Vol. 58, No. 9

INFECTION AND IMMUNITY, Sept. 1990, p. 3020-3028

0019-9567/90/093020-09$02.00/0 Copyright C 1990, American Society for Microbiology

Mutants of Staphylococcal Toxic Shock Syndrome Toxin 1: Mitogenicity and Recognition by a Neutralizing Monoclonal Antibody LORNA BLANCO,' EDMUND M. CHOI,' KATHRYN CONNOLLY,' MICHAEL R. THOMPSON,2 AND PETER F. BONVENTREl* Department of Molecular Genetics, Biochemistry and Microbiology' and Department of Medicine,2 University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0524 Received 11 April 1990/Accepted 23 June 1990

Toxic shock syndrome toxin 1 (TSST-1), a 22-kilodalton protein made by strains of Staphylococcus aureus harboring the chromosomal toxin gene, may elicit toxic shock syndrome in humans. In vitro, TSST-1 induces T cells to proliferate and macrophages to secrete interleukin-1. To conduct a structure-function analysis, point mutations on the TSST-1 gene were generated by site-directed mutagenesis to identify amino acids critical for activity of the toxin. Specific tyrosine and histidine residues were replaced by alanines. Wild-type and mutant TSST-1 gene constructs were expressed in Escherichia coli, and the products were tested for their mitogenic potential and reactivity with a TSST-1 neutralizing monoclonal antibody (MAb 8-5-7). Four of the mutants were similar to the wild type; i.e., the mutant toxins stimulated murine T cells and reacted with MAb 8-5-7 equally as well as the wild type. Two mutants exhibited a decrease in mitogenic activity, but one of these retained the capacity to bind with MAb 8-5-7 while the other was no longer recognized by the same antibody. One double mutant demonstrated minimal mitogenic activity and did not react in enzyme-linked immunosorbent and immunoblot assays with MAb 8-5-7. The data show that specific residues near the carboxy terminus of TSST-1 are essential for mitogenic activity and in forming the epitope recognized by neutralizing MAb 8-5-7.

initiated a detailed study whose goals were to define amino acid sequences essential for expression of mitogenicity and to map epitopes within the toxin molecule recognized by a panel of TSST-1 MAbs of murine origin (7). The strategy adopted was to introduce alterations in the TSST-1 gene on a plasmid vector by site-directed mutagenesis (21). KokanMoore and Bergdoll (18) recently reported that chemical modification of either the histidine or tyrosine residues of TSST-1 resulted in significant loss of mitogenic activity. Therefore, several independent mutations involving one or two of these amino acid residues were constructed in our first effort to conduct a structure-function analysis of TSST-1. The mitogenic activity of the cloned gene products expressed in Escherichia coli (i.e., native and mutant TSST-1) were evaluated. We describe seven TSST-1 mutants possessing either unchanged or significantly reduced mitogenic activity for murine spleen cells. Also, several mutant toxins were altered in epitope configuration so that recognition by MAb 8-5-7 was either reduced or completely lost.

A considerable body of evidence suggests that the staphylococcal toxin toxic shock syndrome (TSS) toxin 1 (TSST-1) plays a pivotal role in the genesis of human TSS (2, 6, 9, 33, 35). Other staphylococcal toxins, primarily staphylococcal enterotoxin B, may also precipitate the illness, particularly in nonmenstrually associated cases (15, 32). A cardinal property of TSST-1 is its potent mitogenic potential for human (10) and murine (30) T lymphocytes. The toxin is also a strong inducer of interleukin-1 (28), tumor necrosis factor, and gamma interferon (16). Recent studies show that TSST-1 and staphylococcal enterotoxins act as superantigens by interaction with T-cell receptors expressing specific V,B elements in conjunction with class II major histocompatibility complex antigens (12, 14, 27, 36). Thus, TSST-1 interacts with a significant percentage of T cells which may activate several immunological pathways. Such a massive stimulation of the immune system by TSST-1 could be responsible for the clinical syndrome which develops subsequent to infection with TSST-1-producing strains of Staphylococcus aureus (8, 9). In a previous study, we showed that the TSST-1-induced illness and mortality in a rabbit model of TSS (29) could be prevented by infusion of a TSST-1-specific monoclonal antibody (MAb) (7). It is likely that in vitro activities of TSST-1 and toxicity in vivo are closely linked phenomena since lymphocyte proliferation and interleukin-1 induction are blocked by a TSST-1 neutralizing MAb (MAb 8-5-7) which also prevents lethality in both toxin (7) and infection (3) rabbit models of TSS. In view of the importance ascribed to neutralizing TSST-1 antibodies in prevention of clinical TSS (6, 35) and the central role apparently played by the toxin in the pathogenesis of the disease (2, 9, 33), we

*

MATERIALS AND METHODS Bacterial strains, plasmids, TSST-1, and TSST-1 antitoxins. Bacterial strains and TSST-1 plasmids were obtained from B. Kreiswirth and R. Novick, Public Health Research Institute, New York, N.Y. The host strain for the plasmid encoding the full-length TSST-1 gene was E. coli AB259 (24). The nucleotide sequence of the TSST-1 gene has been determined (5). Plasmid vector pRN6550 consists of pBR322 into which a DNA fragment containing the entire TSST-1 coding sequence and flanking regions was inserted (20). Purified TSST-1 was provided by J. Parsonnet, Channing Laboratories, Boston, Mass. Details of the purification procedure have been published (28). Several MAbs against purified TSST-1 were originally

Corresponding author. 3020

VOL. 58, 1990

obtained by hyperimmunization of BALB/c mice and subsequent hybridoma formation and dilution cloning (7). One of these, MAb 8-5-7, was used in this study since it neutralizes mitogenicity and interleukin-1 induction of TSST-1 as well as the lethal effects of the toxin administered parenterally to rabbits (7). MAb 8-5-7 was purified from mouse ascites by using the protein A MAPS system (Bio-Rad Laboratories, Richmond, Calif.) Polyclonal rabbit anti-TSST-1 serum was purchased from Toxin Technologies, Madison, Wis.). Construction of TSST-1 gene mutants. The approach used in the generation of mutant TSST-1 proteins is summarized in Fig. 1. A 1.6-kilobase (kb) BglI fragment encoding the TSST-1 gene was isolated from pRN6550 and ligated into the SmaI site of M13mpl9, an E. coli bacteriophage. A silent mutation creating a unique HindIlI restriction endonuclease site was introduced into this vector at position -1 of the TSST-1 leader peptide, using site-directed mutagenesis (construct I) (21). A 20-mer oligonucleotide, 5'CTGCAAAA GCTTCTACAAAC, was used to generate this mutation. A 0.7-kb HindIII-PstI fragment encoding the mature TSST-1 peptide from construct I was subcloned into M13mpl8 to make a cassette for generating the TSST-1 mutants (construct II). Six oligonucleotides in the sense orientation were used to introduce alanine substitutions at histidine and tyrosine residues of TSST-1. The oligonucleotides with the mutations underlined are as follows: 5'CCGAGTCCTGCT GCTAGCCCTGC (51.52); 5'GGAACTGCTATCGCTTTC CAA (80.82); 5'CAAATAGCTGGATTAGCTCGTTCAA (141.144); 5'TTAAAGGCTTGGCCAAAG (115); 5'CGGG TGGTGCTTGGAAA (153); and 5'AAGCCAAGCTACTA GCGA (74), where the numbers in parentheses assigned to the mutants correspond to their amino acid positions in the mature TSST-1 protein. Sequences of the mutant phage clones were determined by using the dideoxy chain termination method (31). A 0.7-kb HindIII-PstI double-stranded DNA fragment containing TSST-1 with mutations was ligated to a plasmid expression vector. The expression vector (construct III) was made by ligating the 1.8-kb EcoRI-PstI fragment from construct I into the EcoRI-PstI site of pUC19H-, a derivative of pUC19 lacking the HindIlI site in the polylinker. The 1.8-kb EcoRI-PstI fragment contains the entire TSST-1 gene with the introduced HindIII site. The wild-type HindIII-PstI fragment was replaced by the 0.7-kb fragment containing the TSST-1 mutation. The complete TSST-1 gene containing specific amino acid changes (construct IV) was confirmed by restriction digest analysis and double-strand sequencing of the entire TSST-1 gene. An additional mutant, 141, was fortuitously obtained from the site-directed mutagenesis reaction used to generate the double mutant 141.144. A summary of the mutants generated in this study is shown in Fig. 2. Mutant TSST-1 DNAs were transformed into competent E. coli AB259 and selected for ampicillin resistance. Recovery of cloned gene products. The gene products were recovered from the periplasmic contents of E. coli AB259 transformed with P17 (wild type) or the TSST-1 mutant plasmids. Proteins secreted into the periplasmic space were obtained by a method described by Kondo et al. (19). Briefly, 50 ml of Luria broth in 250-ml flasks was inoculated from colonies and incubated at 37°C with shaking until early stationary phase. Cells were recovered by centrifugation at 4°C, and the pellet was suspended in 10 ml of sucrose buffer and placed on ice for 15 min. The cells were centrifuged, the pellet was suspended in 2.5 ml of ice-cold distilled water, and the suspension was reincubated on ice for an additional 10 min. This procedure releases the contents of the periplasmic

MUTANTS OF STAPHYLOCOCCAL TSST-1

BglI

3021

Hind III

/ Hind III

P * mutant

a

I

FIG. 1. Construction of the mutant TSST-1 gene. The open and closed areas indicate the signal sequence and mature protein of TSST-1, respectively. The introduced HindlIl site at position -1 is indicated. EcoRI (E), SmaI (S), and PstI (P) are restriction sites in the polylinker. The arrow marks the direction of transcription. For details on the constructs (I to IV), see Materials and Methods.

space of the osmotically shocked bacteria. The preparations ("shockates") were recentrifuged, and the supernatants were filter sterilized and stored at -80°C. By convention, the gene product corresponding to unaltered TSST-1 is identified as P17. The mutant toxins in the periplasmic preparations are identified by the number(s) corresponding to the amino acids replaced by site-directed mutagenesis. Purification of TSST-1 from P17 periplasmic contents. A two-step purification procedure was developed to rapidly purify wild-type TSST-1 from P17 periplasmic preparations.

BLANCO ET AL.

3022

INFECT. IMMUN.

80

1

51.52

I

51 52 74

182

141

107 115

135

1144 153 164

|

115

174 194

YY-HYHH-Y-YYY AA A

74

80.82

13 Y

-

I

AA~ -A

141

AA~

141.144 153

|

1

A-

FIG. 2. Summary of TSST-1 mutants generated by site-directed mutagenesis. The first line represents the mature native TSST-1 protein. The locations of tyrosine (Y) and histidine (H) residues are indicated. Subsequent lines represent the combination of H and Y residues modified to an alanine (A) in the mutants studied. The number assigned to each mutant corresponds to the position of the residue(s) in the 194-amino-acid native toxin.

Briefly, shockates were applied to C18 Sep-Pak cartridges (Waters Associates, Milford, Mass.); the cartridges were washed with buffer containing 10% acetonitrile, and TSST-1 was eluted with 60% acetonitrile-0.1% trifluoroacetic acid buffer. The Sep-Pak served to desalt and enrich the TSST-1 product prior to application to a C18 reverse-phase highpressure liquid chromatography column. TSST-1 was eluted as a single symmetrical peak, separable from other proteins by using a gradient of 10 to 50% acetonitrile in 0.15% trifluoroacetic acid. The fraction containing TSST-1 was centrifuged under vacuum to remove traces of the trifluoroacetic acid buffer, and the purified product was suspended in phosphate-buffered saline prior to further analysis. The purified P17 recombinant toxin was identified as TSST-1 by immunological identity in the competitive enzyme-linked immunosorbent assay (ELISA), using MAb 8-5-7, and by its identical characteristics to those of purified TSST-1 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reverse-phase chromatography. The purity of the high-pressure liquid chromatography-purified TSST-1 was >90% as judged by silver staining of SDSPAGE. A similar purification scheme has been reported for staphylococcal enterotoxin B (34). Mitogen assay. TSST-1 and cloned gene products in the periplasmic contents were evaluated for mitogenic potential in a lymphocyte proliferation assay as described previously (26). Single-cell suspensions of spleens excised from BALB/c mice were adjusted to 5 x 106 splenocytes per ml of RPMI 1640 supplemented with 5% fetal calf serum (Hazleton Biologicals, Lenexa, Kans.); 0.1-ml volumes (5 x 105 cells) were then distributed in 96-well flat-bottom culture plates (Costar, Cambridge, Mass.). T-cell responses were elicited by purified TSST-1 at the concentrations indicated. Concanavalin A (purified concanavalin A provided by David Behnke, Cincinnati, Ohio) was used as a positive control at a concentration of 3 ,ug/ml. Cloned TSST-1 gene products were assayed by measuring lymphoproliferative responses to serial dilutions of the sterile shockates. For neutralization experiments, MAb 8-5-7 at 1.0 mg/ml and dilutions of cloned gene products were mixed prior to their addition to mouse spleen cells. The final volume in each well was 0.2 ml. Cultures were incubated at 37°C for 48 h, after which each well was pulsed with 1.0 ,uCi of [3H]thymidine (specific activity, 37 Ci/mmol; ICN Radiochemicals, Irvine, Calif.) for

an additional 18 h. The cells were collected with an automatic cell harvester (Skatron, Norway) onto glass-fiber filters and air dried. Samples were counted in a liquid scintillation counter to assess incorporation of [3H]-thymidine by lymphocytes. Data are presented as the means of triplicate determinations ± the standard error of measurement.

TSST-1 competitive ELISA. TSST-1 and mutant toxins were measured in a competitive ELISA, using MAb 8-5-7. The preparation and characterization of this neutralizing antibody were as described previously (7). The competitive ELISA is a modification of our original screening assay used to detect MAbs to TSST-1 (unpublished data). Briefly, Immulon II micro-ELISA plates were sensitized with 0.2 pug of purified TSST-1 per well, and residual protein-binding sites were blocked with 1% bovine serum albumin in phosphate-buffered saline (10 mM sodium phosphate [pH 7.2], 0.15 M NaCi). Dilutions were made in 0.1% bovine serum albumin-phosphate-buffered saline. Dilutions of purified TSST-1 or E. coli periplasmic shockates containing native TSST-1 or mutant TSST-1 were mixed and incubated with MAb for 1 h at 37°C. The mixtures were added to wells, and plates were then incubated for 1 h and washed. Subsequently, the plates were incubated with affinity-purified goat anti-mouse antibody (heavy and light chains) conjugated to horseradish peroxidase (Kirkegaard and Perry, Gaithersburg, Md.). After washing, a two-part 2,2'-azinobis(3-ethylbenzthiazoline sulfonic acid) substrate system was used to detect the peroxidase complexes (Kirkegaard and Perry). Each dilution of TSST-1 or periplasmic shockate was incubated in triplicate: two in sensitized wells and one in an uncoated well. The absorbance of the control well was subtracted from that of the sensitized well. Purified TSST-1 yielded a competition curve with a sensitivity of approximately 10 to 20 pg per well (0.5 to 1.0 ng/ml) for native toxin. Detection of TSST-1 and mutant proteins by immunoblot assay. MAb 8-5-7 and polyclonal antiserum originally prepared against native TSST-1 were used to probe the immunodominant regions of TSST-1 and mutant proteins. Purified TSST-1 or periplasmic shockates were subjected to SDSPAGE in 15% acrylamide gels by the method of Laemmli (23). The quantity of mutant TSST-1 in each culture shockate was estimated to be approximately the same as judged by Coomassie brilliant blue R-250 staining profiles. Proteins were electrophoretically transferred to nitrocellulose sheets, which were then stained with Poinceau S to determine fidelity of transfer (22). Residual binding sites were blocked by incubation of the sheets for 1 h at 37°C in 10% nonfat dry milk dissolved in phosphate-buffered saline. After washing, the protein blot was incubated for 1 h at 37°C with either rabbit anti-TSST-1 or purified MAb 8-5-7 diluted in 3% bovine serum albumin-phosphate-buffered saline. Rabbit anti-TSST-1 was detected with protein A-horseradish peroxidase (Kirkegaard and Perry). The murine MAb 8-5-7 was detected with horseradish peroxidase-affinity-purified goat anti-mouse antibody. Immunoblots were developed with 4-chloro-1-naphthol, in conjunction with hydrogen peroxide, following the recommendations of the manufacturer (BioRad Laboratories). RESULTS Generation of mutant TSST-1 genes. A panel of seven mutant TSST-1 genes was generated by using oligonucleotide site-directed mutagenesis. Mutations were introduced at specific histidine and tyrosine residues of TSST-1 toxin.

MUTANTS OF STAPHYLOCOCCAL TSST-1

VOL. 58, 1990

Dilution Factor 128 64 32

1 1F

16 8

4

0 U) .0

A

1

2 3 4 5

1

0.8 0 C) c .0

2

3023

6

7 8

9 10

o

0.6

\A

0.4

~~A

AA\A

0.2

B

0 l

0

l

0.5

1.0

1.5

2.0

2.5

3.0

1

TSST-1 (ng/ml)

2 3 4 5 6 7 8

9 10

FIG. 3. Competitive anti-TSST-1 MAb ELISA measuring TSST-1. The curves compare the reactivity of a purified TSST-1 standard (A), dilutions of periplasmic contents of the toxin-negative E. coli AB259 host strain (0), and E. coli with P17 encoding wild-type TSST-1 (A).

Single or pairwise alanine substitutions in the TSST-1 molecule are indicated in Fig. 2. E. coli AB259 transformants express recombinant toxins in the periplasmic space. The wild-type P17 product and the mutant gene products were identified and assayed for biological function and immunological reactivity by the in vitro assays described in Materials and Methods. Recognition of cloned gene products by ELISA. The TSST-1 competitive ELISA detected immunologically cross-reacting material in dilutions of periplasmic shockates from E. coli AB259 transformed with the P17 (wild-type) plasmid. Figure 3 shows a typical competition ELISA profile for purified TSST-1 and for shockates P17 and AB259, the toxin-negative parent strain. Competitive displacement of MAb 8-5-7 was consistently shown with P17. By ELISA, we measured approximately 40 to 100 jig of TSST-1 per ml of P17 shockate, depending on the preparation. This was consistent with estimates made by SDS-PAGE and mitogenic activity. No TSST-1 was detected in shockates from the parent strain AB259. Periplasmic preparations of each mutant were also tested in the monoclonal ELISA. Four of the mutant TSST-1 behaved identically to the product of P17; these included single-site mutants at 74 and 153 and double mutants at 51.52 and 80.82. Mutant toxin 141 was detectable in the ELISA, while mutation at residue 115 and the double mutation at 141.144 yielded products not recognized by MAb 8-5-7 (data not shown). SDS-PAGE and immunoblotting of cloned gene products. The periplasmic contents from each mutant were compared on Coomassie blue-stained SDS-PAGE with those of the parent strain and the P17 strain encoding TSST-1. Each of the mutant strains and P17 showed the presence of a readily detectable protein band which comigrated with purified TSST-1 (data not shown). No comparable protein band was detected in periplasmic shockates of E. coli AB259. We compared the binding of polyclonal rabbit anti-TSST-1 and neutralizing MAb 8-5-7 with each of the TSST-1 mutants separated on SDS-PAGE. No band was detected in the shockate from E. coli AB259 with either immunoprobe. Both the polyclonal antiserum and MAb 8-5-7 reacted readily with purified TSST-1 and with the recombinant toxin produced by P17. All of the mutant TSST-1 products were detected with polyclonal antiserum. Only mutant 141.144 showed reduced antibody binding (Fig. 4A). A considerable variation in

FIG. 4. Immunoblots of TSST-1, P17, and TSST-1 mutants with monoclonal and polyclonal anti-TSST-1 serum. (A) Polyclonal antitoxin; (B) MAb 8-5-7. Lanes: (A) 1, TSST-1; 2, AB259; 3, P17; 4, 51.52; 5, 74; 6, 80.82; 7, 115; 8, 141; 9, 141.144; 10, 153; (B) 1, 153; 2, blank; 3, 141.144; 4, 141; 5, 115; 6, 80.82; 7, 74; 8, 51.52; 9, P17; 10, TSST-1.

signal was obtained with MAb 8-5-7 (Fig. 4B). Mutants 51.52, 74, 80.82, 141, and 153 were consistently detected and were comparable to the signal obtained with P17. TSST-1 mutant 115 (lane 5) demonstrated reduced binding to MAb 8-5-7, while mutant 141.144 (lane 3) consistently failed to bind this antibody. These results suggest that one or more major antibody-binding epitopes are altered by replacement of these amino acid residues. Mitogenic activity of the cloned TSST-1 (P-17) gene product. Figure 5 provides a comparative titration of the mitogenic potency of purified TSST-1 and the cloned TSST-1 gene product expressed by P17. Purified toxin exhibited mitogenic activity at concentrations as low as 1.0 ng/ml (Fig. 5A). Recombinant TSST-1 in the periplasmic contents of P17 was mitogenic for murine lymphocytes at all dilutions tested (1:400 to 1:25,600) (Fig. SB).

0

a.

a. c

500150 50 30 10 6

3

TSST-1 (ng/ml)

1 0.5

1:4 1:8 1:16 1:32 1:64 1:1281:256

Dilution (x102)

FIG. 5. Comparative mitogen responses of mouse splenocytes to purified TSST-1 and P17. (A) Concentrations of TSST-1 (H) tested were between 500 and 0.5 ng/ml. (B) Periplasmic contents of P17 (-) were tested in twofold dilutions. E. coli AB259 periplasmic contents (OI) served as a negative control.

INFECT. IMMUN.

BLANCO ET AL.

3024

10 0.

500

100

75

50

30

1

0.5

0.25

ng/mI FIG. 6. Mitogenic activity of high-pressure liquid chromatography-purified P17 (M) and comparably processed purified TSST-1 (H).

A small quantity of TSST-1 was purified from a P17 periplasmic preparation by high-pressure liquid chromatography. The recombinant toxin was identical to purified TSST-1 by reactivity to MAb 8-5-7, by SDS-PAGE, and by reverse-phase chromatography elution characteristics. Equivalent amounts of the purified P17 gene product and purified TSST-1 were compared in the mitogen assay. Figure 6 shows the responses of murine lymphocytes stimulated with purified recombinant TSST-1 or native toxin purified from S. aureus culture filtrates. The responses over the concentrations tested were comparable. As another means of confirming that the P17 gene product represented biologically active TSST-1, the neutralization of its mitogenicity by MAb 8-5-7 was assessed. Figure 7A shows the neutralization of TSST-1-induced mitogenesis by MAb 8-5-7. It should be noted that 62.5 ng of TSST-1 represents antigen excess and may explain why total neutralization of mitogenicity was not achieved. Figure 7B shows that MAb 8-5-7 also reduced mitogenic activity of P17 to very low levels. At the highest dilution of P17 tested (1:1,600), the mitogen response was reduced by MAb 8-5-7 to the background level of the untransfected E. coli periplasmic preparation. This dilution of P17 is equivalent to 22.4 ng of TSST-1 per ml as estimated by competitive ELISA (Fig. 3). We conclude from the data presented thus far that the toxin expressed by P17 and TSST-1 are comparable and represent identical entities.

10

A

20

B

8 10-

2125 62.5 250 TSST-1 (ng/ml)

1:400 1:800 1:1600 Dilution

FIG. 7. Neutralization of mitogenicity of purified TSST-1 and P17 by MAb 8-5-7. Symbols: (A) TSST-1 (H); TSST-1 + MAb (ED); (B) P17 (-); P17 + MAb (H1); AB259 (l). The concentration of MAb 8-5-7 used with 1.0 mg/ml.

Dilution (x102)

Dilution (x102)

1:4 1:8 1:16 1:32 1:64 t128 1:256 Dilution Wx102)

1:4 1:8 1:16 1:32 1:64 1:128 1:256 Dilution Wx102)

FIG. 8. Comparative lymphoproliferative responses of mouse splenocytes to mutant TSST-1 gene products. P17 (U) and AB259 (O) are included in all panels as the respective positive and negative controls. The symbol El is (A) mutant 74; (B) mutant 115; (C) mutant 141; or (D) mutant 141.144.

Mitogenic activity of mutant TSST-1 proteins. The data shown in Fig. 8 illustrate contrasting mitogenic responses elicited by the TSST-1 mutants generated by single or double substitutions of histidine or tyrosine residues or both by site-directed mutagenesis. Each panel compares the mitogenicity of the periplasmic contents of P17 with a mutant. Panel A shows that TSST-1 mutant 74 and P17 do not differ in mitogenic potential. Mutants 51.52, 80.82, and 153 behaved similarly to 74: i.e., mitogenic activity comparable to that of P17 (data not shown). The response of mouse lymphocytes to mutant 115 is shown in panel B. In this case, incorporation of [3H]thymidine was significantly reduced. When compared with P17, mitogenicity was reduced by approximately 50%. Modification of the histidine at position 141 of the 194-amino-acid protein also depressed mitogenicity considerably (Fig. 8C). Differences in mitogenic activities of P17 and mutant 141 cannot be ascribed to reduced expression of the mutant by E. coli AB259. When the quantities of P17 and mutant 141 were compared by ELISA, it was evident that the wild type and mutant were produced in comparable amounts (data not shown). A similar comparison was not made with P17 and mutant 115, since the latter was no longer recognized by MAb 8-5-7 in the ELISA. Comparison of P17 and mutants 115 and 141 by Coomassie blue staining of SDS-PAGE gels, however, showed protein bands of comparable intensity (data not shown). Thus, 115 and 141 represent mutations which result in substantial loss of mitogenicity for T cells. The data for mutant 141.144 in panel D show a profound loss in mitogenicity of the altered

MUTANTS OF STAPHYLOCOCCAL TSST-1

VOL. 58, 1990

3025

30

cL

C.)

20 0

aLPS/PB

B 74 115 141 AB259 FIG. 9. Neutralization of mitogenicity of mutant TSST-1 mutants 74, 115, and 141 by MAb 8-5-7. Mitogen responses induced by the three mutant TSST-1 proteins at a dilution of 1/3,200 are shown in the left-hand bar of each pair (E). The right-hand bar of each pair shows the responses cf similar dilutions of the mutants incubated with MAb 8-5-7 (U). AB259 (O) is the negative control.

protein. This would suggest that histidine 141 and tyrosine 144 are critical for the mitogenic activity of TSST-1. Neutralization of mitogenicity of TSST-1 mutants. It was of interest to determine whether neutralizing MAb 8-5-7 could block the mitogenic activity of the TSST-1 mutants. One would predict that those mutants not recognized by MAb 8-5-7 in the ELISA would be unaffected by the antibody in their residual mitogenic activities. Figure 9 shows data obtained with mutants 74, 115, and 141. Mutant 74, which was not modified in mitogenic activity from the wild type (Fig. 8B), was neutralizable by MAb 8-5-7. In contrast, mutant 115, which had a reduced mitogenic potential (Fig. 8C), was not neutralized when incubated with MAb 8-5-7. This is consistent with the observation that mutant 115 did not react with MAb 8-5-7 in the ELISA. Mutant 141 exhibited reduced mitogenic activity (Fig. 8C), but its residual mitogenic activity was neutralized by MAb 8-5-7. This is consistent with the observation that MAb 8-5-7 recognized this mutant protein in the ELISA. The double mutant (141.144), which did not retain significant mitogenic activity, was also not recognized by MAb 8-5-7 in the ELISA (data not shown). These results suggest that tyrosine 144 but not histidine 141 is critical for retention of a conformation promoting effective binding with the neutralizing antibody. The periplasmic contents of E. coli containing TSST-1 and the mutant toxins probably contain trace amounts of bacterial lipopolysaccharide (LPS). Shockates prepared from the host E. coli AB259 stimulated mouse splenocytes so that low-level background counts were seen in the mitogen assays. We attribute this to LPS contamination in the shockates since addition of polymyxin B (11) to the wells prior to pulsing with [3H]thymidine reduced background counts of AB259 to the level obtained with the RPMI 1640 medium control (data not shown). However, since it has been shown that LPS may act synergistically with TSST-1

a. 40000-

20000

0 RPMI

LPS

PB

LPS/PB

FIG. 10. Effect of exogenous LPS and polymyxin B on mitogenic activities of TSST-1 and periplasmic shockates of P17 and mutant 141.144. Symbols: (A) TSST-1 at 1.0 (1B) or 0.1 (U) ,ug/ml; (B) P17 at 1/400 (U) and 141.144 at 1/400 (El) dilution. RPMI, Complete medium, no additives; LPS, RPMI plus E. coli LPS at 1.0 ,ug/ml; PB, RPMI plus polymyxin B at 5.0 ,ug/ml; LPS/PB, RPMI plus E. coli LPS at 1.0 ,ug/ml and polymyxin B at 5.0 ,ug/ml.

(1), several control experiments were performed to ensure that our interpretation of data obtained with P17 and the TSST-1 mutants was valid. The experiments were designed to determine whether exogenously added LPS to shockates or to purified TSST-1 augmented mitogenic activity sufficiently to compromise the data generated with wild-type P17 and the mutant proteins obtained by site-directed mutagenesis. Figure 10 shows data demonstrating clearly that LPS does not augment significantly the mitogenic activity of either TSST-1 or the recombinant products in the E. coli AB259 periplasmic preparations. The mitogenic activity of each sample was tested under four experimental conditions: (i) no additions to the RPMI 1640 medium; (ii) LPS (E. coli serotype 055:85; Sigma Chemical Co., St. Louis, Mo.) added at a final concentration of 1.0 ,ug/ml; (iii) polymyxin B added at a concentration of 5.0 ,ug/ml; (iv) LPS (1.0 ,ug/ml) and polymyxin B (5.0 jig/ml) added. The data show that the mitogenic activity of TSST-1 at the two concentrations tested was comparable in all cases (Fig. 1OA). Recombinant toxin in the P17 shockates also demonstrated essentially the same level of mitogenic activity in the presence or absence of exogenous LPS or polymyxin B or both. The loss of mitogenic activity of mutant 141.144 was apparent with or without the additives to the RPMI culture medium (Fig.

3026

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INFECT. IMMUN.

TABLE 1. Ability of TSST-1 and mutant TSST-1 proteins to bind MAb 8-5-7 or stimulate T-cell proliferation as determined by ELISA, immunoblot, and mitogen assays' Mutant

Mitogen assay

ELISA

Immunoblot

P17 AB259 51.52, 74, 80.82, 153 115 141 141.144

++

++

++

-

-

-

++ +

++

++ +

+

++

++

+/-

-

-

Assays were scored as positive (++), positive but reduced (+), minimal (+/-), or negative (-).

10B). Thus, it can be concluded that the mitogenic activity of P17 can be attributed to intact TSST-1 and that the loss of mitogenic potency of mutant protein 141.144 reflects loss of biological activity resulting from the replacement of histidine 141 and tyrosine 144 by alanine residues. The composite characteristics of the P17 gene product and the mutant toxins are summarized in Table 1. Periplasmic preparations were characterized by their mitogenicity and recognition by MAb 8-5-7 in ELISA and an immunoblotting assay. P17 which expresses unaltered TSST-1 was positive by all three assays and served as the reference against which each of the mutants was evaluated. Four of the mutants were unaltered in their activities when compared with P17. The other three mutants varied in the characteristics demonstrated. All three were significantly reduced in mitogenicity. Mutant 141.144 was not recognized in its native configuration (ELISA) by MAb 8-5-7 or after SDS denaturation (immunoblots). Mutant 115 was also not recognized by MAb 8-5-7 in the ELISA but was recognized weakly in the immunoblot after denaturation. In contrast, mutant 141 retained antibody recognition in both assays in spite of its reduced mitogenic function. DISCUSSION The single most significant characteristic of staphylococci associated with TSS is the production of an exotoxin identified as TSST-1 (2, 8, 9, 33, 35). Delineation of the immunological and physiological pathways leading to the shock syndrome are unrealized objectives. As a step toward that goal, we initiated a structure-function analysis of the TSST-1 protein. The objective was to correlate substitution of alanine for specific histidine or tyrosine residues or both of TSST-1 with retention or loss of mitogenicity for murine T lymphocytes and recognition by a neutralizing MAb (7). In this report, the construction of TSST-1 mutants by sitedirected mutagenesis is described and their respective recombinant gene products are characterized. Structure-function studies of the TSST-1 molecule have been conducted by others. Blomster-Hautamaa et al. (4), Murphy et al. (25), and Kokan-Moore and Bergdoll (17) showed that an internal cyanogen bromide fragment (amino acids 34 to 158) retained mitogenic activity and immunological reactivity to anti-TSST-1 MAbs. Edwin and Kass (13) showed that a papain fragment of TSST-1 corresponding to its carboxy terminus (amino acids 88 to 194) retained biological activities as well as reactivity with several anti-TSST-1 MAbs developed in our laboratory (7). On the basis of these observations, they postulated that the biologically active region of TSST-1 resides in a 71-amino-acid segment encompassed by residues 88 and 158. Kokan-Moore and Bergdoll (18) chemically modified histidine or tyrosine residues of

TSST-1 in an attempt to determine amino acids important for function. They found that modification of these amino acid residues substantially reduced mitogenicity of the toxin. These observations prompted us to construct mutants replacing specific histidine and tyrosine residues located within an internal fragment (34 to 158) between the two methionine residues of TSST-1. TSST-1 possesses nine tyrosine and five histidine residues. Replacing tyrosines at positions 51, 52, 80, and 153 and histidines at positions 74 and 82 with alanines did not alter either biological activity or antibody-binding characteristics of the mutant toxins. Thus, these amino acid residues do not appear to reside at the mitogenic site of TSST-1, nor are they critical for the binding of neutralizing MAb 8-5-7. Three mutations resulted in recombinant products with significantly altered characteristics. Mutant 115 retains approximately half-maximal mitogenic activity of TSST-1 and demonstrates different dose-response characteristics. This mutant toxin was not recognized by neutralizing MAb 8-5-7 in the ELISA and gave a reduced signal by immunoblotting after SDS-PAGE. Since no change was apparent in binding of polyclonal anti-TSST-1 to mutant 115 as detected by immunoblotting, this site may not be immunodominant. Tyrosine 115 may make up part of the binding site for MAb 8-5-7 or the tyrosine may stabilize the conformation of native TSST-1. Kokan-Moore and Bergdoll (18) showed that modification of only 1 to 2 tyrosines of TSST-1 resulted in changes of secondary structure parameters, indicating a conformational change. Thus, such a change in conformation can account for the reduction in the mitogenic activity of mutant 115. Replacement of histidine 141 results in a substantial loss of mitogenic activity. In contrast to mutant 115, 141 retains its binding characteristics with MAb 8-5-7 and polyclonal antiTSST-1. These results suggest that this mutant has lost a determinant critical for mitogenic activity but is distinct from 115 by retention of the epitope recognized by MAb 8-5-7. It is likely that histidine 141 is required for effective interaction of TSST-1 with its cellular receptor (11). The double mutation involving histidine 141 and tyrosine 144 reveals information of fundamental significance. Like mutant 115, 141.144 is not recognized by MAb 8-5-7 in the ELISA and only weakly by immunoblotting. Unlike mutants 141 and 115, however, the double mutant 141.144 lost virtually all mitogenic activity. The reduced affinity of MAb 8-5-7 for 141.144 in this case dramatically parallels the loss of mitogenic activity. These data suggest that tyrosine 144 participates in a common site critical for both mitogenic activity for murine lymphocytes and neutralization by MAb 8-5-7. Since we have demonstrated that this same antibody protects rabbits against the lethal activity of TSST-1 (7), it is likely that these amino acid residues are critical for the activity of TSST-1 in vivo. In view of the apparent importance of tyrosine 144 for the activities of TSST-1, the single mutation of tyrosine 144 is currently being prepared for evaluation. Because of the proximity of tyrosines at 135, 163, and 174 to the region governing mitogenic activity, we also are making additional single and multiple constructs at these loci by site-directed mutagenesis. The strategy we describe to study TSST-1 permits mapping of the active sites of the toxin. Although we have described what appears to be an important functional domain, the pleiotropic effects of TSST-1 on the immune system suggest the possibility for existence of other independent domains which interact with distinct receptors on T cells and accessory cells. A complete structure-function

MUTANTS OF STAPHYLOCOCCAL TSST-1

VOL. 58, 1990

analysis will contribute to the understanding of TSS pathogenesis and may suggest means for prevention and treatment. Our preliminary experiments comparing human and mouse lymphoid cells indicate a striking similarity in their responses to the recombinant mutant toxins (data not shown). Thus, it is likely that the strategy we suggest for the mutational analysis of TSST-1 is valid and will be applicable to studies of human TSS. ACKNOWLEDGMENTS

This study was supported by a gift from Tambrands, Inc., Palmer, Mass. The technical assistance of Jennifer Connolly is acknowledged with thanks. We also thank Barbara Cromer for excellent secretarial help. LITERATURE CITED 1. Beezhold, D. H., G. K. Best, P. F. Bonventre, and M. R. Thompson. 1989. Endotoxin enhancement of toxic-shock syndrome toxin-1 induced secretion of interleukin I by murine macrophages. Rev. Infect. Dis. ll(Suppl.):289-293. 2. Bergdoll, M. S., B. J. A. Crass, R. F. Reiser, R. N. Robbins, and J. P. Davis. 1981. A new staphylococcal enterotoxin, enterotoxin F, associated with toxic shock syndrome Staphylococcus aureus isolates. Lancet i:1017-1021. 3. Best, G. K., D. C. Scott, M. Kling, M. R. Thompson, L. E. Adinolfi, and P. F. Bonventre. 1988. Protection of rabbits in an infection model of toxic shock syndrome (TSS) by a TSS toxin-i-specific monoclonal antibody. Infect. Immun. 56:998999. 4. Blomster-Hautamaa, D., R. P. Novick, and P. M. Schlievert. 1986. Localization of biologic functions of toxic shock syndrome toxin-1 by use of monoclonal antibodies and cyanogen

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Mutants of staphylococcal toxic shock syndrome toxin 1: mitogenicity and recognition by a neutralizing monoclonal antibody.

Toxic shock syndrome toxin 1 (TSST-1), a 22-kilodalton protein made by strains of Staphylococcus aureus harboring the chromosomal toxin gene, may elic...
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