1 mM dithiothreitol. (DTT)' (Serva, Heidelberg, Germany) was added and supernatants harvested by centrifugation at room temperature (Sorvall GS3 rotor,.
Vol. 268, No. 16, Issue of June 5, pp. 11959-11962,1993 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
Staphylococcus aureus&-Toxin PRODUCTION OF FUNCTIONALLY INTACT,SITE-SPECIFICALLY MODIFIABLE PROTEIN BY INTRODUCTION OF CYSTEINE AT POSITIONS 69, 130, AND186* (Received for publication, September 18, 1992, and in revised form, December 29, 1992)
Michael Palmer$, Renate Jursch, Ulrich Weller, Angela Valeva, Karin Hilgert, Michael KehoeQ, and Sucharit Bhakdi From the Institute of Medical Microbiology, University of Mainz, Augustwplutz, W-6500Germany and the §Department of Microbiology, MedicalSchool, University of Newcastle upon Tyne, Newcastleupon Tyne NE2 4HH, United Kingdom
Staphylococcal u-toxin, the prototype of an oligo- groups into the molecule, which in its native form contains merizing, pore-forming cytotoxin, is sensitive to bio- no cysteine (7). We show here that introduction of cysteine chemical modifications and cannot be labeled with bio- at residue 130 generates toxin molecules that can be labeled tin or fluorescein under preservation of its biological with biotin or fluorescein at the sulfhydryl group with full activity. In this study, we have used site-directed mu- preservation of hemolytic activity. Labeling cysteines at two tagenesis to introduce cysteine residues at positions other positions, 69 and 186, reduces activity but still allows 69, 130, and 186. Each mutant was fully and rapidly binding to target cells. Use of these labeled probes renders reactive with several sulfhydryl-specificreagents, in- detection of the toxin possible after cell binding without the dicating superficial location. Coupling of SH-groups use of antibodies. Furthermore, probing the reactivity of the with fluorescein-maleimide or biotin-maleimide was sulfhydryl groups in monomeric, free, and membrane-bound tolerated withoutloss of hemolyticactivity at position toxin renders identification of superficially located residues 130, and the formed hexamerswere visible on target possible. cells by fluorescence microscopy and could be detected on electroblots by reaction with streptavidin-peroxiMATERIALS AND METHODS dase. At the two other positions, modification caused Construction of S. aureus Clones Secreting Cysteine-containing significant loss of activity. However, the labeled proteins still bound to red cells, as shown by fluorescence Toxins-The cloned gene for a-toxin (8)was inserted into M13mp18 and mutagenized according to Eckstein (9); the appropriate reagents microscopy and electroblotting. Intrinsicallylabeled a- were from Amersham (Braunschweig, Germany). Mutant toxin represents a novel tool to study the interaction phagepurchased clones were selected by dideoxysequencing with Sequenase 2.0 of this pore-former with target membranes. (U. S. Biochemical Corp., Bad Homburg v.d.H., Germany). A 660-base pair KpnI fragment from the a-toxin gene of plasmid pDU1212 (IO) was replaced by the corresponding mutant fragments (isolated from replicative form DNA of phages). The resulting plasmids (pAC69,pAC130, and pAC186)were checked by restriction a-Toxin is an exoprotein that is regarded as a major path- analysis in comparison with plasmid pDU1212. ogenicity factor of Staphylococcus aureus (1-3). The toxin is Plasmids were transfected into (a-toxin-defective) S. aureus secreted as a hydrophilic monomer of M , 34,000 that binds DU1090, (restriction-deficient) S. aureus RN4220 serving as interirreversibly to specific acceptor sites present on rabbit eryth- mediate host (10). Integrity of plasmids was verified by restriction rocytes (4, 5) and on human platelets, monocytes, lympho- analysis and detection of a 33-kDa protein in culture supernatants cytes and endothelial cells (1).Six molecules then aggregate by SDS-PAGE. Mutagenesis and sequencing were carried out following the instructo form a transmembrane pore that has been sized to 1-2 nm tions of the respective suppliers. Transfection of S. aurew was done diameter and causes osmotic lysis of erythrocytes (1). as described by Fairweather et al. (10). Transfected clones were For further studies on the interaction with target cells, we selected by means of resistance against chloramphenicol (12.5 mg/ wished to label the molecule with fluorescent or otherwise liter) conferred by pDU1212 and derivatives. Plasmid DNA from S. detectable moieties. Chemical modification of pore-formers aurew was purified by protoplasting cells as for transfection (10) and under preservation of activity is generally a difficult task. processing further as inalkaline lysis preparation of Escherichia coli Further purification was done by extraction with cetyltriThis is not surprising, since the molecules harbor essential plasmids. methylammonium bromide as described in Ref. 11. domains for cell binding, membrane insertion, and pore forCloning procedures were essentially as described (11). mation. Oligomerizing toxins also contain surfaces that are Purification of Mutant Proteins-Staphylococci weregrown in involved in protomer-protomerinteraction. None of these Tryptone soybean broth (Difco) at 37 “C for 16 h. 1mM dithiothreitol functionally importantdomains have been unequivocally (DTT)’ (Serva, Heidelberg, Germany) was added and supernatants harvested by centrifugation at room temperature (Sorvall GS3 rotor, identified in any pore-forming toxin to date (1, 6). 20 min, 8000 rpm). During all subsequent steps, the preparation was After our attempts to produce active toxin labeled with kept a t 4“C.Concentration and buffer exchange into 200ml of amino-reactive biotin or fluorescein derivatives uniformly ammonium acetate (20 mM, pH 6.2; Merck, Darmstadt, Germany) failed, we sought to introduce selectively reactive sulfhydryl with 5 mM DTT were done using a hemofilter (Sartorius, Gottingen, Germany, model SM 40042). After a clearing spin (Sorvall GSA rotor, ’This study was supported by Deutsche Forschungsgemeinschaft 20 min, 12,000 rpm) and filtration (Sterivex 0.22 pm; Millipore, Grant SFB 311 and by the Verband der Chemischen Industrie. The Eschborn, Germany) the solution was applied to an S-Sepharose HR costs of publication of this article were defrayed in part by the 16/10 column using a P2 peristaltic pump (both from Pharmacia, payment of page charges. This articlemusttherefore be hereby marked ‘‘aduertisernnt’’ in accordance with 18 U.S.C. Section 1734 The abbreviations used are: DTT, dithiothreitol; PAGE, polysolely to indicate this fact. acrylamide gel electrophoresis; PBS, phosphate-buffered saline; $ To whom correspondence should be addressed. DTNB, 5,5’-dithiobis(nitrobenzoicacid).
11959
11960
Staphylococcal a-Toxin withModifiable Cysteine
FIG. 1. Purification of a-toxin Cys-130. The supernatant of 3 1 bacterial culture was concentrated and transferred into 200 ml of buffer A (20 mM ammonium acetate, pH 6.2) using a hemofilter. The concentratewas applied to a cation exchange column (S-Sepharose HR 16/10, Pharmacia). Thecolumn was washed with buffer A until the absorption reached the base line, and then the linearconcentration gradient was started. Shown are theabsorption a t 280 nm and the ammonium acetate concentration gradient (from 0.02 to 0.5 M ) as functions of elution volume. The toxin eluted as a single peak at about 0.1 M ammonium acetate. Inset,peak fractions analyzed by SDS-PAGE. The faint bands of high molecular weight represent spontaneously formed hexamers toxin as the only detectable contamination.
0
Freiburg, Germany), equilibrated with ammonium acetate (20 mM, pH 6.2), washed with three column volumes of the latter buffer, and eluted (2 ml/min) with a linear gradient to 0.5 M ammonium acetate (pH 6.2) containing 5 mM DTT (total gradient volume 170 ml). Elution was monitored by measurement of absorbance at 280 nm, and peak fractions were checked by hemolytic titration with rabbit erythrocytes(12) and by SDS-PAGE.Appropriatefractions were pooled, aliquoted, and stored frozen a t -70 "C. Protein concentrations were estimated by measuring the extinction a t 280 nm,using an extinction coefficient of 1.8 for a solution of 1 mg/ml (determined by weighing a batch of lyophilized, purified toxin). Modification of Sulfhydryl Groups-For titration and modification of sulfhydryl groups, proteins were transferred into deaerated phosphate-buffered saline containing 1 mM EDTA, pH 7.4 (PBS/EDTA) using a Sephadex 25 PD-10 column (Pharmacia). N-Methylmaleimide (Sigma, Munchen, Germany) and analogues (fluorescein-maleimide, Pierce; biotin-maleimide, Sigma) were dissolved in dimethylsulfoxide (Merck) anddiluted to 10 mM in PBS/EDTA. Toxin solutions at concentrations of approximately 2 mg/ml were incubated with a 5-fold molar excess of maleimide reagent for 15 min a t room temperature and thereactions stopped by adding DTT toa final concentration of 2 mM. Unbound labels were then removed and the modified proteins transferred into PBS using a PD-10 column. Modification with fluorescein-maleimide was controlled by measuring the extinction a t 490 nm using an extinction coefficient of 78,000 liters/(mol. cm) as specified in the product information supplied by Pierce. 5,5'-Dithiobis(nitrobenzoicacid) (DTNB; Sigma) was dissolved at 10 mM in sodium phosphate buffer, pH 7.0, and added to a final concentration of0.5mM. Incubation of proteins was in 0.1 M Tris, 0.01 M EDTA, pH 8.0, without (for native conditions) or with (for denaturing conditions) 6 M guanidinium chloride (Merck). Reaction yields were quantitated by measuring the extinction at 412 nm. Extinction coefficients were assessed using DTT. For assaying the activity of p-hydroxymercuriphenylsulfonate toward cysteine mutants, thecompound was dissolved a t 1mM in PBS/ EDTA and this buffer used for hemolytic titrations. Wild type toxin was used with the same buffer for comparison. Labeling of Cells-When cells were labeled for fluorescence microscopy, PBS supplemented with 30 mM dextran 4 (Pharmacia) was used throughout for incubations and washing steps to prevent osmotic lysis of cells (12).In the depicted experiments, high concentrations of toxins (0.2 mg/ml) were used in order to obtain easily documentable pictures. Incubations with toxins and with streptavidin-fluorescein isothiocyanate (Sigma, 0.01 mg/ml) were for 30 min at 22 "C. For direct labeling, the cells were incubated with fluoresceinated toxins, washed twice, and subjected to fluorescence microscopy. Wild typetoxin exposed to fluorescein-maleimide in exactly thesame manner as the mutants served as negative control. Indirect labeling was undertaken with toxins biotinylated a t their sulfhydryl groups. In these experiments, two negative controls were included. First, wild type toxin was reacted with biotin-maleimide under identical conditions. Second, the cysteine mutants were first reacted with DTNB toblock sulfhydryl groups and post-reacted with biotin maleimide. Erythrocytes were incubated with the respective
60
120
180
2io
300
Elution Volume (ml)
toxin preparation. Cells were subsequently washed twice and postincubatedwith streptavidin-fluorescein isothiocyanate. Cells were finally washed twice and inspected by fluorescence microscopy. Processing of red cell membranes, SDS-PAGE, and electroblotting followed published procedures (12). Blots were developed with streptavidin-peroxidase (Amersham) diluted 1:lOOO in PBS. RESULTSANDDISCUSSION
All proteins were easily purified using the same conditions as developed for wild type toxin,except that DTTwas present throughout the preparation in order to protect sulfhydryl groups (Fig. 1).Specific hemolytic activity of all three mutant toxins was identical to thatof wild type toxin (50,000 hemolytic units/ml). Cysteines inMutantProteinsAre Accessibleto SH-reagents-Sulfhydryl groups were titrated with DTNB under native and denaturing conditions.each In case, reactions were complete within seconds, yielding the expected number of one SH-group permolecule. Treatment with 2 mM N-methylmaleimide for 15 min, followed by addition of a molar excess of DTT and buffer exchange on a PD-10 column, removed all DTNB-titratable SH-groups. Thus, in all instances the SHgroups were accessible to DTNB and N-methylmaleimide under native conditions. Wild type toxin reacted neither with DTNB (as demonstrated by absence of absorption a t 412 nm) nor withmaleimide derivatives. Fluorescein-maleimide modification and SDSPAGE yielded fluorescent protein bands with cysteine mutants butfailed to doso with wild type toxin (data not shown). Cys-130 toxin could be labeled with fluorescein-maleimide to an extent of 95% as assessed by absorbance at 490 nm. Labeling could be blocked almost completely (6% residual labeling) by pretreatment with 1 mM DTNB for 15 min at room temperature and was zero with wild type toxin. Thus, labeling was sulfhydryl-specific. Activity of Modified Toxins-The functional consequences of coupling differed, however. With a-toxin Cys-130, neither reaction with small compounds (N-methylmaleimide, DTNB) nor reaction with larger ones such as p-hydroxymercuriphenylsulfonate,fluorescein-maleimide, or biotin-maleimide altered the hemolytic titer. The hemolytic titer of Cys-69 was hardly affected by the small compounds butreduced about 8-fold by biotin-maleimide and nearly abolished by fluorescein-maleimide. The hemolytic titer of a-toxin Cys-186 was uniformly reduced about 4-8-fold by all compounds tested. The cysteine mutants alsodiffered in their ability to form
Staphylococcal with &-Toxin
Modifiable Cysteine
11961
dimers connectedby intermolecular disulfide bridges (dimers which were cleavable by DTT (not shown). In addition, nonare neverobserved with wild typetoxin). Cys-69 formed heated samples also displayed aggregates of high molecular disulfide bonds at room temperature when reacted with half weight that could be dissociated by boiling. Demonstration of Cell-bound Labeled Toxins-Cell-bound molar equivalent of DTNB (not shown). With Cys-130 and Cys-186 toxins, dimers formed efficiently only upon heating toxins could be detected using various techniques. Fluoresto 50 "C. Fig. 2 illustrates the results obtainedwith the latter ceinated toxinswere directly detected on erythrocytesosmotically protected by dextran 4 with fluorescence microscopy mutants. Samples A and B were analyzed directly, whereas samples C and D were boiled in SDS prior toelectrophoresis. (Fig. 3A). Biotinylated proteinswere visualized on thewashed In all cases, dimers of approximately 68 kDa were observed, cells by incubation with streptavidin-fluorescein. When biotinylated Cys-69 toxin was used, post-staining with streptavidin-fluorescein resulted in bright fluorescence of the membranes (Fig. 3C). Thus, residue 69 must be superficially locatedinthemembrane-boundtoxin. Cys-186 toxin also yielded clear fluorescence (not shown). -68kd By contrast, cells carrying the biotinylated Cys-130 toxin and post-treated with streptavidin-fluorescein exhibited very weak membrane fluorescence (not shown). The possibility -34kd ~- . that thisresidue becomes membrane-embedded, rendering the biotin label inaccessible to streptavidin-fluorescein in intact A B C D FIG.2. SDS-PAGE of cysteine-containing a-toxin mutants. cells, is being considered. Negative controls were performed to demonstrate thespecToxin solutions were heated a t 50 "C to promote dimerization, and samples were then boiled in presence of SDS to dissociate any ificity of fluorescent labeling. First, wild type toxin was renoncovalent aggregates. A and C, Cys-130-toxin; B and D, Cys-186- acted with fluorescein-maleimide and employed in identical toxin. A and B , unheated samples; C and D,samples boiled in SDS. experiments. Cells remained free of fluorescence (Fig. 3R). The respective dimersexhibited slightly differentelectrophoretic mobility, probably due to different conformations. Noncovalent ag- Second, cysteine mutants were reacted with DTNB prior to gregates were dissociated by boiling, whereas dimers could only be incubation with biotin-maleimide. Cells laden with these toxins also failed to exhibit significant fluorescence (Fig. 30). cleaved by DTT (notshown).
w
FIG. 3. Fluorescence microscopy of osmo-protected red cells treated with labeled toxins. A, rabbit erythrocytes treated with atoxin Cys-130 that was labeled at its sulfhydryl-group with fluorescein-maleimide. B , corresponding negative control with wild type toxin that had been exposed to fluorescein-maleimide. C, red cells laden with biotinylated Cys-69 toxin and reacted with streptavidin-fluorescein. D, corresponding negative control (cells were treated with Cys-69 toxin that had been reacted with DTNR prior to biotin-maleimide, and then exposed to streptavidin-fluorescein).
11962
Staphylococcal a-Toxin withModifiable Cysteine 200kd-
-
34kd-
A B C
FIG.4. Electroblotted SDS-PAGE, developed with streptavidin-peroxidase. Rabbit red cells were lysed with SH-biotinylated toxins and the membranes solubilized with SDS. A, negative control(membranes from cells treated with unlabeled wild type toxin). B, membranes from cells lysed withbiotinylated Cys-130 toxin; sample not boiled. Toxin is present mainly as hexamer. C , same aslune B; sample boiled for 5 min in presence of SDS. Hexamers are dissociated to monomers. Note that biotin is reactive with streptavidin in both the toxin monomer and hexamer, indicating superficial location.
Finally, biotinylated andcell-bound proteins were also visualized following membrane solubilization and SDS-PAGE by development of electroblots with streptavidin-peroxidase. Fig. 4 depicts the results obtained with Cys-130 toxin. When samples were not boiled, the toxin was detected mainly in hexamer form. Dissociation to the monomer occurred after boiling (Fig. 4). Thesefindingsdemonstratedthatbiotin molecules bound to Cys-130 were accessible on the surfaces of both the monomer and the hexamer. While biotinylated Cys-69 exhibited the same behavior, only faint staining of hexamers was observed with Cys-186 toxin (not shown). CONCLUSION
In sum, this study documents the successful construction of intrinsically and homogeneously labeled a-toxin derivatives. At the same time,residues 69,130, and 186 can now be
located to the surface of the nativemonomer. Biotin attached to residue 69 isaccessible on monomers and also onhexamers in both solubilized and membrane-bound forms. Residue 69 must thus be located superficially in the extramembranous part of the pore. Biotin attached to the sulfhydryl group of Cys-130 is reactive with streptavidine onelectroblots, revealing superficial location of Cys-130 on hexamers as well. Intriguingly, this biotin residue seems to be poorly reactive on osmotically protected, intact cells, raising thepossibility that Cys-130 becomes buried in the membrane. It is easy to anticipate that the use of these and other similarly constructed toxin mutants containingcysteine residues a t defined locations will facilitate future studies on the molecular interaction of a-toxin with membranes and target cells. Ackmwledgments-We are grateful to Dr. A. Bornemann (Institute of Neuropathology, Mainz University) for performing fluorescence photography and to M. Messner for skillful technical assistance. REFERENCES
1. Bhakdi, S., and Tranum-Jensen, J. (1991)Microbiol. Reu. 55,733-751 2. Bernheimer, A. W. (1974)Biochim. Biophys. Acta344.27-50 3. McCartney, C. A., and ATbuthnott,J. P. (1978)in Bacterial Toxins and Cell Membranes (Jeljaszewlcz, J., and Wadstrom, T., eds) pp. 89-127,Academic Press Inc.. London
4. Cassidy, P., and Harshman, S. (1976)Biochemistry 15,2348-2355 5. Hildebrand, A., Pohl, M., and Bhakdi,S. (1991)J. Riof. Chem. 266,17195173nn
6. BI&KZY S., and Tranum-Jensen, J. (1987)Reu. Physiol. Biochem. P h r macof. 107,147-223 7. Gray, G. S., and Kehoe, M.(1984)Infect. Immun. 46,615-618 8. Kehoe, M.,Duncan, I., Foster, T., Fairweather, N., and Dougan, G . (1983) Infect. Immun. 41. 1111-1115 9. Taylor, J. W., Ott, J., and Eckstein,F.(1985)Nucleic Acids Res. 13,87648785 10. Fairweather, N., Kennedy, S., Foster, T., Kehoe, M., and Dougan, G . (1983) Infect. Immun. 41, 1112-1117 11. Ausubel. F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1988)Current Protocols in Molecular Biology, Wiley-Intersclence, New York
12. Bhakdi, S., Muhly, M., and Fussle, R. (1984)Infect. Immun. 46,318-323
