INFECTION AND IMMUNITY, May 1994, p. 1843-1847(0 1 9-9567/94/$04.00+0Copyright © 1994, American Society for Microbiology

Site-Directed Mutagenesis of the Alpha-Toxin Gene ofStaphylococcus aureus: Role of Histidines in Toxin

Activity In Vitro and in a Murine Model

Vol. 62, No. 5

BARBARA E. MENZIES* AND DOUGLAS S. KERNODLEDivision of Infectiolus Diseases, Departmenit of Medicine, Vanderbilt Universiy School of Medicine, Nashville,

Tennessee 37232, and Department of Veterans Affairs Medical Cente,; Nashville, Tennessee 37212

Received 16 November 1993/Returned for modification 6 January 1994/Accepted 24 February 1994

Staphylococcus aureus alpha-toxin is a membrane-damaging exoprotein that oligomerizes to form transmem-brane pores. Chemical modification of histidines with diethylpyrocarbonate has been shown to reduce thehemolytic activity of alpha-toxin, suggesting that one or more of the histidine residues is important for toxinfunction. To individually assess the functional importance of each of the four histidine residues (residues 35,48, 144, and 259), we used oligonucleotide-directed mutagenesis of the cloned alpha-toxin gene to replace eachhistidine with leucine. The mutant toxins were expressed in S. aureus and evaluated for hemolytic activity in vitroand for lethality in an intraperitoneal murine model. Substitution of histidine 35 with leucine produced amutant toxin (H35L) without hemolytic or lethal activity. Mutant toxins H48L, H144L, and H259L exhibited 7,16, and 46%, respectively, of the hemolytic activity of wild-type toxin. Immunoblotting of purified H35L toxinincubated with liposomal membranes demonstrated intact membrane binding and hexamer formation that wasclearly detectable but reduced compared with that of the wild-type toxin. This suggests that hexamer formationalone is not sufficient for the expression of alpha-toxin activity. The nature of the defect underlying the lack ofactivity of the H35L mutant toxin remains to be elucidated but may involve failure of the hexamer to span thelipid bilayer to form a transmembrane pore or a change in the internal surface and permeability characteristicsof the pore.

Staphylococcal alpha-toxin is a cytolytic exoprotein pro-duced by most pathogenic strains of Staphylococcus aureus andis considered a major virulence factor (1, 20). This pore-forming toxin has been well characterized in the laboratory asa potent hemolysin and as a causative factor in dermonecrosisand lethality in laboratory animals (7, 26). In addition, it exertscytotoxic effects on mammalian cells such as cultured endothe-lial cells (28), human platelets (4), and human monocytes (3).The cytolytic property of alpha-toxin has been studied

extensively in rabbit erythrocytes and in artificial membranesystems (11, 14, 27). There is general agreement that poreformation is a sequential process involving hexamer assemblyfrom monomeric toxin units. The molecular domains or resi-dues required for these events have not been identified defin-itively despite previous studies which have involved the use ofmonoclonal antibodies against peptide domains (13, 16) andchemical modification (8, 9).The alpha-toxin gene has been cloned, and the DNA and

amino acid sequences have been reported (12) and revised(30). The polypeptide chain consists of 293 amino acid residueswith four histidines located in positions 35, 48, 144, and 259.Chemical modification of the histidine residues of alpha-toxinby diethylpyrocarbonate results in reduced hemolytic activity(23). Both membrane binding and oligomerization appear tobe impaired. However, the relative contribution of each histi-dine in the events leading to transmembrane pore formationand the relevance to in vivo toxicity have not been determined.Oligonucleotide-mediated site-directed mutagenesis for substi-

Corresponding author. Mailing address: Division of InfectiousDiseases, A-3310 MCN, Vanderbilt University Medical Center, Nash-ville, TN 37232-26t)5. Phone: (615) 327-4751, ext. 5495. Fax: (615)343-6160.

tution of single amino acids offers a means of assessing thefunctional role of each of the histidine residues. We show thatintroduction of the nonpolar leucine residue for each of thefour histidines results in mutant toxins with different degrees ofimpairment in hemolytic activity in vitro and that these differ-ences correlate with reduced lethal activity in mice.

(This work was presented at the 33rd Interscience Confer-ence on Antimicrobial Agents and Chemotherapy, New Or-leans, La., 17 to 20 October 1993.)

MATERIALS AND METHODS

Materials and media. Restriction endonucleases, T4 DNAligase, and Sequenase version 2.0 T7 DNA polymerase werepurchased from United States Biochemical Corp. (Cleveland,Ohio). The Muta-Gene M13 kit (Bio-Rad Laboratories, Her-cules, Calif.) was used for site-directed mutagenesis. Oligonu-cleotides used for primers in site-directed mutagenesis andsequencing were synthesized on a Cyclone Plus automaticDNA synthesizer (Millipore Corp., Bedford, Mass.) by theDNA core facility, Department of Molecular Physiology andBiophysics, Vanderbilt University. Purification of mutagenicoligonucleotides was performed by using the C-18 Sep-pak(Millipore) chromatographic method. Escherichia coli strainswere cultivated in LB and 2 x YT media prepared as describedby Sambrook et al. (24). S. aureus strains were propagated intryptic soy broth and agar (Difco Laboratories, Detroit, Mich.).Chloramphenicol (Sigma Chemical Co., St. Louis, Mo.), andampicillin and erythromycin (United States BiochemicalCorp.) were used for selection under appropriate conditions.

Bacterial strains and plasmids. E. coli MV1 190 supplied byBio-Rad in the Muta-Gene kit was the recipient strain for themutated alpha-toxin gene-vector. The restrictionless S. aulreulsstrain RN4220 was obtained from R. Novick (Public Health

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1844 MENZIES AND KERNODLE

Research Institute, New York, N.Y.). S. aureus DU1090 wasprovided by T. J. Foster (Trinity College, Dublin, Ireland) andis an alpha-toxin-negative strain produced by insertional inac-tivation of the cloned alpha-toxin gene, hla, followed by allelicreplacement of the chromosomal wild-type gene (20). S. aureusWood 46 (ATCC 10832) was purchased from the AmericanType Culture Collection (Rockville, Md.). Plasmids pDU1150and pDU1212 contain the cloned alpha-toxin gene from strainWood 46 and were provided by T. J. Foster (10, 17). PlasmidpCW59 in RN4220 was provided by K. G. H. Dyke (Oxford,England).

Site-directed mutagenesis. A 3.0-kb Hindlll-EcoRI frag-ment of the cloned alpha-toxin gene from pDU1150 in E. coliK-12 (17) was excised and inserted into M13mpl8 for in vitromutagenesis by the Kunkel method (18). Twenty-base oligo-nucleotide primers were constructed such that a single basechange would produce a codon change that resulted in ahistidine-to-leucine substitution. The desired single-nucleotidesubstitution in the genes encoding the four mutant toxins wasconfirmed by dideoxynucleotide sequencing (25).

Construction of S. aureus clones. To construct S. aureusstrains which produced the mutant toxins, we used a protocolsimilar to that of Fairweather et al. in generating the E. coli-S.aureus shuttle plasmid pDU1212, which carries wild-type hla(10). First, the 3.0-kb HindIII-EcoRI fragment containing amutagenized hla (mhla) gene was ligated to pBR322. An E.coli-S. aureus shuttle vector plasmid was then formed in E. coliMV1190 by ligation of the pBR322-mhla construct to the3.0-kb HindIll fragment of pCW59, an S. aureus-B. subtilisshuttle plasmid encoding chloramphenicol resistance. Therecombinant shuttle vector containing mhla was harvested,purified, and transformed via protoplasts into S. aureusDU1090 (hla inactivated and erythromycin resistant) by usingS. aureus RN4220 as an intermediate host (21). Chloramphen-icol (10 ,ug/ml) and erythromycin (10 ,ug/ml) were used forselection of clones. Plasmid DNA from S. aureus clones wasprepared by using lysostaphin and the alkaline lysis procedure(24). Plasmids containing the mhla constructs were checked byrestriction analysis for comparison with plasmid pDU1212.

Purification of wild-type and mutant alpha-toxins. S. aureusDU1090 strains which had been transformed with shuttleplasmids containing the wild-type and mutant hla were culti-vated in 500 ml of tryptic soy broth for 16 h at 150 rpm at 37°Cwith chloramphenicol (12.5 ,ug/ml) and erythromycin (10 ,ug/ml). Each mutant toxin was purified from culture supernatantsby using the controlled-pore glass method of Cassidy andHarshman (6, 15). After elution from the column, fractionsexhibiting hemolytic activity were pooled and concentratedwith a Centriprep-10 device (Amicon, Beverly, Mass.) in 0.01M sodium phosphate buffer (pH 7.2). The specimens wereanalyzed for purity by using a sodium dodecyl sulfate (SDS)-12% polyacrylamide gel stained with Coomassie blue (Bio-Rad). Protein concentration was determined by the A280, using1.1 as the milligram-per-centimeter extinction coefficient (15).Hemolytic activity determination. Hemolytic titration was

performed as described by Bernheimer (2). Briefly, serialdilutions of purified toxin in 0.5 ml of diluent (0.01 M sodiumphosphate-buffered saline with 0.1% albumin [pH 7.2]) wereincubated with 0.5 ml of a 1% suspension of washed rabbiterythrocytes at 37°C for 30 min. The inverse of the tubedilution resulting in 50% hemolysis determined by the A545 isthe number of hemolytic units (HU) per ml of test solution.Assays were performed twice, and the titer was expressed asthe mean of the two values.

Preparation of multilamellar liposomes. Multilamellar lipo-somes were prepared from a mixture of egg yolk phosphati-

dylcholine (Avanti Polar Lipids, Alabaster, Ala.) and choles-terol (Sigma) in a 2:1 molar ratio in chloroform. The solutionwas dried by rotoevaporation at 40°C to form a thin film oflipid on a round-bottom flask. The lipid film was dispersed inphosphate-buffered saline (PBS; pH 7.2) to give a final phos-pholipid concentration of 3 pLmol/ml. This suspension wasmechanically agitated on a wrist-action shaker for 2 h. Theliposomes were extruded once through a 0.4-pLm-pore-sizepolycarbonate membrane and twice through a 0.2-,um-pore-size polycarbonate membrane (Nuclepore, Pleasanton, Calif.)alternating with five freeze-thaw cycles to generate a homoge-neous formulation.Assembly of toxin on liposomes. A mixture of toxin and egg

yolk phosphatidylcholine-cholesterol liposomes (0.1 p.mol oflipids) in PBS (1 ml) was incubated at 37°C for 1 h. Liposome-bound toxin was collected by centrifugation at 23,000 x g for20 min at 4°C, washed twice with PBS, centrifuged, andsolubilized in 1% SDS at 25°C. Samples were subjected toSDS-polyacrylamide gel electrophoresis (SDS-PAGE; 10%acrylamide) without boiling prior to electrophoresis exceptwhen indicated. The gel proteins were electrophoreticallytransferred to nitrocellulose membranes. After the membraneswere blotted, they were blocked with 0.5% Tween 20 inTris-buffered saline (10 mM Tris, 500 mM NaCl [pH 8.0]) andthen incubated for 1.5 h with polyclonal anti-alpha-toxin serum(1:100 dilution) raised against S. aureus Wood 46 alpha-toxinhexamer in immunized rabbits. The blot was developed withgoat anti-rabbit immunoglobulin G-alkaline phosphatase con-jugate (Boehringer Mannheim Biochemicals, Indianapolis,Ind.) at a 1:2,000 dilution by using nitroblue tetrazolium and5-bromo-4-chloro-3-indolyl phosphate as the substrate.Murine lethal studies. All in vivo experiments were ap-

proved by the institutional committee for animal care. Maleand female NIH Swiss mice, weighing 20 to 24 g each, wereinjected intraperitoneally (i.p.) with microgram quantities offilter-sterilized purified toxin in 1-ml volumes by using 25-gauge needles and tuberculin syringes. At the time of i.p.injection, buprenorphine, at a dose of 2 mg/kg of body weight,was administered intramuscularly to minimize any potentialsuffering. Death by 24 h was used as the end point, and thetime in minutes from i.p. injection until death was recordedprecisely. The chi-square analysis with Yate's correction wasused to determine the statistical significance of differences inthe number of deaths of mice in each set. For some experi-ments, Kaplan-Meier plots were analyzed with NCSS software(version 5.5; Hintze JL, Kaysville, Utah), and the Peto/Wil-coxon test was used to determine the statistical significance.

RESULTS

Site-directed mutagenesis and transformations. By usingoligonucleotide-directed mutagenesis, the codon for each ofthe four histidines of alpha-toxin was changed such thatleucine was substituted, resulting in four mutant toxins (H35L,H48L, H144L, and H259L). Dideoxynucleotide sequencingconfirmed these substitutions and excluded any spurious mu-tations elsewhere in the genes for the four mutant toxins. Eachof the four mutant toxin genes and the wild-type toxin genewere incorporated individually into an E. coli-S. aureus shuttleplasmid and inserted into the alpha-toxin-negative S. aureusstrain DU1090 by using protoplast transformation.

Purification of toxins. Alpha-toxin from each of the trans-formed DU1090 strains was purified by adsorption chromatog-raphy (6, 15). SDS-PAGE of each of the purified mutant toxinsand wild-type toxin identified a single 33-kDa band migrating

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STAPHYLOCOCCUS AUREUS ALPHA-TOXIN MUTANTS 1845

a b c d e f g 100

90

80-i

>. 70

c 60

50

[1] 4040

cc 30lLI

20

10

021.5-14.4w

0 3

FIG. 1. Coomassie brilliant blue-stained SDS-12% polyacrylamidegel of approximately 2 p.g each of purified toxin specimens. Lanes: a,standard (Wood 46 alpha-toxin); b, low-molecular-mass markers (withvalues shown to left of gel); c to g, purified toxins from transformed S.aureus DU1090 strains, namely, wild type (lane c), H35L (lane d),H48L (lane e), H144L (lane f), and H259L (lane g).

alongside the purified native alpha-toxin from the prototypicstrain Wood 46 (Fig. 1).

Hemolytic activity of wild-type and mutant alpha-toxinspurified from S. aureus DU1090. The specific activities of all ofthe purified mutant alpha-toxins were less than that of the wildtype. Wild-type toxin expressed in transformed DU1090 dem-onstrated a specific hemolytic activity of 26,250 HU/mg, avalue comparable to those previously reported (15). The H35Lmutant toxin was devoid of hemolytic activity, with a sensitivityby this assay of less than 1 HU/mg. H48L and H144L toxinsexhibited 7 and 16% of the specific activity of the wild-typetoxin, respectively (i.e., 1,895 and 4,268 HU/mg of protein).H259L toxin exhibited the least reduction in hemolytic activity,with an activity of approximately 46% of that of wild-type toxin(i.e., 12,130 HU/mg of protein).

Lethal studies in mice. The lethal activities of the wild-typetoxin and three of the mutant toxins (H35L, H48L, andH259L) were assessed after i.p. injections in mice (Table 1).Four groups containing seven to eight mice in each groupreceived 10 jig of either wild-type toxin or one of the mutanttoxins. Studies were repeated on a different day, and the resultswere combined for analysis. Of the 16 mice that received 10 ,ugof wild-type toxin, 14 (88%) had expired by 24 h. In contrast,no deaths occurred among 16 mice that received 10 ,ug of theH35L toxin or 15 mice that received 10 ,ug of H48L toxin.

TABLE 1. Lethal activity of mutant toxinsa

AaxD(,ug) No. of mice expired/Alpha-toxin Dose totalgnob

Wild type 10 14/16H35L 10 0/16C

100 0/8H48L 10 0/15c

100 718H259L 10 14/16e

a Lethality at 24 h in mice receiving i.p. injection of purified wild-type andmutant alpha-toxins.

b For values compared with wild type values P was <0.005 (c), =0.513 (d), and=0.593 (e) as determined by chi-square test (Yate's corrected).

HOURSFIG. 2. Kaplan-Meier plot of survival over time in mice post-i.p.

injection of 10 ,ug of wild-type toxin and 10 ,ug of H259L toxin. Eachgroup consisted of eight mice. The curves were significantly different(P, <0.002; Peto/Wilcoxon test).

Although the total number of mice that expired after receivingthe H259L or wild-type toxin was identical, the time from i.p.injection until death was longer for mice receiving the H259Ltoxin (Fig. 2).Groups of eight mice were challenged further with the H35L

and H48L toxins at 100-,ug doses (Table 1). All of the mice thatreceived the H35L toxin survived; however, seven of the eightmice injected with the H48L toxin expired.Toxin assembly in liposomes. Using multilamellar liposomes

composed of egg yolk phosphatidylcholine and cholesterol, wecompared the membrane binding and oligomerization of wild-type toxin and H35L toxin by using SDS-PAGE immunoblot-ting. Polyclonal anti-alpha-toxin serum raised against S. aureusWood 46 hexamer permitted detection of both the hexamericand monomeric forms of alpha-toxin. After incubation of 0.1,umol of egg yolk phosphatidylcholine-cholesterol liposomeswith 5 p,g of toxin and of another sample with 10 ,ug of toxin,the membranes were washed twice to remove toxin that wasnot membrane bound. After SDS-PAGE immunoblotting ofthe membranes without boiling, monomeric and hexameric

kW205-

116m-80i

49-

32

27

A BWT H35L WT H35L

FIG. 3. SDS-PAGE immunoblots of wild-type (WT) and H35Ltoxin hexamer formation in liposome membranes. Liposome-boundwild-type and H35L mutant toxins were recovered following incuba-tion of toxin at concentrations of 5 (A) and 10 (B) tLg/ml. Numbers tothe left of the figure indicate molecular mass standards (in kilodaltons).

kD

97.4-66.2k

42.7k

31.0-

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toxins were observed in both wild-type and H35L toxin samples(Fig. 3). Whereas the intensity of the monomeric bands (33kDa) was similar to that of the wild type, the intensity of thehexameric band (200 kDa) of the H35L toxin was moderatelyreduced. This finding was confirmed in triplicate. To confirmthat the 200-kDa band seen with H35L-bound liposome rep-resented the hexameric form, another sample was boiled for 5min in the presence of 2% SDS and 5% 2-mercaptoethanolprior to PAGE, demonstrating dissociation of the toxin hex-amer to the monomeric form (data not shown).

DISCUSSION

In this study, we used oligonucleotide-directed mutagenesisto construct alpha-toxin mutants, each harboring a singlesubstitution of histidine to leucine. Each of the four modifiedtoxins were expressed, purified, and assayed for hemolyticactivity against rabbit erythrocytes and for lethal activity inmice. We show that a single amino acid substitution ofhistidine 35 is sufficient to inactivate both the hemolytic andlethal activities of the toxin. Substitution of histidine 48, 144,and 259 produced mutant toxins with less impaired hemolyticactivity, and we observed a near-direct correlation between thehemolytic and lethal activities of the mutant toxins. In general,our results support the suggestions of Menestrina et al. (19)that all four histidine residues are required for maximalexpression of hemolytic activity.Of the four mutant toxins, H35L toxin appears to be the

most interesting in that both hemolytic and lethal activitieswere fully ablated. We sought to define the nature of the defectunderlying the loss of biological activity by using a protein-freeliposome model to assess toxin membrane assembly. Previousstudies have shown that alpha-toxin interacts with multilamel-lar liposomes to form hexameric structures with resultantmembrane permeabilization (11, 31). Our SDS-PAGE immu-noblotting demonstrates that H35L toxin retains the ability tobind in a model membrane system; however, oligomerizationinto hexamers is moderately reduced compared with that ofwild-type toxin.

That the H35L toxin is able to oligomerize to hexamericstructures must be reconciled with its lack of activity. There isgeneral agreement that hexamer formation is required formembrane damage; however, recent studies have shown thathexamer formation is not always accompanied by membranedamage. For example, investigators have shown that proteo-lytically nicked toxin forms hexamers in the membranes ofnucleated cells without causing cell damage (5, 22). Additionalevidence has arisen from the finding of Walker et al. thatN-terminal truncation mutants of alpha-toxin missing 2 to 22amino acids are able to form oligomers on erythrocytes butlack hemolytic activity (29). It has been proposed that thealpha-toxin hexamer must assume a change in conformation asit penetrates the membrane, thereby exposing critical residuesthat serve to line the pore channel (14). Nonlytic hexamerssuch as those formed by H35L toxin may not be able to achievethe necessary change in conformation to penetrate the mem-brane sufficiently or may alter the electrostatic forces of thepore channel such that it remains closed. In addition, H35Ltoxin may also form unstable membrane-bound hexamers sincemoderately reduced hexamer formation was observed in themodel membrane system when compared with that of the wildtype.Our data show that histidine 35 of alpha-toxin is critical to

the biological activity of alpha-toxin. Clearly, other studies areneeded to define the structure of hexameric transmembranepores. The availability of these mutant toxins and the creation

of others from other single amino acid substitutions should aidin determining structure-function relationships of alpha-toxinon a molecular level.

ACKNOWLEDGMENTS

We thank Sidney Harshman for useful discussions and criticalreview of the manuscript and Rama Voladri for technical adviceregarding oligonucleotide-directed mutagenesis. We are also apprecia-tive of the assistance of Hans Schreier in the preparation of liposomes.

This work was supported by a merit review grant from the ResearchService of the Department of Veterans Affairs.

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