Staphylococcal Scarlet Fever: Role of Pyrogenic Exotoxins
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INFECTION AND IMMUNITY, Feb. 1981, p. 732-736 Vol. 31, No. 2
0019-9567/81/020732-05$02.00/0
Staphylococcal Scarlet Fever: Role of Pyrogenic Exotoxins
PATRICK M. SCHLIEVERT
Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, California 90024
Staphylococcal pyrogenic exotoxin (PE) types A and B were tested for their
role in production of a scarlatiniform rash. The PEs elicited minimal skin reactions
after intracutaneous injection into animals not presensitized to the toxins. In
contrast, erythematous and edematous rashes were produced after administration
of either PE to animals presensitized to homologous toxin. After 3 to 4 days, the
erythematous areas showed membranous desquamation. Staphylococcal PEs also
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enhanced delayed and Arthus hypersensitivity skin reactions developed against
unrelated proteins; the reactions subsequently desquamated. In addition, animals
previously sensitized to either staphylococcal PE type developed scarlatiniform
rashes after challenge with heterologous staphylococcal or any group A strepto-
coccal PE. The data suggest that staphylococcal PEs produce scarlet fever-like
rashes comparable to group A streptococcal PEs and that all PE types contain a
common core moiety against which delayed hypersensitivity may be developed.
Syndromes closely related to classical group all strains of S. aureus obtained from patients
A streptococcal scarlet fever have resulted from with either syndrome elaborated PEs, and the
infection with Staphylococcus aureus. Stevens PEs have been used in experimental animals to
describes three individuals with staphylococcal produce many manifestations ofthe diseases (17;
scarlet fever, none of whom had a streptococcal Schlievert, in press).
infection (20). Other investigators described The purpose of this investigation was to assess
scarlet fever in patients with staphylococcal os- the role of staphylococcal PEs in production of
teomyelitis (1), wrist infection (6), or leg abscess the scarlatiniform rash. In addition, PEs from
(10). In two instances, injection of culture super- both S. aureus and group A streptococci were
natant fluids of the corresponding S. aureus tested for the presence of the shared common
strain into Dick-positive persons resulted in core moiety.
scarlatiniform rashes (1, 10).
Previously, it was suggested that group A MATERIALS AND METHODS
streptococcal pyrogenic exotoxins (PEs) produce All reagents and glassware used for preparation of
the classical scarlet fever rash by enhancing PEs and biological assays were maintained pyrogen
acquired skin reactivity to streptococcal anti- free.
gens (15). This included enhancement of hyper- Bacteria. Staphylococcal PEs were obtained from
sensitivity skin reactions developed against the the Hanisburg strain of S. aureus (17). Group A
"common core" moiety shared by all three strep- streptococcal PEs were prepared after culture of
tococcal PE types (3, 8, 9, 15). Based on these strains 594, T18P, and T19P (16). Microorganisms
data, it was concluded that erythrogenic toxin were maintained in the lyophilized state in the pres-
represents the enhancement of hypersensitivity ence of whole defibrinated fresh rabbit blood.
reactions by PEs (15). Toxin preparation. PEs were prepared after cul-
ture of the organisms in a dialyzable beef heart me-
Recently, two serologically distinct staphylo- dium (22). PEs were purified by differential precipi-
coccal PEs were purified and characterized bio- tation with ethanol and resolubilization in either ace-
chemically and biologically (17; P. M. Schlievert, tate-buffere saline (8) or distilled water (17), followed
Biochemistry, in press). These toxins share by preparative thin-layer isoelectric focusing (14).
many important properties with the group A Toxin concentrations were measured by the microbi-
streptococcal PEs (2, 3, 5, 12, 13, 17-19) and may uret protein assay (23).
therefore contribute to the development of Animals. Darkly pigmented rabbits weighed 2.0 to
staphylococcal scarlet fever. In addition, it was 2.5 kg.
proposed that the staphylococcal toxins may Assay for pyrogenicity. Assays for the capacity
of PEs to produce fever were performed by the 4-h
contribute to the development of two severe method of Kim and Watson (8, 22). The dose of PEs
scarlet fever-like syndromes, Kawasaki disease used in tests for pyrogenicity was 20 MPD-4/kg and
and toxic shock (P. M. Schlevert, M. Chai, M. represented 20 jg of protein per kg of staphylococcal
T. Osterholm, and D. W. Watson, submitted for PE A and 90 ,Lg per kg of type B toxin. (MPD-4
publication). The basis for the proposal was that equaled the dose of PE required to produce a 0.5°C
732VOL. 31, 1981 STAPHYLOCOCCAL PEs 733
average fever response in five rabbits 4 h after intra- thematous and edematous skin reactions after
venous injection.) intracutaneous challenge with homologous toxin
Immunizations. Animals were sensitized to puri- (Table 1). The reaction diameters obtained in
fied protein derivative (PPD) by subcutaneous injec- these animals were different from those obtained
tion of 1 ml of an equal parts mixture of phosphate-
buffered saline (0.005 M sodium phosphate, 0.15 'M in nonsensitized animals at the P < 0.001 level
NaCl, pH 7.0) and complete adjuvant containing heat- of significance. The skin reactions in the sensi-
killed Mycobacterium tuberculosis (Difco Laborato- tized animals were comparable in appearance to
ries, Detroit, Mich.). After 2 weeks, animals showed the enhanced reactivity obtained in a previous
an average skin test response of 24-mm diameter 24 h study in which group A streptococcal PEs were
after intracutaneous challenge with 250 test units of used as sensitizing and challenge toxins (15). At
PPD (Mantoux; Connaught Laboratories, Ltd., Wil- 3 to 4 days after challenge with the staphylococ-
lowdale, Ontario, Canada). cal toxins, membranous desquamation around
Rabbits were sensitized to staphylococcal PEs by the challenge sites was observed.
subcutaneous injection of 200 ug of toxin emulsified in When the rabbits were resensitized with
1 ml of incomplete adjuvant (Difco Laboratories).
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Sensitization of rabbits to bovine serum albumin staphylococcal PEs and then challenged again
(BSA) was accomplished by administering 1 mg of with homologous toxin, the average skin reaction
protein subcutaneously emulsified in incomplete ad- of some of the animals was diminished (Table
juvant. Rabbits were considered sensitized when small 2). This reduction in skin reactivity in these
local Arthus reactions were elicited after intracutane- rabbits corresponded to the development of im-
ous challenge with 350 jig of BSA. munity to the pyrogenicity of the PEs (Fig. 1).
Skin testing. Rabbits were skin tested with antigen The remaining animals again showed significant
injected intracutaneously, 0.1 ml per animal in phos- skin reactivity (Table 2) and also were not im-
phate-buffered saline. Test doses were: PEs, 50 tag; mune to the pyrogenicity of the challenge PE
PPD, 250 test units; BSA, 350 jLg. Student's t test
analysis of paired or unpaired normally distributed (Fig. 1).
data was used to assess differences between means of Staphylococcal PEs were tested for capacity
control and experimental skin reaction diameters. to enhance hypersensitivity skin reactions de-
veloped against unrelated proteins in rabbits.
RESULTS Animals presensitized to PPD showed signifi-
Initial studies were done to assess the capacity cant enhanced skin reactivity on one flank when
of staphylococcal PEs to induce the scarlatini- challenged with PPD plus PE (Table 3), com-
form rash in rabbits without prior sensitization pared to the other flank which received PPD
to the toxins (Table 1). Slight skin reactions alone. Exotoxin alone given to rabbits presensi-
were obtained which were pink and somewhat tized to PPD elicited skin reactions comparable
edematous, whether the challenge toxin was to those shown in Table 1 and were subtracted
staphylococcal PE type A or B. The weak reac- from values obtained after challenge with PPD
tivity may reflect prior infection with S. aureus. plus PE.
Animals which were presensitized to either Staphylococcal PEs also enhanced Arthus
staphylococcal PE type showed significant ery- skin reactions to BSA (Table 4). Animals ac-
tively immunized against BSA showed signifi-
cantly enhanced skin reactivity when challenged
TABLE 1. Skin test reactivity of rabbits with and on one flank with BSA plus PEs compared to
without prior sensitization to PEs challenge with BSA alone. Exotoxins alone ad-
PE type Skin re-
action TABLE 2. Skin reactivity of rabbits presensitized
No. of diam twice to PEs and challenged with homologous toxin
rabbits (mm) ±
Sensitizinga Challengeb tested SEc No. of rab- Skin reaction
after 24 PE type- bits tested diam (mm) ± SE
h after 24 h
_d Staphylococcal A 5 5±4 Staphylococcal A 2 57 ± 2
Staphylococcal A Staphylococcal A 5 54 ± 4 Staphylococcal A 3 19 ± 4
- Staphylococcal B 5 7±6 Staphylococcal B 1 45
Staphylococcal B Staphylococcal B 5 43 ± 3 Staphylococcal B 4 17 ± 3
-
Anfimals were presensitized with staphylococcal PEs (200
jig) emulsified in incomplete adjuvant and givenPE. subcutane-
a
gsg
Dose, 50 administered intracutaneously in 0.1
ml of phosphate-buffered saline. Animals were presen-
ously 2 weeks before challenge with homologous sitized with staphylococcal exotoxins (200 iLg/injec-
I
Dose of challenge PE, 50 itg administered intracutane-
ously in 0.1 ml of phosphate-buffered saline. tion), emulsified in incomplete adjuvant and given
'SE, Standard error. subcutaneously 2 and 4 weeks before challenge with
, None. homologous PE.734 SCHLIEVERT INFECT. IMMUN.
lococcal PE type showed significant erythema-
No./Group tous and edematous skin reactions after chal-
U
lenge with heterologous staphylococcal or group
2 A streptococcal PEs. After 3 to 4 days, membra-
U 1.5/ nous desquamation of the reaction sites was
observed. The data indicate that delayed hyper-
sensitivity raised against one PE type conferred
w
1.0 / hypersensitivity on all other PEs thus far iden-
a- tified.
w
DISCUSSION
0.5 The development of the scarlatiniform rash
3 was previously shown to result from enhance-
ment of acquired hypersensitivity skin reactions
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4 by PEs of group A streptococci (8, 9, 15). Thus,
0 1 2 3 4
HOURS TABLE 4. Skin reactivity of rabbits to BSA
FIG. 1. Pyrogenicity of staphylococcal. PEs in rab- enhanced by PEs
bits preimmunized against homologous t4 oxin. Intra-
venous challenge doses of PEs were: A (C), 0), 20 pg/ No. of Skin reaction
kg; B (5, *), 90 pg/kg. Number of rabbfits in each Skin test material rabbits ± SE after
ate
group is indicated on the right side of the? figure. tested 24h
BSAa 5 24 ± 3
TABLE 3. Skin reactivity of rabbits t LoPPD BSAa + staphylococcal Ab 5 46 ± 3C
enhanced by PEs BSAa + staphylococcal Rb 5 40 ± 4C
Skin re- a
BSA concentration, 350 ,ig.
No. of action b PE dose, 50 jig.
Skin teist material rabbits dia Sinfi
tested (mm) ± cance
(P)C c Skin reactivity values obtained when PE was ad-
SE after ministered to normal rabbits were subtracted from
24h values shown.
PPD (250TU)b 5 15 ± 3 PVOL. 31, 1981 STAPHYLOCOCCAL PEs 735
erythrogenic toxin and a positive Dick reaction then monthly for a total of 7 months. None of
were defined in terms of two factors: (i) PE in the animals survived longer than the 7-month
the absence of neutralizing antibody, and (ii) period. The underlying cause of lack of specific
pre-existing delayed or Arthus hypersensitivity responsiveness is not known, but the phenome-
developed against a streptococcal antigen which non may explain why approximately 25% of pa-
was coadministered with PE. Further, PEs were tients with toxic shock syndrome have recurrent
shown to contain a shared common core moiety, episodes (4) and continue to manifest the scar-
against which delayed hypersensitivity can be latiniform rash. Studies are presently under way
developed, as well as serologically specific pri- to assess immune responsiveness in individuals
mary toxic components, which are responsible with one, two to three, or numerous episodes of
for enhancing skin reactivity (8, 9, 15). There- toxic shock syndrome.
fore, once delayed hypersensitivity was devel- ACKNOWLEDGMENTS
oped against one PE type, the scarlet fever rash This investigation was supported by Public Health Service
could be produced by any streptococcal PE type research grant AI-16691 from the National Institute of Allergy
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without further sensitization. and Infectious Diseases.
In this study, data were presented which in- The technical assistance of Heinrich Kolbel is gratefully
dicate that staphylococcal PEs also produce er- acknowledged.
ythematous and edematous rashes by the en- LITERATURE CITED
hancement of acquired hypersensitivity. This 1. Aranow, H., Jr., and W. B. Wood, Jr. 1942. Staphylo-
property is consistent with the PEs' being re- coccal infection simulating scarlet fever. J. Am. Med.
sponsible for the clinical signs of staphylococcal Assoc. 119:1491-1495.
scarlet fever and supports a recent proposal that 2. Barsumian, E. L., C. M. Cunningham, P. M. Schliev-
the staphylococcal PEs may contribute to the ert, and D. W. Watson. 1978. Heterogeneity of group
A streptococcal pyrogenic exotoxin type B. Infect. Im-
development of Kawasaki disease and toxic mun. 20:512-518.
shock syndrome (Schlievert et al., submitted for 3. Barsumian, E. L, P. M. Schlievert, and D. W. Wat-
publication), both of which must be distin- son. 1978. Nonspecific and specific immunological mi-
guished from classical streptococcal scarlet fever togenicity by group A streptococcal pyrogenic exotox-
ins. Infect. Immun. 22:681-688.
(4, 7, 11, 21). Also, data were presented which 4. Center for Disease Control. 1980. Toxic-shock syn-
demonstrated that all PEs, whether streptococ- drome-United States. Morbid. Mortal. Weekly Rep.
cal or staphylococcal, shared the common core 29:229-230.
component of the toxins. This observation is 5. Cunningham, C. M., and D. W. Watson. 1978. Sup-
pression of the antibody response by group A strepto-
significant since it suggests that scarlet fever or coccal pyrogenic exotoxin and characterization of the
related diseases may be produced up to five celis involved. Infect. Immun. 19:470-476.
times, twice by S. aureus and three times by 6. Feldman, C. A. 1962. Staphylococcal scarlet fever. N.
group A streptococci, and that prior sensitization Engl. J. Med. 267:877-878.
7. Kawasakri, R., F. Kosaki, S. Okawa, I. Shigematsu,
with one PE type is sufficient for development and H. Yanagawa 1974. A new infantile acute febrile
of the scarlatiniform rash after the host encoun- mucocutaneous lymph node syndrome (MLNS) pre-
ters either the homologous or any heterologous vailing in Japan. Pediatrics 54:271-276.
PE. The shared common core of all PE types 8. Kim, Y. B., and D. W. Watson. 1970. A purified group
A streptococcal pyrogenic exotoxin. Physicochemical
also is consistent with reports that Dick-positive and biological properties including the enhancement of
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It is noteworthy that few individuals who de- 9. Kim, Y. B., and D. W. Watson. 1972. Streptococcal
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