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* Department of Infectious Diseases, Faculty of Medicine, Imperial College School of Science, Technology, and Medicine, London, United Kingdom; and
Department of Molecular Medicine, School of Medicine, University of Auckland, Auckland, New Zealand
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, lymphotoxin-,
IFN-
, IL-1 and -8) from human cells, when cocultured with
streptococcal supernatants. The potent effects of SMEZ were apparent
even though transcription and expression of SMEZ were barely
detectable. Human V
8+ T cell proliferation in response
to S. pyogenes was SMEZ-dependent. Cells from HLA-DQ8
transgenic mice were 3 logs more sensitive to SMEZ-13 than cells from
HLA-DR1 transgenic or wild-type mice. In the mouse, SMEZ targeted the
human V8+ TCR homologue, murine V11, at the expense
of other TCR T cell subsets. Expression of SMEZ did not affect
bacterial clearance or survival from peritoneal streptococcal infection
in HLA-DQ8 mice, though effects of SMEZ on pharyngeal infection are
unknown. Infection did lead to a rise in V11+ T cells in
the spleen which was partly reversed by disruption of the
smez gene. Most strikingly, a clear rise in murine
V4+ cells was seen in mice infected with the
smez- mutant S.
pyogenes strain, indicating a potential role for SMEZ as a
repressor of cognate anti-streptococcal
responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The purpose of the current study was to address the contribution made by SMEZ, in a physiological context, to the immunological effects of GAS both in vitro and in vivo; the study was made possible by use of isogenic streptococcal strains which differred only in ability to produce SMEZ.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An M89 Streptococcus pyogenes clinical necrotizing fasciitis isolate (H293) with toxin genotype–streptococcal pyrogenic exotoxin (spe)a-, spec-, streptococcal superantigen (ssa)-, spei-, speg+, speh+, spej+, mitogenic factor (mf)+, smez+–was used in all experiments. Genotypes of strains were determined by PCR and sequencing confirmed that the strain carried the smez-13 allele as predicted by M serotype. M typing was performed by the Central Public Health Laboratory (Colindale, U.K.). Streptococci were cultured in Todd Hewitt broth with 0.2% yeast (THY) (Oxoid, Basingstoke, U.K.), or RPMI 1640 containing 10% FCS, 2 mM glutamine (Life Technologies, Paisley, U.K.), or on horse blood agar (Oxoid) at 37°C, aerobically. S. pyogenes strain H305 is an M1 serotype organism and was used to derive probes for smez.
Supercompetent Escherichia coli-SCS1 cells (Stratagene, La Jolla, CA) were grown in Luria Bertrani broth aerobically at 37°C. Kanamycin (Sigma-Aldrich, Poole, U.K.) was used at a concentration of 50 µg/ml for the selection of E. coli transformants. Erythromycin (Ery) lactobionate (Abbott Laboratories, Dublin, Ireland) was used at 1 µg/ml for streptococci.
Reagents
All chemical reagents were purchased from Sigma-Aldrich. Restriction enzymes were from Life Technologies. rSMEZ alleles were prepared as previously described (3); anti-SMEZ serum was raised in rabbits immunized with rSMEZ-1 and -2.
Transformations
A 331-bp internal fragment corresponding to bases 9–340 of the
630-bp smez-1 coding sequence was amplified from genomic DNA
extracted from H305 using primers smez1 and smez2 (Table I
). Primers were designed using the
published smez-2 sequence aligned to the M1 genome database,
to allow recognition between different smez alleles
(2, 4). The temperature-sensitive shuttle vector
pGhostaph1 was created to facilitate cloning in
E. coli by ligation of the kanamycin resistance gene
3'-aminoglycoside phosphotransferase type 1 (Aph1) from pUC4K to the
BamHI site of pGhost5+ (Appligene,
Illkirch, France). The 331-bp smez fragment was then
cloned into the EcoRI site of pGhostaph1 in
SCSI supercompetent E. coli. Restriction enzyme
analysis of plasmid DNA from one kanamycin-resistant transformant
revealed that three copies of the 330-bp smez fragment had
ligated to pGhostaph1; this plasmid was designated
pGhostsmez and was used to transform the S.
pyogenes strain, H293. pGhostsmez transformants were
cultured on Ery-containing THY agar plates at 30°C for 36–48 h;
pGhostsmez is replicative in Gram-positive bacteria at
30°C. Transformed colonies were then inoculated into THY broth (+Ery)
and cultured at 30°C for 24–36 h. The cultures were then transferred
to 37°C, and incubated for 12–14 h. At 37°C, the plasmid is
nonreplicative and may integrate into the chromosome at the
smez locus. Cultures were then streaked onto THY + Ery
plates. The replicative plasmid, pDL413, was used to control for
transformation efficiency as before (5); transformation
efficiencies of
103–104/µg plasmid DNA
were typically observed.
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Southern analysis was performed on XbaI-cut genomic DNA extracted from the parent strain H293 and an Ery-resistant transformant, H377, using a 331-bp digoxygenin (DIG)-labeled smez probe (amplicon prepared using primers smez1 and smez2). Blots were stripped and reprobed as described previously with a 673-bp DIG-labeled aph1 probe (corresponding to nucleotides 838-1531 of the pUC4K sequence, amplified using primers Aph1 and Aph2) (6). PCR analysis of parent strain H293 and mutant H377 was performed using the primers smezP (which corresponds to a nucleotide sequence 69-bp upstream of the coding sequence for smez on the M1 genome database) and smezR (which anneals to nucleotides 601–618 of the smez-2 gene) in addition to primer pair smez1/smezR.
Growth analysis of bacterial isolates
The ability of the parent strain H293 and mutant strain H377 to grow in both broth and whole blood was compared in standard conditions. Growth in THY was monitored over 12 h by measurement of OD600, while growth in heparinized murine blood was monitored over 3 h in a rolling whole blood opsonophagocytosis assay as described previously (5). Growth in blood was expressed as fold increase in CFU (i.e., total CFU after 3 h/CFU inoculated). Quantitation was performed by plating out dilutions onto blood agar plates.
RNA and Western analysis
Northern analysis of RNA obtained at mid-log, late-log, and
stationary growth phases from parent strain H293 and mutant strain H377
was performed as described previously using the DIG-labeled 330-bp
smez PCR product as a probe; control hybridizations were
performed using DIG-labeled probes for mf (or DNaseB)
(7). For RT-PCRs, 10 µg of streptococcal RNA, prepared
from each strain at each growth phase, was treated with DNase I,
Amplification Grade (Sigma-Aldrich) for 15 min at room temperature.
DNase I was inactivated by heating for 10 min at 65°C. This RNA was
used for reverse transcription (RT) using the Superscript II
Reverse Transcription kit (Life Technologies) as per the supplier’s
protocol, either in the presence or absence of reverse transcriptase (5
µg DNase I-treated RNA per tube). A single 20-µl RT reaction was
performed for each strain at each growth phase; 2 µl of each RT
reaction was then used as template DNA for amplification using primers
smez1/smezR, MF1/MF2, and SPEG1/SPEG2 (Table I
). Amplification (28
cycles) was performed using an annealing temperature of 55°C.
For Western analysis, 10 µl of cell-free THY bacterial culture supernatant was subjected to SDS-PAGE under reducing conditions, electroblotted onto nitrocellulose, and incubated with a 1/10,000 dilution of rabbit polyclonal anti-SMEZ serum, before development using the ECL system (Amersham Pharmacia Biotech, Piscataway, NJ). rSMEZ-13, 10 ng, was run as a positive control.
Preparation of human PBMC
Blood (20 ml) was drawn from healthy donors and diluted 1/2 in 0.9% saline. The diluted blood was overlaid on equal volumes of Ficoll-Paque (Amersham Pharmacia Biotech) and centrifuged at 800 x g for 35 min. The interface, rich in lymphocytes, was removed and washed once in HBSS at 800 x g for 10 min. The cells were finally resuspended in 10 ml of RPMI 1640 medium containing 10% FCS, 2 mM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin (Life Technologies).
Preparation of murine spleen cells
Spleens were dissected out and immediately immersed in 10 ml RPMI 1640 containing 0.05 mM 2-ME. They were then dissociated into single cell suspensions in sterile petriplates and passed through a 70-micron nylon cell strainer (BD Biosciences, Oxford, U.K.) to remove particulate matter. Cells were then resuspended in RPMI 1640 medium containing 10% FCS, 2 mM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin (Life Technologies) and 0.05 mM 2-ME.
Preparation of culture supernatants
Cell-free culture supernatants used for the PBMC or splenocyte stimulation assays were obtained from overnight cultures of H293 or H377 in RPMI 1640 containing 10% FCS, 2 mM glutamine without antibiotics. Bacterial counts of the H293 and H377 broths as estimated by viable counts were similar (H293 = 2.4 x 107 CFU/ml; H377 = 3.6 x 107 CFU/ml). Cultures were filter-sterilized and supernatants were stored at -20°C. No endotoxin could be detected in RPMI supernatants using the Limulus assay.
Proliferation and cell stimulation
Human PBMC (2 x 105 cells/well) or murine spleen cells (4 x 105 cells/well) in a volume of 200 µl, were stimulated with culture supernatants (diluted to 1% and 0.1%) or rSMEZ-13 (0.1–10,000 pg/ml) at 37°C, 5% CO2. Control stimulants included Con A (10 µg/ml), rSMEZ-1 (0.1–10,000 pg/ml), and staphylococcal enterotoxin B (SEB, 10–1000 pg/ml). Tritiated thymidine (1 µCurie) was added to each well after 60 h and cells were harvested at 72 h onto filter mats in a 1295-004 Betaplate 96-well cell harvester (Wallac and Berthold, Milton Keynes, U.K.). cpm were measured in a 1205 Betaplate liquid scintillation counter (Wallac and Berthold). For cytokine assays, human PBMCs were stimulated with a 1% bacterial supernatant or 0.1 ng/ml rSMEZ-13. Cell-free tissue culture supernatants from stimulated human PBMCs were collected and frozen at -20°C before cytokine analysis.
TCR-V repertoire analysis in vitro
Human PBMC (2 x 106 cell/ml) or
murine spleen cells (1–10 x 106 cell/ml)
were stimulated with streptococcal culture supernatants (1% dilution)
or rSMEZ-13 (10 ng/ml) for three days at 37°C in the presence of 5%
CO2 in 0.5 ml RPMI 1640. After 3 days, 0.5 ml
fresh RPMI was added to each well along with 20 ng/ml rIL-2 (R&D
Systems, Abingdon, U.K.), and incubated for a further 3 days. Human
PBMC or murine spleen cells were washed once and resuspended in 1x
PBS, 1% BSA (Sigma-Aldrich) buffer. Cells (4 x
105 to 1 x 106) were
stained with FITC or PE-labeled Ab and incubated for 45 min at 4°C in
the dark. Human cells were double-stained with PE-conjugated
anti-human TCR-V2, V8 and V12 (BD Biosciences) together
with either FITC-conjugated anti-human CD4 or anti-human CD8
(Sigma-Aldrich) and analyzed by flow cytometry. Murine spleen cells
were double-stained with PE-conjugated Abs specific to murine V2, 3,
4, 5.1, 7, 8.1, 10, 11, and 13 (BD Biosciences), along with
FITC-conjugated Abs to either murine CD4 or CD8 (Sigma-Aldrich). Cells
were washed thoroughly with 2 ml PBS-BSA buffer and used for FACS
analysis. FACS analysis was performed in a FACSCalibur (BD
Biosciences). Gated events (20,000) were counted for each sample.
Animals
C57BL/10.DQ8 transgenic mice carrying genomic constructs for DQA1*0301 and DQB*0302 and FVB/N.DR1 transgenic mice carrying genomic constructs for DRA1*0101 and DRB1*0101 have been previously described (8, 9). For in vitro comparison of responsiveness to SMEZ, C57/BL10.DQ8 and FVB/N.DR1 transgenic mice both were used on a matched syngeneic (C57BL/10 x FVB/N)F1 background. Transgenic mice were genotyped as previously described. Age and weight-matched HLA transgene-negative littermates were used as controls. Groups of transgenic mice used in in vivo experiments were age-, sex-, and weight-matched. All animals received food and water ad libitum. All animal procedures were conducted within local and home office ethical guidelines.
Immunological effects of SMEZ during infection
Parent strain H293 and mutant strain H377 were grown overnight in 100 ml THY at 37°C, in the absence of any antibiotic. Bacteria were washed once and resuspended in 3–4 ml of 0.9% saline such that the OD600 was identical between strains. Suspensions were adjusted to 109 CFU/ml. Bacterial suspension (0.2 ml) was administered i.p. to groups of C57BL/10.DQ8 transgenic mice identical in sex, age, and weight. To address whether SMEZ affected survival in this model of peritoneal infection, groups of nine C57BL/10.DQ8 transgenic mice were infected i.p. with either H293 or H377 and survival was monitored over 7 days.
To investigate the immunological effects of SMEZ during sepsis, groups of five C57BL/10.DQ8 mice were infected i.p. with either H293 or H377; 48 h after infection, blood was drawn by cardiac puncture for estimation of blood bacterial counts. In separate experiments, serum was obtained for cytokine estimation (48 and 60 h after infection) and SMEZ quantification (60 h only) by centrifugation of cardiac puncture blood samples at 5000 x g for 10 min. Peritoneal aspirates were also collected from killed infected mice for bacterial quantification (48 h), cytokine analysis (48 and 60 h), and SMEZ quantification (60 h only); 2 ml sterile saline was injected into the peritoneum, massaged for 1 min, and then 0.5–1 ml fluid was withdrawn from the peritoneal cavity. The aspirate was centrifuged at 5000 x g for 10 min and the supernatant was frozen at -20°C. Bacterial counts were quantified by plating of dilutions of serum or lavage fluid onto blood agar. The stability of the smez mutation in in vivo-passaged H377 bacteria was determined by analysis of DNA obtained from 25 separate streptococcal isolates subcultured from the peritoneal aspirates of H377-infected mice; isolates were initially cultured on blood agar without antibiotic, then replica-plated onto THY + Ery. Genomic DNA was isolated from each isolate and analyzed by PCR, using primer pairs smezP/smezR and smez1/smez2.
Proliferation bioassay for SMEZ production in vivo
Sera and peritoneal aspirates from H293- and H377-infected mice were coincubated at 1/10, 1/100, and 1/1000 dilutions with human PBMCs from a single donor in standard proliferation assays. Results were compared with parallel studies using rSMEZ-13 (0.1 fg/ml to 100 ng/ml) as the stimulant. The relationship between rSMEZ-13 concentration and proliferation was plotted and used to semiquantitatively assess the concentrations of SMEZ in serum and peritoneal aspirate.
TCR-V repertoire analysis in vivo
For analysis of the TCR-V T cell subsets expanded during
infection, groups of five C57BL/10.DQ8 transgenic mice were infected
i.p. with H293 (1.52 x 108 CFU/mouse) or
H377 (5.3 x 108 CFU/mouse). Mice were
killed 48 h after infection and spleens from groups of infected
transgenic mice were dissected out and dissociated into single cell
suspensions in RPMI 1640. Spleen cells were stained immediately with
Abs against murine V4, V8, or V11 and murine CD4 and the
proportion of stained cells was quantitated by flow cytometry as
described above.
Cytokine measurements
Both human and murine cytokine quantitation was done by ELISA, using paired Abs purchased from R&D Systems. Standard curves were set up with the respective recombinant cytokines (R&D Systems); all cytokine measurements were made within sensitivity ranges of individual assays.
Statistics
The Mann-Whitney U or ANOVA tests were used for
comparison between groups in all in vitro experiments. Values of
p 
0.05 were considered significant. Survival was
compared using the log rank test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A single Ery transformant was obtained after culture at 37°C and
this was designated strain H377. Southern analysis demonstrated a
difference between XbaI-cut genomic DNA from the parent
strain H293 and H377, when probed with smez (Fig. 1B). The single 2.7-kb
smez band seen in XbaI-cut genomic DNA from H293
was replaced by two bands of 5.5 and 6.5 kb in H377 DNA, confirming
that pGhostsmez had integrated at the smez locus
as shown in Fig. 1A, causing an insertional duplication of
the smez gene. All further work was performed using this
strain. Probing with the aph1 probe further confirmed the
insertion of the plasmid at the smez locus. Aph1
cohybridized with smez to the 6.5-kb band as expected (data
not shown).
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Analysis of bacterial growth in vitro
There was no difference between the growth rates of H293 and H377 when cultured in THY (not shown). Furthermore, the two strains did not differ significantly in their ability to grow in whole murine blood when cultured over 3 h at 37°C (H293: 31.2-fold increase, H377: 41.1-fold increase). In addition, there were no differences in DNase production, hemolysis, or capsule production when colonies were observed on solid medium.
Transcription and expression of smez is highly restricted in vitro
Northern analysis failed to detect a smez transcript
from either strain at any phase of growth. This was in contrast to
mf transcripts which were easily detected at late-log and
stationary phases in RNA from both strains (not shown). Transcripts for
smez could only be detected by RT-PCR, using primers smez1
and smezR (which anneal to the 5' and 3' ends of the smez
coding sequence). This yielded a faint, but distinct, 608-bp band in
H293 total RNA from the late-log phase of growth only, which was not
seen for H377 (Fig. 1C). Transcripts for speg and
mf did not differ between H293 and H377.
Although rSMEZ-13 yielded a clearly defined band, Western blotting using polyclonal anti-SMEZ antiserum failed to detect a specific SMEZ band in 10 µl supernatant samples from H293, even when supernatants were concentrated 10-fold.
SMEZ is the major mitogen secreted by S. pyogenes
H293 culture supernatants induced a dose-dependent mitogenic
response in human PBMC from three donors. The H377 culture supernatant
demonstrated a marked reduction in mitogenicity (Fig. 2A). Hence, the mitogenic
activity of H293 in human PBMC is largely attributable to
SMEZ.
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B). To
estimate the concentration range of SMEZ-13 produced in vitro in broth
culture by H293, the mitogenic activity of the
SMEZ+ culture supernatant was compared with that
of pure rSMEZ-13. It was concluded that the concentration of SMEZ in
H293 broth could be as low as 4–5 pg/ml.
SMEZ is responsible for S. pyogenes-induced
expansion of human V8-positive T cells in vitro
SMEZ is known to stimulate V8+ human
cells (1, 2, 10), though V8+
human T cell stimulation has also been attributed to a number of other
streptococcal products (11). To investigate the
contribution of SMEZ to the overall V8-stimulatory effect of GAS,
human PBMC were stimulated with culture supernatants from H293
(SMEZ+) and H377 (SMEZ-).
H293 supernatant induced expansion of V8+ both
in the CD4+and CD8+ T cell
populations (Table II). The increase in
V8+ T cells was eliminated when using H377
supernatant. This effect was specific to V8+ T
cells, as there were no differences observed in proportions of
V2+ or V12+ T cells
between H293- and H377-stimulated PBMC cultures (data not shown).
Furthermore, there were no differences observed in proportions of
V2+ or V12+ T cells
between supernatant-stimulated PBMC cultures and cultures incubated in
medium alone.
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Human PBMC from three donors were stimulated with H293, H377,
rSMEZ-13 or medium alone for 6, 24, 48, and 72 h and the
production of IFN-, TNF-, TNF-, IL-1, and IL-8 was measured
by ELISA. There was a time-dependent increase in production of all the
cytokines, except TNF-, up to 72 h from H293-stimulated PBMCs.
For TNF-, there was an increase up to 48 h, followed by a
subsequent decrease. For all the cytokines studied, H293 supernatant
stimulated production of significant levels of cytokines compared with
both the H377 and medium controls (Fig. 3, left panels).
Surprisingly, cytokine release from PBMCs simulated with supernatant
from H377 was no different to cells incubated with medium alone. rSMEZ
induced a time-dependent increase in production for all cytokines,
similar to that observed for the culture supernatants, except for
IFN-, which decreased after a 48-h incubation (Fig. 3, right
panels).
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To study the immunological effects of SMEZ in vivo, it was
essential to establish the murine cell types that were most sensitive
to SMEZ by examination of the mitogenic activity of rSMEZ-13 in vitro.
HLA-DQ8 transgenic murine spleen cells were at least 3 logs more
sensitive to rSMEZ-13 than wild-type murine cells, yielding a
detectable mitogenic response even at 0.1 pg/ml SMEZ (Fig. 4A). Spleen cells from HLA-DR1
transgenic mice were less sensitive to SMEZ than HLA-DQ8 cells
(requiring 10 pg/ml of rSMEZ-13 for proliferation); these marked
differences persisted even when cells were coincubated with rSMEZ-1
instead of rSMEZ-13 (data not shown). In contrast, HLA-DQ8 and HLA-DR1
transgenic spleen cells demonstrated no significant differences in
responsiveness to the staphylococcal superantigen SEB. SEB
concentrations of 100 pg/ml produced significant proliferation in
transgenic cells, while wild-type cells required 3 logs more SEB to
proliferate (not shown). Overall, spleen cells from HLA-DQ8 transgenic
mice were more sensitive to rSMEZ-13 than HLA-DR1 transgenic or
wild-type murine spleen cells.
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B). Hence, the mitogenic activity of H293
was specifically detectable in murine cells expressing HLA-DQ8 and the
activity was wholly attributable to SMEZ. Therefore, all further murine
experiments were performed using C57BL/10.DQ8 mice.
SMEZ-13 specifically expands the murine T cell V11+
subset; V11+ T cell expansion in S.
pyogenes-stimulated murine spleen cells is wholly attributable
to SMEZ
To study the murine V-T cell subsets expanded by SMEZ-13,
spleen cells from C57BL/10.DQ8 mice were stimulated with pure rSMEZ-13
(10 ng/ml). Significant expansion of V11+
CD4+ T cells was seen, while there was a marked
reduction in all the other V-bearing T cell subsets studied,
compared with unstimulated controls (Table III).
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-T cell subsets stimulated in vitro with H293 and H377
culture supernatants were also studied. Again, there was a significant
expansion of the V11+ T cells, and a marked
reduction in all the other V-bearing T cell subsets in the
H293-stimulated splenocytes compared with H377-stimulated and
-unstimulated controls (Table III). Surprisingly, spleen cells
stimulated with H377 supernatant did not show any change in proportions
of V-T cell subsets compared with the unstimulated control,
consistent with the lack of mitogenicity of H377 in murine spleen cell
culture. Role of SMEZ in peritoneal streptococcal infection
Peritoneal bacterial clearance 48 h after the onset of i.p.
infection did not differ between mice infected with H293 (median
1.4 x 103 CFU/ml, range 30–1.33 x
104 CFU/ml) and mice infected with H377 (median
1.1 x 102 CFU/ml, range 0–4.0 x
104 CFU/ml) (Fig. 5A). Colonies (25 of 25)
cultured from H377-infected mice were Ery-resistant and PCR analysis
confirmed that the smez mutation was stable in all
isolates.
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B). At 48
h, only one of five mice in each group was bacteremic, consistent with
previous data using this bacterial strain. Cytokine levels in serum and peritoneal aspirates from H293- and H377-infected mice
TNF-, IFN-, and IL-6 levels were measured in peritoneal
aspirates obtained from H293- and H377-infected C57BL/10.DQ8 mice, 48
or 60 h after infection, in separate experiments. TNF- and
IFN- levels were higher in the H293-infected groups compared with
the H377-infected group in both the experiments (at 48 and 60 h),
though these differences were not significant. IL-6 levels were higher
in the H293-infected group compared with the H377-infected group at
both time points; differences in IL-6 levels were statistically
significant (Mann-Whitney U test) (Fig. 6). Levels of cytokines in serum were at
the lower limits of detection; there were no detectable differences
between the two groups.
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Sera and peritoneal aspirates obtained at 60 h from
C57BL/10.DQ8 mice infected with H293 or H377 were used to stimulate
single donor human PBMC proliferation. Peritoneal aspirates from four
of five H293-infected mice stimulated significant proliferation of
human PBMCs, whereas aspirates from H377-infected mice and control
peritoneal lavage fluid did not. Thus, the mitogenic activity of
peritoneal lavage fluid from H293-infected mice could be attributed to
SMEZ (Fig. 7A). Serum from
three of five H293-infected mice induced significant proliferation of
human PBMCs compared with H377-infected mice and control mouse serum,
though this difference was not significant as not all H293-infected
mice had detectable mitogenic activity in serum (Fig. 7B).
The data confirmed that SMEZ was produced at the site of infection in
H293-infected mice. The relation between rSMEZ-13 concentration and
donor PBMC proliferation, measured as cpm tritiated thymidine
incorporation, was plotted (not shown) and was used to derive rough
estimates for SMEZ concentrations in peritoneal lavage fluid (median
600 fg/ml, range 0–200 pg/ml) and serum (median 1 pg/ml, range 0–7
ng/ml). As SMEZ in the peritoneum had been diluted by lavage,
concentrations of SMEZ at the serosal surface of i.p. organs may be 40-
to 50-fold higher than those found in lavage fluid. Body fluids contain
several cytokines which may alter the SMEZ-induced proliferative
response in vitro, and these figures must be regarded as estimates
only.
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-bearing T cell subsets expanded during
infection
Intraperitoneal infection with S. pyogenes H293 led to
an increase in the proportion of V11+ T cells
in murine spleen. There was a small but statistically significant
decrease in the V11+ T cell population of
H377-infected mice, compared with those of the H293-infected mice.
There were no significant differences in the proportions of
V8+ T cells in spleens from both groups of
mice. Interestingly, a small but statistically significant increase in
V4+ T cells in H293-infected mice was
dramatically enhanced when mice were infected with the SMEZ-negative
strain H377 (Table IV).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In stark contrast to other secreted proteins made by S.
pyogenes, transcripts for smez were almost
undetectable, in keeping with the extremely low concentrations of SMEZ
protein produced in broth culture. Whereas transcripts for other
secreted proteins are maximally detected at stationary phases of growth
(7), transcripts for smez were only detected by
RT-PCR at late-log phase; whether this reflects mRNA instability or
constitutive repression of the smez promoter is unknown.
This feature was not unique to the M89 serotype studied, as transcripts
for smez were also not detected in the M1 serotype strain
H305 used in previous studies. In both cases, mf and
emm transcripts were easily detected by Northern
hybridization. As smez is less than 20 kb from the
mga virulence regulon which regulates expression of
genes such as emm, it is possible that regulation of this
superantigen is interconnected with regulation of virulence factors
involved in bacterial survival in the host. Despite the low levels of
SMEZ protein present in supernatant from H293, this study demonstrates
that the mitogenic activity of streptococcal supernatant in human PBMC
culture is largely attributable to SMEZ. Indeed, proliferation of
V8-expressing Jurkat cells is the most reliable and sensitive method
of detecting SMEZ production by streptococci (3). These
findings contrast with our previous studies, which demonstrated that MF
(shown to be DNaseB) did not contribute to the mitogenic activity of
the same M89 strain (6). In an earlier study, we
demonstrated that SPEA contributed very little to the overall mitogenic
activity of an M1 strain in human PBMC culture, despite SPEA
concentrations in excess of 500 ng/ml in culture supernatants from the
parent strain (5). We conclude that the tiny amounts of
SMEZ present in culture supernatants from H293, calculated by bioassay
to be 
5 pg/ml, are largely responsible for the mitogenic activity of
this strain.
Native preparations of a wide range of streptococcal proteins,
including M protein, SPEA, SPEB, and MF were previously reported to
selectively stimulate human T cells bearing TCR V8
(11, 12, 13, 14, 15). More recently a previously unknown streptococcal
superantigen, SPEX (identical to SMEZ) was shown to underlie the
potent stimulation of human V8+ T cells
attributed to SPEB and MF (16, 17). As the quantities of
SMEZ required for stimulation of human T cells are 2–3 log-fold less
than other streptococcal toxins and as SMEZ copurifies with other
streptococcal proteins (10), we speculated that all the
TCR V8-stimulatory activity of S. pyogenes could be
attributed to SMEZ, or contamination with SMEZ. Using an isogenic
mutant streptococcal strain, deficient in SMEZ production, this work
confirms that SMEZ is responsible for all detectable S.
pyogenes supernatant-induced TCR V8 human T cell proliferation.
Streptococcal supernatant from both the parent and smez
mutant strain failed to induce detectable expansion of human T cells
bearing TCR V2, the subset targeted by the recently characterized
superantigen SPEJ (18, 19), consistent with the low level
of residual mitogenicity in the smez mutant strain.
The studies described demonstrate unequivocally that SMEZ is the single
most important stimulus to cytokine production from S.
pyogenes-stimulated human mononuclear cells. Indeed, supernatant
from the smez- mutant did not elicit
detectable cytokine production from human cells, when compared with
medium alone. This may be because SMEZ is the only significant stimulus
present in supernatant which can lead to cytokine production, or, more
likely, because SMEZ is an essential cofactor which synergizes with
other streptococcal proteins. Very recently, it was reported that a
native preparation of the SMEZ-16 allele was 10-fold more active than a
native preparation of SPEA when cytokine production from human
mononuclear cells was quantified (20). The
cytokine-stimulating role of a range of streptococcal secreted proteins
has been extensively reported (21, 22); data from the
current study underpin the value of examining protein function in a
physiological context, using supernatants from isogenic strains which
are free of endotoxin contamination. We and others have shown that
levels of cytokines such as TNF- (lymphotoxin-) and IFN- are
produced by superantigen-stimulated human mononuclear cells in a
time-dependent manner, peaking at 72–96 h after stimulation,
consistent with the pattern of superantigen-induced T cell mitogenesis
(23, 24, 25). Despite the potent promitogenic activity of
SMEZ, the rapidity of SMEZ-dependent cytokine production, seen at
24 h, and the SMEZ-dependent induction of monokines such as IL-1
and IL-8 was unexpected. The possibility that the smez
mutant had sustained a nonspecific phenotypic change during genetic
manipulation was considered but discounted, because expression of other
virulence factors was unchanged, and because the strains grew equally
well in both broth and whole blood. It was felt unlikely that
expression of recognized superantigens such as SPEG would be affected
by the mutation, as the gene encoding SMEZ is distant from genes
encoding other secreted toxins. Indeed, transcription of mf
and speg was unaffected by the mutation. Although
smez is adjacent to dpp and a cluster of genes
regulated by mga, none of these latter virulence factors is
associated with T cell mitogenesis. Transcripts for the
mga-regulated virulence factor emm89 are
detectable by Northern blotting to equal degrees in both H293 and H377
while transcription of dpp is not detectable in the M89 strain
described in this study (M. Unnikrishnan, unpublished observations).
Furthermore, the abrogation of cytokine induction was specific to
secreted proteins made by the smez-
mutant, as the smez mutation did not affect TNF- or
IFN- induction by whole washed heat-killed streptococci (S.
Sriskandan, not shown).
Investigation of the role played by superantigens in bacterial
pathogenesis has been hampered by a lack of disease models which are
superantigen-sensitive and which can be investigated using readily
available immunological tools. We recently demonstrated that mice
expressing human HLA-DQ transgenes were highly sensitive to SPEA, both
in vitro and in vivo, compared with wild-type mice. Although
HLA-DQ-dependent immune activation during invasive infection with a
SPEA-producing M1 S. pyogenes was largely dependent on SPEA
production in this artificial system, mortality was unaffected by
disruption of the gene encoding SPEA (26). From an
evolutionary standpoint, it is likely that expression of superantigens
favors bacterial survival or dissemination within the host range,
rather than destruction of the host and consequent abortive infection.
In this study, HLA transgenic mice were used to investigate the role
played by the ubiquitous streptococcal superantigen SMEZ in
pathogen-host interactions. SMEZ binds exclusively to the MHC class II
-chain in a zinc-dependent manner (27). APCs which are
transfected with HLA-DQ-B1*0302 or HLA-DQ-B1*0401 present rSMEZ-13 to
Jurkat cells more efficiently than cells which express HLA-DR1 (T.
Proft, unpublished observations). Spleen cells from HLA-DQ8 transgenic
mice (expressing HLA-DQ-B1*0302) were clearly more
responsive to rSMEZ-13 than HLA-DR1 transgenics, and subsequent in vivo
work was therefore conducted in this murine strain.
In vivo experiments using an i.p. model of infection demonstrated that SMEZ did not contribute to mortality or impede bacterial clearance in this site. However, the peritoneum is an unusual setting for GAS infection and one cannot exclude a role for SMEZ in the pathogenesis of mucosal or skin surface infection, carriage, or reinfection. In particular, assessment of the role of SMEZ in nasopharyngeal streptococcal infection is the focus of future work. One major aim of the experiments described was to investigate whether SMEZ was produced during infection. Data obtained using peritoneal lavage fluid and serum from mice infected with isogenic streptococcal strains differing only in SMEZ production demonstrated that mitogenic activity in these fluids during infection is attributable to SMEZ. In separate work, we have detected a highly potent mitogen in the serum of some patients with streptococcal toxic shock and mice infected with M1 S. pyogenes; mitogenic activity was inhibited by polyclonal anti-SMEZ serum, but not by polyclonal antisera to other streptococcal superantigens, confirming that the dominant promitogenic toxin produced during invasive infection is SMEZ (T. Proft, M. Unnikrishnan, S. Sriskandan, manuscript in preparation).
IL-6 levels were measured in S. pyogenes-infected mice as a
parameter of severity of sepsis, as levels are known to correlate with
mortality (28). TNF- and IFN- were measured, as
these proinflammatory cytokines can mediate outcome in
superantigen-exposed mice (29). Consistent with previous
studies of streptococcal sepsis, significant elevation of cytokines was
not seen in serum of infected mice. In contrast, it was possible to
detect local production of cytokines in the peritoneum of mice infected
with the parent streptococcal strain; this was reduced in those
infected with the smez- mutant strain.
Although the entire murine T cell repertoire could not be investigated
in this study, it is likely that murine TCR V11 is the principal
target of SMEZ. Murine V11 is the closest homologue of human V8
and demonstrates 71–77% identity at the nucleotide level
(30). Infection with S. pyogenes led to an
increase in V11+ T cells in murine spleen,
which was reduced in mice infected with the smez mutant.
Coupled with the cytokine data, the results are consistent with an in
vivo superantigen response. However, remarkably, mice infected with the
smez mutant demonstrated a clear increase in the proportion
of CD4+V4+ cells at
48 h. Skewing of the T cell repertoire was not seen in spleen
cells cultured in vitro with supernatants from the smez
mutant strain. Thus, the in vivo change in T cell repertoire is
unlikely to be a residual effect of other superantigens expressed
by the smez mutant. CD4+ V-specific
Ag-specific immune responses can be detected early in the course of an
infection (31). We speculate that exposure to SMEZ may
suppress expression of TCR V subsets which are of importance in the
development of cognate responses to streptococcal Ags. The development
of isogenic streptococcal mutants which differ only in toxin production
and HLA class II transgenic murine models which are
superantigen-sensitive are critical steps toward understanding the role
played by superantigens in evasion of the host immune response.
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2 Address correspondence and reprint requests to Dr. Shiranee Sriskandan, Department of Infectious Diseases, Faculty of Medicine, Imperial College School of Science, Technology, and Medicine, Hammersmith Hospital, Du Cane Road, London, W12 0NN, U.K. E-mail address: s.sriskandan{at}ic.ac.uk
3 Abbreviations used in this paper: GAS, group A streptococci; SMEZ, streptococcal mitogenic exotoxin Z; SPE, streptococcal pyrogenic exotoxin; SSA, streptococcal superantigen; MF, mitogenic factor; THY, Todd Hewitt Yeast broth; Ery, erythromycin; Aph, 3'-aminoglycoside phosphotransferase; DIG, digoxygenin; RT, reverse transcription; SEB, staphylococcal enterotoxin B.
Received for publication October 11, 2001. Accepted for publication June 28, 2002.
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), or RPMI (----) only. Right panels,
Time-dependent cytokine production in human PBMC in response to 100
pg/ml rSMEZ-13 (
). A, IFN-
