacterial protoplasts and spheroplasts, mitochondria, lysosomes, lipid spherules, and eryth-

5 1098 NOLTE AND KAPRAL rocytes from all species tested (1, 12, 13). This wide spectrum of activity suggests that the toxin interacts with some material common to most membranes. A toxin with the ability to interact with hydrophobic ligands would not require a specific membrane receptor, but only the hydrophobic regions of the lipid bilayer. In this regard, Kapral has provided evidence that delta-toxin interacts with phospholipids and fatty acids (9, 10). The interaction between toxin and these lipids presumably occurs through hydrophobic bonding. Delta-toxin has also been characterized by its rather low specific activity (1 HD.-w is equivalent to 10,ug of toxin). Recently, Chao and Birkbeck (2) proposed the use of cod erythrocytes to provide a more sensitive hemolytic assay for the toxin and reported a specific activity of 1,200 HD,,o/mg in their system. The authors reported that this system suffers from the fact that fish erythrocytes are not readily available and that they deteriorate rapidly at 370C. We found that by suspending human erythrocytes in isotonic sucrose instead of saline, a specific activity of 2,800 HDrO/mg was obtained. The release of substantial amounts of protein from erythrocyte membranes by treatment with a low-ionicstrength medium has been reported (4). It is possible that loosely bound membrane proteins are eluted from erythrocytes when suspended in isotonic sucrose and that these proteins when removed from the membrane somehow render it more susceptible to the action of delta-toxin. If such proteins possessed exposed hydrophobic moieties, they might interfere with the insertion of toxin into the membrane. The enhancement of delta-toxin hemolytic activity by certain longchain fatty acids (10) might involve shielding of such sites on the erythrocyte surface. ACKNOWLEDGMENT This investigation was supported by Public Health Service grant AI-7826 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED INFECT. IMMUN. 1. Bernheimer, A. W Interactions between membranes and cytolytic bacterial toxins. Biochim. Biophys. Acta 344: Chao, L. P., and T. H. Birkbeck Assay of staphylococcal 8-haemolysin with fish erythrocytes. J. Med. Microbiol. 11: Colacicco, G., M. K. Basu, A. R. Buckelew, and A. W. Bernheimer Surface properties of membrane systems: transport of staphylococcal delta toxin from aqueous to membrane phase. Biochim. Biophys. Acta 465: Fairbanks, G., T. L Steck, and D. F. H. Wallach Electrophoretic analysis of the major polypeptide of the human erythrocyte membrane. Biochemistry 10: Heatley, N. G A new method for the preparation and some properties of staphylococcal delta haemolysin. J. Gen. Microbiol. 69: Hjerten, S Some general aspects of hydrophobic interaction chromatography. J. Chromatogr. 87: Hjerten, S Hydrophobic interaction chromatography of proteins on neutral adsorbents, p In N. Catsimpoolas (ed.), Methods of protein separation, vol. 2. Plenum Publishing Corp., New York. 8. Kantor, H. S., B. Temples, and W. V. Shaw Staphylococcal delta hemolysin: purification and characterization. Arch. Biochem. Biophys. 151: Kapral, F. A Inhibition of Staphylococcus aureus delta hemolysin by phospholipids. Proc. Soc. Exp. Biol. Med. 141: Kapral, F. A Effect of fatty acids on Staphylococcus aureus delta-toxin hemolytic activity. Infect. Immun. 13: Kapral, F. A., and M. M. Miller Product of Staphylococcus aureus responsible for the scalded-skin syndrome. Infect. Immun. 4: Kreger, A. S., and A. W. Bernheimer Disruption of bacterial protoplasts and spheroplasts by staphylococcal delta hemolysin. Infect. Immun. 3: Kreger, A. S., K.-S. Kim, F. Zaboretsky, and A. W. Bernheimer Purification and properties of staphylococal delta hemolysin. Infect. Immun. 3: Nolte, F. S., and F. A. Kapral EBinding of radiolabeled Staphylococcus aureus delta toxin to human erythrocytes. Infect. Immun. 31: