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Departments of
*
Microbiology and
Biochemistry, University of Minnesota Medical School, Minneapolis, MN 55455; and
Wyeth Vaccines, West Henrietta, NY 14586.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both group A streptococci and Staphylococcus aureus produce potent exotoxins that belong to the family of pyrogenic toxin superantigens (PTSAgs) (10). PTSAgs simultaneously bind to class II MHC molecules and to the Vß chain of the TCR outside of the conventional peptide groove (11). Through this interaction, these potent toxins subvert the normal immune response by forming a TCR-superantigen-MHC ternary complex (12) that stimulates T cells bearing the appropriate Vß region (13). Because there are a limited number of Vßs and various superantigens recognize a subset of these (e.g., SPE C binds primarily to Vß2; (13)), as many as 20% of all T cells can be stimulated. This results in extensive T cell proliferation with the consequent massive release of cytokines (14, 15, 16, 17, 18) which are believed to cause the most severe features of the illness.
The streptococcal pyrogenic exotoxins (SPEs), also known as erythrogenic toxins or scarlet fever toxins, include the serologically distinct types A, B (cysteine protease), C, F (DNase), G, and H, as well as streptococcal superantigen (19) and streptococcal mitogenic exotoxin Z (SMEZ) (20). Except for SPE B and SPE F, all of the other characterized PTSAgs are believed to share a similar three-dimensional structure (21, 22, 23, 24, 25, 26). SPEs have been implicated as causative agents of STSS (2) and are responsible for the fever, rash, and severe clinical manifestations associated with scarlet fever. In particular, SPE A and SPE C are highly associated with severe disease (27), although this association is not 100%. For example, in a study that evaluated 16 consecutive cases of severe invasive streptococcal disease, of 9 patients who developed STSS, 8 died. Of the 16 isolates, 15 made either SPE A or SPE C, and the 1 isolate that did not make either PTSAg came from a patient with borderline invasive disease (erysipelas) (4). Production of recently characterized exotoxins (28) similar to SPE A and SPE C may account for cases of STSS that are not associated with these two toxins. However, PTSAgs are clearly not solely responsible for invasive streptococcal disease. Cysteine protease (29) is invariably present in all clinical isolates and has been shown to be necessary for disease in a mouse model of soft tissue infection (30), and other factors are likely to be involved.
Epidemiologically, SPE C is not as well characterized as SPE A, primarily due to the higher association of SPE A with invasive disease and the poor immunogenicity of SPE C. SPE C is believed to exist as a dimer although the MHC binding site normally seen with other PTSAgs (31, 32) is thought to be contained within the dimer interface (13, 33). A second MHC binding site present on SPE C, analogous to the MHC binding site of staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin E (SEE) (34), is thought to bind through zinc (33). Due to the dimerization of SPE C, this may represent a novel mechanism of PTSAg-mediated T cell stimulation where dimerized toxin can cross link two TCR and two MHC class II molecules simultaneously (13). Based on mutational analysis of staphylococcal enterotoxin B (SEB) that indicated the N terminus was critical for T cell activation (35), Yamaoka et al. (36) randomly mutated 14 residues at the N terminus of SPE C in an attempt to localize residues important for activity. That study indicated that residues Tyr15, Ala16, and Tyr17 were likely involved in TCR recognition, although the TCR binding domain of SPE C has yet to be completely mapped.
STSS and other invasive streptococcal disease often occurs in patients with no underlying disease. Along these lines, various groups have shown that a lack of protective immunity to the SPEs may contribute to host susceptibility (37, 38, 39). For example, patients with invasive disease had lower titers of anti-M1 and anti-streptococcal superantigen-neutralizing Abs compared with healthy controls (39). In another report, analysis of 132 clinical isolates from invasive episodes of streptococcal disease over a 12-year period revealed that although the isolates showed great genetic diversity, the speA gene was strongly associated with STSS (38). Furthermore, this study demonstrated a complete lack of SPE A-neutralizing Abs in patients with STSS and a low level of SPE A-neutralizing Abs in patients with uncomplicated bacteremia compared with uncomplicated erysipelas (38). These studies indicate that the lack of immunity to the PTSAgs is a strong risk factor for disease and that vaccine toxoids of the SPEs may protect against invasive disease and STSS.
Our research goals include the development of vaccine toxoids such that the immune system can target the causative toxin. In this study, we utilized the three-dimensional structure of SPE C (33) compared with other PTSAg structures, to predict residues important for binding to the TCR or class II MHC, or important for dimer formation. Single residues were mutated and assayed for the loss of mitogenic activity, and amino acids important for biological activity were altered to design double- and triple-site mutants of SPE C. These double- and triple-site mutants were evaluated for pyrogenicity, superantigenicity, toxicity, immunogenicity, and their ability to protect against SPE C-induced STSS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bacterial strains used in this study included Escherichia
coli XL1-blue (Stratagene, La Jolla, CA) and DH5
(Life
Technologies, Gaithersburg, MD) as cloning strains, and E.
coli BL21 (DE3) (Novagen, Madison, WI) was used for expression of
recombinant proteins. S. pyogenes T18P was used for
purification of wild-type SPE C in some experiments (40).
The plasmids used in this study included pMIN521 (41) as
template for the speC gene, and pET-28 (Novagen) was used as
the cloning vector for expression constructs. Kanamycin and ampicillin
were both used at 50 µg/ml for E. coli containing
appropriate plasmids. For cloning experiments, E. coli was
grown in Luria-Bertani broth (42). For expression of SPE C
molecules, E. coli and S. pyogenes were grown in
dialyzed bovine heart medium (43). Strains were stored at
-70°C in their appropriate broth supplemented with 20% glycerol
(v/v) or freeze dried.
Cloning and site-directed mutagenesis
To predict SPE C residues that were important for interaction
with the TCR or with MHC class II molecules, the SPE C structure
(33) was aligned with the structures of toxic shock
syndrome toxin 1 (TSST-1) (23, 25), SEB (21),
and SEC (26). Residues on SEC that are known to interact
with TCR (44) or residues on TSST-1 and SEB that are known
to interact with MHC class II (31, 32) were used to
predict amino acids on SPE C at similar positions. Residues
Asp12,
Tyr15, and
Tyr17 were predicted to be located close to
residues that interact with the TCR and residues
His35 and Asn38 were
predicted to be close to residues that interact with MHC class II
molecules. For the initial generation of single-site mutants, the
previously cloned wild-type speC gene (pUMN521) was used.
The inverse PCR Quik Change mutagenesis procedure (Stratagene) was used
to introduce specific codon changes to create the individual mutants
D12A, Y15A, Y17A, H35A, and N38D. With this strategy, mutant
oligonucleotide primers (Table I
) were
used to replicate both strands of the entire plasmid using the high
fidelity Pfu polymerase, while incorporating the desired
mutation. The original template DNA was removed by digestion with the
restriction enzyme DpnI that digests only methylated DNA and
the remaining PCR products were transformed into E. coli
DH5. Each gene was sequenced to confirm that the correct mutation
was incorporated. The double mutant Y15A/N38D was also constructed in a
similar manner. To allow for more efficient expression, the Y15A/N38D
mutant was amplified by PCR using primers SPE C-forward
(5'-CCCATGGCAGACTCTAAGAAAGACATTTCGAATG-3') and SPE
C-reverse
(5'-CCCGGATCCTTATTTTTCAAGATAAATATCGAAATG-3') and
cloned into the NcoI and BamHI sites (underlined
in primers) of pET-28 resulting in pLP637. The triple mutant
Y15A/H35A/N38D was constructed from pLP637 to create pJKM79 using the
inverse PCR method with forward and reverse H35A/N38D primers. Due to
the close proximity of these two mutations, the primers used to add the
H35A mutation to pLP637 contained both H35A and N38D mutations. The
wild-type speC (45) gene was also amplified
from plasmid pUMN521 using primers SPE C-forward and SPE C-reverse and
cloned into pET-28 resulting in pJKM80. To allow for high level
production of the recombinant SPE C molecules, pLP637, pJKM79, and
pJKM80 were transformed into the expression host E. coli
BL21 (DE3).
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The Y15A/N38D and Y15A/H35A/N38D mutants were expressed from
E. coli BL21 (DE3) containing the appropriate recombinant
plasmid by growth in dialyzed bovine heart medium to
OD600 0.5 and induced with 0.2 mM isopropyl
ß-D-thiogalactoside. Cells were grown for an additional 3–4 h, and
recombinant toxin was precipitated and purified as previously described
(43, 46). Briefly, culture supernatants were precipitated
with 4 volumes of absolute ethanol. Precipitates were resolubilized in
pyrogen-free water, and purification was achieved by successive
preparative flatbed isoelectric focusing using pH gradients of 3.5–10
and 6–8. Wild-type SPE C was produced from S. pyogenes T18P
(40) or from E. coli BL21 (DE3) containing
pJKM80 as described above. Purified proteins were analyzed for purity
on SDS-10% polyacrylamide gels. Toxins were dialyzed extensively in
pyrogen-free water and stored lyophilized until used.
T cell proliferation assays
Isolated human PBMCs or rabbit spleen cells were cultured in RPMI medium supplemented with 2% FCS and L-glutamine (2 mM) at 2 x 105 cells/well. Serial 10-fold dilutions of purified wild-type or mutant SPE C proteins were added to the wells in quadruplicate. Plates were incubated in 7% CO2 at 37°C for 72 h, at which time each well received 1 µCi/well [3H]thymidine (Amersham, Arlington Heights, IL). After another 18 h, cells were harvested on to fiberglass filters (Whatman, Maidstone, England), and [3H]thymidine incorporation was assessed. Background was considered as counts from cells not treated with toxin (17).
Miniosmotic pump model of toxic shock syndrome (TSS)
The miniosmotic pump model of TSS was used to assess lethality of the various mutant toxins (47, 48). Miniosmotic pumps (Alza Pharmaceuticals, Palo Alto, CA) were preloaded with 500 µg wild-type or mutant toxin in PBS (pH 7.2). These devices are designed to release a constant amount of toxin over a period of 7 days to mimic exposure of the animal to toxin during an actual infection. American Dutch belted rabbits were anesthetized with ketamine and xylazine, and preloaded miniosmotic pumps were implanted s.c. via a small incision in the left flank and closed through primary intention. Animals were monitored for symptoms of TSS and mortality was recorded for 15 days.
Enhancement of endotoxin shock model of TSS
The PTSAgs also have the ability to sensitize rabbits to the lethal effects of endotoxin induced shock. To address this activity, American Dutch belted rabbits were pretreated with sublethal doses of wild-type or mutant SPE C (5 µg/kg body weight) administered i.v. by the marginal ear vein (43). After 4 h, animals were challenged i.v. with a sublethal dose (10 µg/kg) of endotoxin from Salmonella typhimurium. Animals were monitored for symptoms of TSS, and mortality was recorded for 2 days.
Immunogenicity of recombinant SPE C molecules
In 2 independent experiments, 10 healthy American Dutch belted rabbits were prebled to determine preimmune serum titers. In each experiment, five rabbits were immunized with 25 µg of either the Y15A/N38D or the Y15A/H35A/N38D SPE C mutant at 2-week intervals (total of 3 immunizations) in IFA. Rabbits were bled at wk 7, and convalescent anti-SPE C titers were determined by ELISA with use of 1 µg wild-type SPE C to coat each well. Also on wk 7, animals immunized with the Y15A/N38D mutant were challenged with wild-type SPE C using the endotoxin enhancement model of TSS, whereas animals immunized with the Y15A/H35A/N38D mutant were challenged with 500 µg wild-type SPE C using the miniosmotic pump model of TSS. As a control, three rabbits were also immunized similarly with wild-type SPE C and titers were determined.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and predictions of
binding sites for the TCR and MHC class II molecules. Mitogenic
activity of the single-site mutants was tested with use of crude
extracts from E. coli added to rabbit splenocytes. Residues
that had significantly reduced activities were used to generate the
double- and triple-site toxoids. The individual mutations were not
studied in detail except that crude extracts had reduced mitogenic
activity compared with wild-type SPE C. Structurally, we predicted five
solvent-exposed residues (Asp12,
Tyr15, Tyr17,
His35, and Asn38) to be
important for recognition of the TCR or the MHC class II or for dimer
formation. Preliminary studies revealed that D12A did not have reduced
activity. The Y17A SPE C mutant did have decreased mitogenic activity;
however, the single-site mutants Y15A, H35A, and N38D showed the
greatest reductions. Based on these activities, the double (Y15A/N38D)-
and triple-site (Y15A/H35A/N38D) mutants were generated and cloned into
the high level expression vector pET-28. The wild-type SPE C gene was
also cloned into this vector. After purification, 5 mg per liter
were routinely obtained from this system (Fig. 2).
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) or human PBMCs (Fig. 4), the activity of both multiple residue
toxoids was reduced to background levels compared with wild-type SPE C.
Recombinant wild-type SPE C and wild-type SPE C obtained from S.
pyogenes T18P behaved similarly in all assays (data not shown).
Both toxoids were nonpyrogenic compared with wild-type SPE C (Table II), further indicating that this
activity is linked to superantigenic activity. The lack of pyrogenic
activity of both toxoids also indicated that there were no significant
amounts of contaminating endotoxin in the preparations (this method has
a lower detection limit of 5 ng/ml endotoxin). Of critical importance,
rabbits challenged with either toxoid exhibited no signs of toxicity in
the two rabbit models of TSS (Table III).
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). Under the same
conditions, immunization with wild-type SPE C resulted in titers
similar to immunization with the SPE C toxoids (Table IV). Animals
immunized with the double-site mutant were challenged with wild-type
SPE C in the endotoxin enhancement model of TSS. Immune animals were
completely protected from this challenge, whereas all nonimmune animals
succumbed (Table IV). Animals immunized with the triple-site mutant
were challenged with wild-type SPE C in the miniosmotic model of TSS.
Again, all immune animals were protected from this challenge whereas
nonimmune animals succumbed (Table IV).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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helix and interact with
the TCR (44). Residues have been mutated in the N-terminal
region of SPE C to examine the role of superantigenicity in erythema
and found that mutants at positions Tyr15,
Ala16, and Tyr17 had
reduced mitogenicity and that erythema was likely attributable to T
cell-stimulatory activity (36). Based on structural
comparisons, our current data, and the previous mutagenesis results
(36), it was concluded that Tyr15 is
a critical residue for interaction with the TCR.
Interestingly, many of the residues analogous to the MHC class II
-chain-binding residues of SEA and SEB are buried in the SPE C dimer
interface (13, 33). Residues His35
and Asn38 from SPE C are analogous to residues in
the OB fold in TSST-1 and SEB, which are known to interact with MHC
class II (31, 32). On the basis of this structural data,
we predicted residue Asn38 to be important for
MHC interaction although the N38I mutant reported earlier did not show
altered activity (36). Although it is unlikely that a
single mutation would disrupt dimer formation completely, whether this
loss of activity depends on dimer formation or MHC class II interaction
is not yet known. It is not known what effect these mutations had to
the overall structure of the SPE C toxoids, although stability and
reactivity of these molecules to Ab did not appear to be altered.
Furthermore, the mutated residues were predicted to be solvent exposed,
and alanine substitution would be expected to remove the side chain
without altering conformation of the main chain or imposing any extreme
steric or electrostatic effects (50). In our previous
structure studies of TSST-1 mutants, changing such surface-exposed
residues to alanine did not affect the overall structure
(51).
SEA and SEE (34) as well as staphylococcal enterotoxin D (25, 49) all appear to have a second, higher affinity MHC class II binding site that is zinc dependent. With SPE C, MHC class II binding is believed to occur through a zinc binding site similar to that seen in SEA (24, 33). Mutational analysis within this region in SPE C has not been done but would be important to assess this region for MHC class II binding.
The SPEs were formerly known as scarlet fever or erythrogenic toxins because they were thought to be the causative agents of the scarlet fever rash. It is now known that the SPEs have a far more complex role in the pathogenesis of streptococcal disease. Purified SPEs cause STSS in rabbits, and various epidemiological studies have indicated that the SPEs are significantly associated with STSS (38, 52, 53). Streptococcal M1 and M3 serotypes cause the majority of invasive disease, and most M1 and M3 types produce SPE A. It has been shown that a widely disseminated SPE A-producing M3 clone has caused invasive disease in Ohio (54), North Carolina (55), Japan (56), and Minnesota (57) during 1994 and 1995. Musser et al. (9) also demonstrated that this same clone caused frequent cases of invasive disease before 1993. Our data further show that wild-type SPE C purified from group A streptococci or recombinant SPE C from E. coli produced highly lethal activity in the rabbit model.
In a study of soft tissue infection, recombinant SPE A purified from
Staphylococcus aureus was used to immunize rabbits followed
by s.c. challenge with SPE A-positive M1 or M3 streptococci
(58). Of the SPE A-immune animals, 16 of 19 survived
whereas only 4 of 20 nonimmune animals survived. Surviving animals
showed soft abscess formation with a high influx of PMNs whereas
nonimmune rabbits lacked inflammation. These authors proposed that
toxin neutralization prevented TNF- release due to
superantigenicity, which was believed to down-regulate chemotactic
receptors on PMNs to inhibit infiltration (59). Indeed, we
believe a major evolutionary role for the SPEs is to allow for
colonization by preventing infiltration of PMNs.
Other researchers have used a SPE A knockout M1 streptococcal strain and have argued that SPE A is not an important virulence factor in streptococcal fasciitis or myonecrosis (60). Using the SPE A-deficient strain, virulence was not diminished in a mouse model of bacteremia and myositis. Unfortunately, the mouse is not a good in vivo model because mice are highly resistant to the lethal effects of the PTSAgs. Furthermore, the strain with the disrupted speA allele retained nearly identical mitogenic activity compared with the wild-type strain, indicating that this strain produces another uncharacterized PTSAg.
Except for speB and speF, the presence of PTSAgs in Staphylococcus aureus and group A streptococci are variable traits. Not all group A streptococci produce either SPE A or SPE C, but expression of at least one of these two toxins is highly associated with invasive disease (27). SPE A is normally more highly associated with invasive disease, although this is not always the case. A study in a Memphis children’s hospital in the 1980s indicated that in this geographical region, the presence of the speC gene was more highly associated than the speA gene in clinical isolates from patients with bacteremia (27). Other novel streptococcal PTSAgs have been characterized including SMEZ and SMEZ-2 as well as SPE G, SPE H, and an incomplete gene for SPE J (28). If these novel PTSAgs are expressed at sufficient quantities, they could produce the same consequences as SPE A or SPE C.
In summary, it appears that the increased incidence and severity of streptococcal disease are likely due to both host factors and the changes in group A streptococci. Cases of noninvasive disease such as pharyngitis may serve as a reservoir for the dissemination of virulent strains to susceptible people (61). To reduce disease, regional surveillance of throat cultures may serve as an alternative to alert the medical community about the potential for invasive group A streptococcal illness (62). However, a functional vaccine may significantly reduce or eliminate severe invasive streptococcal disease and could also show benefits to less severe disease states such as pharyngitis, impetigo, or rheumatic fever. In this view, we have constructed a pentamutant of SPE A that is protective when used as an immunogen in rabbits challenged with wild-type SPE A (64). Here we have described the construction of two toxoid mutants of the SPE C superantigen that are protective against challenge with wild-type SPE C in two rabbit models of TSS. Both mutants were inactive for other biological activities such as fever induction and superantigenicity. Only five amino acids were initially targeted for mutagenesis, yet three of these mutations significantly altered superantigenic activity which demonstrated the capability of the initial structural predictions. The failure of the D12A mutation to show an effect on superantigenicity was not surprising in that although the various structurally characterized PTSAgs have common structures, they are not identical, and certainly these structural variations are responsible for difference biological activities. Collectively, these data indicate that these two toxoids may be useful for inclusion with other vaccine candidates to help protect against invasive infection, scarlet fever, and STSS by group A streptococci.
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Acknowledgments |
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, and Dinesha Walek
(MicroChemical Facility, University of Minnesota) for performing HPLC
purification. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Patrick Schlievert, Department of Microbiology, University of Minnesota Medical School, 420 Delaware Street SE, Minneapolis, Minnesota 55455.
3 Abbreviations used in this paper: STSS, streptococcal toxic shock syndrome; PTSAg, pyrogenic toxin superantigen; SPE C, streptococcal pyrogenic exotoxin C; SPEs, streptococcal pyrogenic exotoxins; SMEZ, streptococcal mitogen exotoxin Z; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B; SEC, staphylococcal enterotoxin C; SEE, staphylococcal enterotoxin E; TSST-1, toxic shock syndrome toxin 1; TSS, toxic shock syndrome.
Received for publication December 20, 1999. Accepted for publication June 6, 2000.
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chain in stabilizing TCR-superantigen- MHC class II complexes. Immunity 10:473.[Medline]
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