Download Regulation of Toxic Shock Syndrome Toxin

Mol. Cells, Vol. 7, No.1 , pp. 28-33
Regulation of Toxic Shock Syndrome Toxin-l Gene
in Staphylococcus aureus
lun-Hee W00 3,4 * , Yang Soo Kim 3,4 and Seung Duk Hwang l ,2
IDepartment of Internal Medicine and 2Hyonam Kidney Laboratory, Soon Chun Hyang University, Asan 337-880, Korea; 3Division of Infectious Diseases, Asan Medical Center and
4College of Medicine, University of Ulsan, Seoul 138-040, Korea
(Received on October 2, 1996)
Staphylococcus aureus produces various proteins in response to discrete signals from the
external environment like many other pathogenic microorganisms. Certain staphylococcal
exoproteins including toxic shock syndrome toxin-l (TSST-l) are secreted according to the
stimuli (rom the environment, and the quantity synthesized is influenced by a number of
different parameters. Using a transposon TnSSl-mediated mutagenesis, a mutanat (RN
6390) defective in TSST-l from synthesis was constructed. TSST-l from wild strain and
mutant strain were purified and quantitated from culture supernatants of Staphylococclls
aureus. The mutant strain RN 6390 produced only 2% of TSST-l compared with ,that produced by the wild strain RN4282. Southern blot hybridization with a tst (TSST-l gene)
probe indicated that the inactivated chromosomal locus is distinct from the tst. These
results suggest that transposition by TnSSl inactivated a chromosomal locus whose
activity was essential for the expression of the TSST-l gene.
Staphylococclls aureus is the etiology of diverse infectious diseases including pyogenic and non-pyogenic or toxin mediated illness. Toxic shock syndrome (TSS) is one of the non-pyogenic infections
and is caused by one or more toxins at the site of a
localized, often relatively asymptomatic or unnoticed
infection with any toxigenic strain of Staphylococcus
aureus. The most common etiology of TSS is toxic
shock syndrome toxin-1 (TSST-1), a protein with a
molecular mass of about 22,000 Da, secreted by S.
aureus, although recent data also implicate staphylococcal enterotoxin A (SEA) in less than 10% of
the TSS cases (Parsonnet and Kasper, 1992; Shin et
at., 1995).
Staphylococcus aureus synthesizes a number of extracellular proteins which play a major role in the
pathogenesis of staphylococcal disease. Mutations
which affect exoprotein production are often pleiotropic (Bjorklind and Arvidson, 1980; Cheung et al.,
1992; Coleman 1981; Smeltzer et at., 1993), suggesting that exoprotein genes may be coordinately regulated in some cases.
After eighteen years of enthusiastic research, we
now know much on TSST-l. Its structural gene (tst)
has been cloned and sequenced, many of its biological and physico-chemical properties have been
determined, and a bevy of immunostimulatory properties have been assigned to it (Kappler, 1989;
Kreiswirth, 1989; Marrack and Kappler, 1990; Todd
* To whom correspondence should be addressed.
et al., 1978; Woo et al., 1996b).
Recent advances in the understanding of bacterial
pathogenesis revealed that groups of virulence genes
are coordinately regulated in response to challenging
stimuli from the natural and artificial environments.
A bacterium may tailor its repertoire of products to
suit its needs in a given environment, the transition
from a natural reservoir to the complex internal milieu of a mammalian host, the transition from conditions of nutrient-excess to those of nutrient-depletion, and so on. There is a good example of this
phenomenon in Vibrio cholerae, in which cholera toxin as well as other proteins are under the control of
the ToxR locus (DiRita, 1992). The alterations of pH,
osmolarity, temperature, and the presence of certain
free amino acids are the factors that influence toxin
production.
Mallonee et al. (1982) previously reported that insertion of the erythromycin-resistance transposon Tn
551 into a chromosomal locus called hla virtually eliminated production of extracellular alpha hemolysin
by S. aureus ISP546.
With the development of recent cloning of several
exoprotein structural genes (Lofdahl et al., 1983;
Kehoe et at. , 1983; Kleckner, 1977; Pattee, 1981;
Shortie, 1983), it has become possible to explain the
The abbreviations used are: BHI, brain heart infusion; Cm,
chloramphenicol; ELISA, enzyme-linked immunosorbent assay; TSS, toxic shock syndrome; TSST-l , toxic shock syndrome toxin-I ; Tn , transposon ; UV, ultraviolet ray .
@ 1997 The Korean Society for Molecular Biology
Vol. 7 (1997)
Jun-Hee Woo et al.
mechanisms of regulation of exoprotein gene expression. We investigated the existence of a locus on
the S. aureus chromosome that controls the synthesis
of TSST-l.
Materials and Methods
Plasmids and strains
Plasmid pRD 11 00 was constructed as described previously by us CWoo et ai. , 1996a). In short, a tripartite
plasmid, pRDllOO, for the expression of TSST-1 in S.
au reus was assembl ed in several steps CWoo et al. ,
1996a). The PCR was used to create on SaLI-EcoRI tst
cloning cassette. Oligonucleotide primer's 5'-GGCCGTCGACTAAAGTCATATTTCACGG-3' and 5'-CCCGAATTCGCGTTATAAAGATAAAAGG-3' for TSST-1
were prepared.
S. aureus ISP546 (gift of J. Lee, Harvard University) is the original Tn551-induced mutant isolated
by Mall onee et al. (1982). S. aureus RN4282 is a naturally occurring toxic shock strain. S. aureus RN6390
is the Tn551-containing transformant mutant strain
made from RN4282. S. aureus RN4220 is the daughter strain of S. au reus NTCC8325 after UV radiation
nitrosoguanidine mutagenesis that contains neither the
TSST-l gene nor a plasmid (Table 1).
Transformation of S. aureus
Protoplast transformation of S. aureus was performed as described (Pattee, 1992). Transformants
were selected at 32 °C on BHI (Brain heart infusion)
agar containing 100 IJ.g of erythromycin per m!.
Insertional mutagenesis
Transformation was performed for the introduction
of Tn551 into RN4282. S. aureus ISP546, harboring
plasmid temperature-sensitive pRN3208 carrying Tn
551 with an erythromycin resistance determinant, was
plated at different cell concentrations on BHllerythromycin (100 lJ.g/ml) agar and incubated at 42 °C for 2
days as described (Kornblum et al., 1986; Pattee,
29
1992). Colonies were replica plated onto BHIICdN03
(0.25 mM) and incubated at 42 °C for 16 h to eliminate bacteria that had retained the whole plasmid.
Colonies that failed to grow in the presence of cadmium nitrate, which indicates the loss of pRN3208,
were selected. Each selected colony was grown in
BHI/erythromycin (100 1J.g/m1) at 32°C and analyzed
further phenotypically .
Immunoblot analysis of toxin
To identify TSST-1 , toxin samples from exponentially growing cells and stationary cells were assessed by SDS/PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) (14%), followed by
Coomassie staining and Western blotting. For Western blotting, at least 0.5 IJ.g of toxin from each
species was applied per lane. Immuno-reactive bands
were visualized by sequential incubation of the blots
with polyclonal leporine anti-TSST-1 antiserum
(Toxin Technology, Sarasota, FL, U.S.A.) and 1 IJ.Ci
25
of C I] protein A (Amersham Corp., Arlington
Heights, IL, U.S.A.), followed by autoradiography
overnight at - 70 °C using Kodak XRP-1 film
(Eastman Kodak, Rochester, NY, U.S.A.) and one
Cronex intensifying screen.
Culture and harvest
One hundred twenty-five ml of 5 X BHI broth
(BHI 185 gil in pyrogen-free water) was placed into
-40 em length of dialysis tubing (Spectra/Por 2,
molecular weight cutoff 2500, 54 mm diameter; Spectrum Medical Industries, Los Angeles, CA, U.S.A.).
Air was expelled, and the tube was knotted twice.
The tube was placed in a 1-1 Ehrlenrueyer flask along
with 312 ml of pyrogen-free water, and the flask and
contents were sterilized in an autoclave.
After the flask had cooled, 193 IJ.I of chloramphenicol (Cm) (34 mg/rul stock in 100% ethanol)
was added, and the mixture was allowed to equilibrate by diffusion. At steady-state, the medium was
therefore - 1.4 X BHI dialysate and 15 lJ.g/ml Cm.
The BHI-containing dialysis tubing "sausage" was
Table 1. Bacterial strains and plasmids
Strains
S. aureus
NTCC8325
ISP546
RN4282
RN4220
RN6390
Plasmids
pRN3208
pRDllOO
Relevant characteristics
8325 pig-l31 hla-316::Tn551
Original Tn551 mutant
A wild-type blood isolate, naturally
occurring toxic shock strain
From 8325 after UV treatment and
nitrosoguanidine mutagenesis
From RN4282 after Tn55 1 insertion
pI258 blaZ401 cad-52 seq-36
Staphylococcal shuttle plasmid consisted
of E. coli pUC19 with HindIII deleted,
SaLI-Eco RI (stH, and B. subtiltis pBD64
Sources
Gift of Jean Lee
Mallonee et aI. , 1982
Pattee, 1992
Novick et al., 1979
This study
Kornblum et aI. , 1986
Woo e ( al., 1996a
30
Regulation of TSST-l in S. aureus
left in place. Two ml of an overnight seed culture of
S. aureus, either wild strain RN4282 or mutant strain
RN6390, was inoculated into 300 ml of culture
media (1.4 x BHI dialysate and 15 j.1g/ml Cm) at 37
·C , 100 rpm for 7 h used as exponentially growing
cells, and inoculated for -20 h until saturation as stationary cells.
Purification of TSST-I
TSST -1 from wild type or mutant strain was prepared from culture supernatants of S. aureus grown
in BHI dialysate, and toxin was purified by a combination of ultrafiltration and dye (Red A) affinity
chromatography, as follows:
The cells at exponential and stationary phases were
removed by centrifugation, and the supernatant fluid
was loaded on a 200 ml Red A column (Amicon,
Beverly MA, U.S.A) which had been preequilibrated
with 20 mM-phosphate buffer, pH 6.5 . The column
was washed with six to eight volumes of equilibration buffer followed by elution with the same
volume of 60 mM-potassium phosphate buffer, pH 6.5
(Passalacqua et ai., 1992). Toxin was eluted at room
temperature with 800 ml of 150 mM potassium phosphate buffer, pH 6.5. The elution was monitored by
UV absorbance at 280 nm. The fractions containing
TSST-1 were washed extensively with pyrogen-free
water, and concentrated across a YM-lO ultrafiltration
membrane. Between purification of TSST-l from different S. aureus strains RN4282 and RN6390, the
Red A column was purged with 8 M urea.
Quantitation of TSST-I
For the determination of the toxin content of crude
samples, we used the competitive enzyme-linked immunosorbent assay for TSST-1 (TSST-1 ELISA) as
described previously (Parsonnet et al. , 1985). TSST-1
samples of a known concentration for the standard
CUI yes
were purchased from Toxin Technology
(Sarasota, FL, U.S.A). All determinations were performed at least twice, and the results were averaged.
Southern blot analysis
To test whether Tn551 was inserted into the TSST-1
structural gene, chromosomal DNA from wild-type
and mutant cells was analyzed by Southern blot hybridization.
For hybridization analysis by Southern blotting,
whole-cell DNA was isolated from the mutant strain
and a wild type by a modification of the method of
Sambrook et al. (1989). The procedure was adapted
for S. aureus by substituting lysostaphin (Sigma, U.S.
A) (final concentration, 500 j.1g/ml) for lysozyme.
DNA preparations were digested to completion with
restriction enzyme Clal, electrphoresed in a 0.9%
agarose gel. After pre-hybridization, DNA on the nitrocellulose membrane was allowed to hybridize with
a radiolabeled 32P-labeled 297 bp BamHI-HindII tstspecific DNA fragment (Blomster-Hautamaa et al.,
Mol. Cells
1986) at 65 ·C overnight, washed twice with 2 x
SSPE with 0.1% SDS at room temperature for 10
min each and once with 1 x SSPE (1 x SSPE is 0.15
M NaCl, 0.015 M sodium phosphate, pH 7.4, 1 mM
EDTA) with 0.1% SDS at 65 ·C for 15 min, and finally autoradiographed.
Expression of tst by the regulatory locus
To investigate the expression of the cloned tst,
transformation of S. aureus RN4220 with pRDllOO
was performed. For the quantitation of the toxin concentration of crude culture supernatants from S. aureus RN4220 with or without plasmid pRDllOO, we
used the competitive enzyme-linked immunosorbent
assay for TSST-l (TSST-l ELISA).
Results
Insertional mutagenesis of toxigenic S. aureus
Growing the transformed bacteria first at 32 ·C and
then at 42 ·C and selecting erythromycin-resistant
colonies yield colonies with Tn551 transposed to the
host chromosome. Transposon mutagenesis was initially unsuccessful. To overcome possible restriction
barriers and problems related to plasmid replication
in wild type S. aureus RN4282, transposition was performed using Tn551 first into RN4220, a S. aureus
mutant that is defective in one or more restriction systems. pRN3208 (RN4220-modified Tn551) was able
to transform wild-type RN4282. The presence of
pRN3208 (containing Tn551) in RN4282 was confirmed by restriction enzyme analysis of DNA The
resultant mutant is RN6390. The identity of this mutant, which was made from a single transposition,
was again confirmed by limited DNA sequencing.
Quantitation of TSST-I from wild type and mutant
strain
The TSST-l produced from culture supernatants of
exponential cells of S. aureus RN4282 was 0.6 j.1g/ml
in amount and the amount of TSST-l from culture supernatants of exponential cells of RN6390 was 0.7
j.1g/ml. However, TSST-l produced from culture supernatants of stationary cells of S. aureus RN4282
was 25.6 j.1g/ml and the amount of TSST-l from culture supernatants of stationary cells RN6390 was 0.6
j.1g/ml.
Immunoblot analysis of toxin from the mutant and
wild type
S. aureus wild type RN4282 and the mutant strain
RN6390 grow at similar rates in liquid medium and
exponential cultures have almost the same protein profiles as determined by SDS/PAGE with Coomassie
stain (Fig. 1).
However, analysis of stationary-phase culture
shows that the mutation results in a marked decrease
in the production of TSST-1 proteins. The difference
in expression of TSST-l from the wild type and the
31
Jun-Hee Woo et at.
Vol. 7 (1997)
1
2
1
Figure 1. Electrophoretic analysis of protein synthesized
by S. aureus RN4282 and RN6390. SDSIPAGE with
Coomassie staining showed the same protein profile from
lane 1, exponentially growing cells of RN4282 as from
lane 2, exponential cells of RN6390.
1
2
3
2
Figure 3. Southern blot analysis of DNA from lane 1, RN
4282 and lane 2, RN6390. The identity of the hybridization patterns indicates the gene was not the site of
the insertion .
and wild-type RN4282 using the probe 32P-Iabeled
297 bp BamHI-HindII tst-specific DNA fragment indicates that the site of the insertion was not inside the
tst gene (Fig. 3 ).
Expression of tst by the regulatory locus
The expression of the cloned TSST-l gene from S.
aureus RN4220 containing pRDllOO was determined
Figure 2. Immunoblot analysis of stationary phase culture
supernatants of RN4282 and RN6390. SDSIPAGE with immunoblot showed the mutation lane 2, stationary cells of
RN6390 resulted in a remarkable decrease in the production of TSST-1 compared with lane 3, stationary cells
of RN4282. Lane 1 is standard TSST-1 (from Tox Tech, FI,
U.S.A.)
by the help of the competitive enzyme-linked immunosorbent assay for TSST-1. The transformation
of S. aureus RN4220, a strain that contains neither
the TSST-l gene nor a plasmid, with the recombinant
plasmid pRDllOO containing the cloned tst resulted
in the production of 51.7 Ilg of toxin per mJ of culture supernatant of stationary cells. However, transformants derived from RN6390 produced 0.5 Ilg per ml
of supernatant, even though the recombinant plasmid
was stably maintained at an equivalent copy number
in the two strains. The results indicate that a regulatory locus is required for expression of the cloned TSST-l gene.
Discussion
mutant strain is clearly illustrated by an immunoblot
analysis using polyclonal leporine anti-TSST-l antibodies (Fig. 2).
Southern blot hybridization
In order to ensure that Tn551 had integrated into
the chromosome of RN4282, the mutant RN6390 and
the wild type RN4282 were analyzed by Southern
blot hybridization . The identity of the hybridization
patterns obtained with DNA from mutant RN6390
In S. aureus, the impact of discrete environmental
conditions on exoprotein expression has been known
for some time. Certain staphylococcal exoproteins are
synthesized in the post-exponential phase, and the expression is influenced by a number of parameters..
With the recognition of the toxic shock syndrome as
a toxin-mediated staphylococcal disease, and with appreciation of the strong association between risk of
menstrual TSS and the use of super-absorbent tam-
32
Regulation of TSST-l in S. aureus
pons, came the suggestion that tampon use somehow
changes the vaginal microenvironment and thereby
enhances production of TSST-l. Evidence was accumulated that synthetic fibers in those brands of tampons most strongly associated with an increased risk
of TSS were strong chela tors of divalent cations, and
that in response to the magnesium reduction so-produced, resident vaginal staphylococci augmented their
production of TSST-1 (Kass et al., 1987).
The first genetic attempt to study exoprotein regulation in staphylococci was initiated by the observation that a Tn551 transposon insertion, originally
isolated as a mutation affecting enterotoxin A production, actually had a pleiotropic, exoprotein-minus
phenotype (Mallonee et al., 1982). Insertion of the
erythromycin-resistance transposon Tn551 into a chromosomal locus called hla virtually eliminated production of extracellular alpha hemolysin by S. aureus
ISP 546.
The hla locus mapped between the purB and ilv
loci and was linked to determinants that affect the
synthesis of enterotoxin A and ~-lactamase .
Like many pathogenic bacteria, S. aureus exercises
precise regulatory control over the expression of its
virulence factors. Though incompletely defined, it is
likely that this control is due to the interaction of several different loci, and that one or more of them
responds to specific environmental cues. Our staphylococcal regulatory locus impacts on the level of
expression of TSST-1 , which seems responsible for
approximately eighty percent of the cases of TSS.
Insertion of Tn551 into the S. aureus chromosome
of strain RN4282, a wild type naturally occurring toxigenic strain isolated from a patient's blood, resulted
in the alteration of expression of TSST-1 (RN6390).
RN 4282 and RN6390 grow at similar rates in liquid
medium and exponential cultures have almost the
same protein profiles. The analysis of stationaryphase culture shows that the mutation results in a
marked decrease in the expression of TSST-1 (Fig. 2).
This difference must also be related to the Tn551 insertion and is consistent with the small production of
TSST-1 by the mutant strain.
Southern blot hybridization with the tst probe indicated that location of Tn551 insertion into mutant
RN6390 was distinct from tst. The identity of the hybridization patterns obtained with DNA from mutant
S. aureus and wild-type cells indicates that the site of
the insertion was not inside the TSST-1 gene (Fig. 3).
This suggests that Tn551 was probably inserted into
a locus involved in the regulation of TSST-l.
As well as genetic evidence, the phenotypic investigation of wild ~ RN4282 and mutant strain
RN6390 provided the same explanation. The expression of TSST-1 from a mutant strain was fiftyfold less than that from the wild type. Because the
overall patterns of protein produced and the chromosomal DNA of tst were not altered, RN6390 was
probably the mutant of the regulatory locus of the
Mol. Cells
chromosome.
In summary, a regulatory locus for the TSST-1
gene existed in th e staphylococcal chromosome and
appeared to be required for expression of the staphylococcal TSST-1 gene. The targets of regulation
may be contained within transposable genetic elements. The marked reduction in the production of
TSST-1 in place of no production in mutant cells
may raise another possible regulatory element. Recent
evidence implies the probable existence of at least
another regulatory locus. The fact that TSST-1 is produced only in a stationary phase suggests that this regulatory locus may regulate genes for accessory proteins. The present study investigated the identification
of the genes responsible for TSST-1 regulation and
hopes to define and clone this regulatory locus. Experiments will be in progress to characterize the structure and mechanism of the action of this locus.
Acknowledgments
This work was supported by grants from the Genetic Engineering Research Fund of the Korean Ministry
of Education to I.-H. Woo and partly from the Hyonam Kidney Fund, Soon Chun Hyang University .
We deeply appreciate the help of Dr. Shin Yung
Kee and Ms. Eun Sun Rha.
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